Note: Descriptions are shown in the official language in which they were submitted.
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Modified Clostridial Toxins with Enhanced Translocation Capabilities and
Altered Targeting Activity For Non-Clostridial Toxin Target Cells
[01] This Non-Provisional Patent Application claims priority pursuant to 35
U.S.C. 119(e) to U.S.
Provisional Patent Application Serial No. 60/807,059 filed July 11, 2006,
which is hereby incorporated by
reference in its entirety.
[02] All patents and publications cited in this application are hereby
incorporated by reference in their
entirety.
[03] The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins
(BoNTs), BoNT/A, BoNT/B,
BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and Tetanus neurotoxin (TeNT), to
inhibit neuronal
transmission are being exploited in a wide variety of therapeutic and cosmetic
applications, see e.g.,
William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OF BOTULINUM TOXIN
(Slack, Inc., 2004).
Clostridial toxins commercially available as pharmaceutical compositions
include, BoNT/A preparations,
such as, e.g., BOTOX (Allergan, Inc., Irvine, CA), Dysport /Reloxin ,
(Beaufour Ipsen, Porton Down,
England), Linurase (Prollenium, Inc., Ontario, Canada), Neuronox (Medy-Tox,
Inc., Ochang-myeon,
South Korea) BTX-A (Lanzhou Institute Biological Products, China) and Xeomin
(Merz Pharmaceuticals,
GmbH., Frankfurt, Germany); and BoNT/B preparations, such as, e.g.,
MyoBlocT"'/NeuroBlocT"' (Elan
Pharmaceuticals, San Francisco, CA). As an example, BOTOX is currently
approved in one or more
countries for the following indications: achalasia, adult spasticity, anal
fissure, back pain, blepharospasm,
bruxism, cervical dystonia, essential tremor, glabellar lines or hyperkinetic
facial lines, headache,
hemifacial spasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral
palsy, multiple sclerosis,
myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismus and
VII nerve disorder.
[04] A Clostridial toxin treatment inhibits neurotransmitter release by
disrupting the exocytotic process
used to secret the neurotransmitter into the synaptic cleft. There is a great
desire by the pharmaceutical
industry to expand the use of Clostridial toxin therapies beyond its current
myo-relaxant applications to
treat sensory-based ailment, such as, e.g., various kinds of chronic pain, as
well as non-neuronal based
disorders, such as, e.g., pancreatitis. One approach that is currently being
exploited to expand Clostridial
toxin-based therapies involves modifying a Clostridial toxin so that the
modified toxin has an altered cell
targeting capability for a non-Clostridial toxin target cell. This re-targeted
capability is achieved by
replacing a naturally-occurring targeting domain of a Clostridial toxin with a
targeting domain showing a
selective binding activity for a non-Clostridial toxin receptor present in a
non-Clostridial toxin target cell.
Such modifications to a targeting domain result in a modified toxin that is
able to selectively bind to a non-
Clostridial toxin receptor (target receptor) present on a non-Clostridial
toxin target cell (re-targeted). A
modified Clostridial toxin with an altered targeting activity for a non-
Clostridial toxin target cell can bind to
a target receptor, translocate into the cytoplasm, and exert its proteolytic
effect on the SNARE complex of
the non-Clostridial toxin target cell.
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[05] Non-limiting examples of modified Clostridial toxins with an altered
targeting activity for a non-
Clostridial toxin target cell are described in, e.g., Keith A. Foster et al.,
Clostridial Toxin Derivatives Able
To Modify Peripheral Sensory Afferent Functions, U.S. Patent 5,989,545 (Nov.
23, 1999); Clifford C.
Shone et al., Recombinant Toxin Fragments, U.S. Patent 6,461,617 (Oct. 8,
2002); Conrad P. Quinn et
al., Methods and Compounds for the Treatment of Mucus Hypersecretion, U.S.
Patent 6,632,440 (Oct.
14, 2003); Lance E. Steward et al., Methods And Compositions For The Treatment
Of Pancreatitis, U.S.
Patent 6,843,998 (Jan. 18, 2005); Stephan Donovan, Clostridial Toxin
Derivatives and Methods For
Treating Pain, U.S. Patent Publication 2002/0037833 (Mar. 28, 2002); Keith A.
Foster et al., Inhibition of
Secretion from Non-neural Cells, U.S. Patent Publication 2003/0180289 (Sep.
25, 2003); J. Oliver Dolly et
al., Activatable Recombinant Neurotoxins, WO 2001/014570 (Mar. 1, 2001); Keith
A. Foster et al., Re-
targeted Toxin Conjugates, International Patent Publication WO 2005/023309
(Mar. 17, 2005); and Lance
E. Steward et al., Multivalent Clostridial Toxin Derivatives and Methods of
Their Use, U.S. Patent
Application No. 11/376,696 (Mar. 15, 2006). The ability to re-target the
therapeutic effects associated
with Clostridial toxins has greatly extended the number of medicinal
applications able to use a Clostridial
toxin therapy. As a non-limiting example, modified Clostridial toxins
retargeted to sensory neurons are
useful in treating various kinds of chronic pain, such as, e.g., hyperalgesia
and allodynia, neuropathic
pain and inflammatory pain, see, e.g., Foster, supra, (1999); and Donovan,
supra, (2002); and Stephan
Donovan, Method For Treating Neurogenic Inflammation Pain with Botulinum Toxin
and Substance P
Components, U.S. Patent 7,022,329 (Apr. 4, 2006). As another non-limiting
example, modified Clostridial
toxins retargeted to pancreatic cells are useful in treating pancreatitis,
see, e.g., Steward, supra, (2005).
[06] The present invention provides novel Clostridial toxins that greatly
extended the number of
therapeutic applications that can exploit the advantages offered by current
Clostridial toxin therapies.
These modified Clostridial toxins comprise, in part, a translocation
facilitating domain that enhances the
process by which a light chain from a modified toxin translocates into the
cytoplasm of a target cell and
enzymatically modify its target SNARE substrate. Thus modified Clostridial
toxins with an altered
targeting activity for a non-Clostridial toxin target cell, such as those
disclosed in, e.g., Foster, supra,
(1999), Dolly, supra, (2001), Donovan, supra, (2002), Shone, supra, (2002),
Foster, supar, (2003), Quinn,
supra, (2003), Foster, supra, (2005), Steward, supra, (2005) and Steward,
supra, (2006) comprising a
translocation facilitating domain disclosed in the present specification,
exhibit increased potency at the
non-Clostridial toxin target cell. These and related advantages are useful for
various clinical, therapeutic
and cosmetic applications, such as, e.g., the treatment of neuropathic
disorders, eye disorders, pain,
muscle injuries, headache, cardiovascular diseases, neuropsychiatric
disorders, endocrine disorders,
cancers, otic disorders, as well as, other disorders where administration of a
modified Clostridial toxin with
an altered targeting activity for a non-Clostridial toxin target cell to an
individual can produce a beneficial
effect.
BRIEF DESCRIPTION OF THE DRAWINGS
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[07] FIG. 1 shows a schematic of the current paradigm of neurotransmitter
release and Clostridial toxin
intoxication in a central and peripheral neuron. FIG. 1A shows a schematic for
the neurotransmitter
release mechanism of a central and peripheral neuron. The release process can
be described as
comprising two steps: 1) vesicle docking, where the vesicle-bound SNARE
protein of a vesicle containing
neurotransmitter molecules associates with the membrane-bound SNARE proteins
located at the plasma
membrane; and 2) neurotransmitter release, where the vesicle fuses with the
plasma membrane and the
neurotransmitter molecules are exocytosed. FIG. 1 B shows a schematic of the
intoxication mechanism
for tetanus and botulinum toxin activity in a central and peripheral neuron.
This intoxication process can
be described as comprising four steps: 1) receptor binding, where a
Clostridial toxin binds to a Clostridial
receptor and initiates the intoxication process; 2) complex internalization,
where after toxin binding, a
vesicle containing the toxin/receptor complex is endocytosed into the cell; 3)
light chain translocation,
where multiple events result in the release of the active light chain into the
cytoplasm; and 4) enzymatic
target modification, where the active light chain of Clostridial toxin
proteolytically cleaves its target SNARE
substrate, such as, e.g., SNAP-25, VAMP or Syntaxin, thereby preventing
vesicle docking and
neurotransmitter release.
[08] FIG. 2 shows the domain organization of naturally-occurring Clostridial
toxins. The single chain form
depicts the amino to carboxyl linear organization comprising an enzymatic
domain, a translocation
domain, a HCN translocation facilitating domain and a Hcc targeting domain.
The di-chain loop region
located between the translocation and enzymatic domains is depicted by the
double SS bracket. This
region comprises an endogenous di-chain loop protease cleavage site that upon
proteolytic cleavage with
a naturally-occurring protease, such as, e.g., an endogenous Clostridial toxin
protease or a naturally-
occurring protease produced in the environment, converts the single chain form
of the toxin into the di-
chain form. As depicted above the single-chain form, the Hcc targeting domain
comprises the (3-trefoil
domain which comprises in an amino to carboxyl linear organization of an a-
fold, a(34/(35 hairpin turn, a(3-
fold, a(38/(39 hairpin turn and a y-fold.
[09] FIG. 3 shows a ribbon diagram of BoNT/A illustrating the modular three-
dimensional structure of the
light chain (LC) comprising the enzymatic domain, the heavy chain HN domain
comprising the
translocation domain, the heavy chain HCN domain comprising the translocation
facilitating domain and
the heavy chain Hcc domain comprising the targeting domain.
[010] FIG. 4 shows modified Clostridial toxins with an enhanced translocation
capability and an altered
targeting activity located at the amino terminus of the modified toxin. FIG.
4A depicts the single
polypeptide form of a modified Clostridial toxin with an amino to carboxyl
linear organization comprising
an altered targeting domain, a translocation domain, a translocation
facilitating domain and an enzymatic
domain,, with the di-chain loop region depicted by the double SS bracket. A
proteolytic cleavage site (P)
within a di-chain loop region is located between the translocation
facilitating and enzymatic domains.
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Upon proteolytic cleavage with a P protease, the single chain form of the
toxin is converted to the di-chain
form. The P protease site can be a Clostridial toxin endogenous protease
cleavage site or a non-
Clostridial toxin exogenous protease cleavage site. Spacers can be placed
between the targeting and
translocation domains, the translocation and translocation facilitating
domains, translocation facilitating
and enzymatic domains or any combination thereof. FIG. 4B depicts the single
polypeptide form of a
modified Clostridial toxin with an amino to carboxyl linear organization
comprising an altered targeting
domain, an enzymatic domain, a translocation domain and a translocation
facilitating domain, with the di-
chain loop region depicted by the double SS bracket. A proteolytic cleavage
site (P) within a di-chain
loop region is located between the enzymatic and translocation domains. Upon
proteolytic cleavage with
a P protease, the single chain form of the toxin is converted to the di-chain
form. The P protease site can
be a Clostridial toxin endogenous protease cleavage site or a non-Clostridial
toxin exogenous protease
cleavage site. Spacers can be placed between the targeting and enzymatic
domains, the enzymatic and
translocation domains, translocation and translocation facilitating domains or
any combination thereof.
[011] FIG. 5 shows modified Clostridial toxins with an enhanced translocation
capability and an altered
targeting activity located between two other domains. FIG. 5A depicts the
single polypeptide form of a
modified Clostridial toxin with an amino to carboxyl linear organization
comprising an enzymatic domain,
an altered targeting domain, a translocation domain and a translocation
facilitating domain, with the di-
chain loop region depicted by the double SS bracket. A proteolytic cleavage
site (P) within a di-chain
loop region is located between the enzymatic and targeting domains. Upon
proteolytic cleavage with a P
protease, the single chain form of the toxin is converted to the di-chain
form. The P protease site can be
a Clostridial toxin endogenous protease cleavage site or a non-Clostridial
toxin exogenous protease
cleavage site. Spacers can be placed between the enzymatic and targeting
domains, the targeting and
translocation domains, the translocation and translocation facilitating
domains or any combination thereof.
FIG. 5B depicts the single polypeptide form of a modified Clostridial toxin
with an amino to carboxyl linear
organization comprising a translocation domain, a translocation facilitating
domain, an altered targeting
domain and an enzymatic domain, with the di-chain loop region depicted by the
double SS bracket. A
proteolytic cleavage site (P) within a di-chain loop region is located between
the translocation facilitating
and targeting domains. Upon proteolytic cleavage with a P protease, the single
chain form of the toxin is
converted to the di-chain form. The P protease site can be a Clostridial toxin
endogenous protease
cleavage site or a non-Clostridial toxin exogenous protease cleavage site.
Spacers can be placed
between the translocation and translocation facilitating domains, the
translocation facilitating and
targeting domains, the targeting and enzymatic domains or any combination
thereof.
[012] FIG. 6 shows modified Clostridial toxins with an enhanced translocation
capability and an altered
targeting activity located at the carboxyl terminus of the modified toxin.
FIG. 6A depicts the single
polypeptide form of a modified Clostridial toxin with an amino to carboxyl
linear organization comprising
an enzymatic domain, a translocation domain, a translocation facilitating
domain and an altered targeting
domain, with the di-chain loop region depicted by the double SS bracket. A
proteolytic cleavage site (P)
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within a di-chain loop region is located between the enzymatic and
translocation domains. Upon
proteolytic cleavage with a P protease, the single chain form of the toxin is
converted to the di-chain form.
The P protease site can be a Clostridial toxin endogenous protease cleavage
site or a non-Clostridial
toxin exogenous protease cleavage site. Spacers can be placed between the
enzymatic and
translocation domains, the translocation and translocation facilitating
domains, the translocation
facilitating and targeting domains or any combination thereof. FIG. 6B depicts
the single polypeptide form
of a modified Clostridial toxin with an amino to carboxyl linear organization
comprising a translocation
domain, a translocation facilitating domain, an enzymatic domain and an
altered targeting domain, with
the di-chain loop region depicted by the double SS bracket. A proteolytic
cleavage site (P) within a di-
chain loop region is located between the translocation facilitating and
enzymatic domains. Upon
proteolytic cleavage with a P protease, the single chain form of the toxin is
converted to the di-chain form.
The P protease site can be a Clostridial toxin endogenous protease cleavage
site or a non-Clostridial
toxin exogenous protease cleavage site. Spacers can be placed between the
translocation and
translocation facilitating domains, the translocation facilitating and
enzymatic domains, the enzymatic and
targeting domains or any combination thereof.
[013] FIG. 7 shows a ribbon diagram of BoNT/A illustrating the boundary
regions of the HCN domain
comprising the translocation facilitating domain. FIG. 7A depicts the amino-
terminal boundary region.
FIG. 7B depicts the carboxyl-terminal boundary region.
DETAILED DESCRIPTION
[014] Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,
Clostridium baratii and
Clostridium butyricum are the most widely used in therapeutic and cosmetic
treatments of humans and
other mammals. Strains of C. botulinum produce seven antigenically-distinct
types of Botulinum toxins
(BoNTs), which have been identified by investigating botulism outbreaks in man
(BoNT/A, /B, /E and /F),
animals (BoNT/C1 and /D), or isolated from soil (BoNT/G). While all seven
botulinum toxins (BoNT)
serotypes have similar structure and pharmacological properties, each also
displays heterogeneous
bacteriological characteristics. In contrast, tetanus toxin (TeNT) is produced
by a uniform group of C.
tetani. Two other species of Clostridia, C. baratii and C. butyricum, also
produce toxins similar to BoNT/F
and BoNT/E, respectively.
[015] Clostridia toxins possess approximately 35% amino acid identity with
each other and share the
same functional domain organization and overall structural architecture.
Clostridial toxins are each
translated as a single chain polypeptide of approximately 150 kDa that is
subsequently cleaved by
proteolytic scission within a disulfide loop by a naturally-occurring
protease, such as, e.g., an endogenous
Clostridial toxin protease or a naturally-occurring protease produced in the
environment (see FIG. 2).
This posttranslational processing yields a di-chain molecule comprising an
approximately 50 kDa light
chain (LC) and an approximately 100 kDa heavy chain (HC) held together by a
single disulfide bond and
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noncovalent interactions. It is widely held that the mature di-chain molecule
comprises three functionally
distinct domains: 1) an enzymatic domain located in the LC that includes a
metalloprotease region
containing a zinc-dependent endopeptidase activity which specifically targets
core components of the
neurotransmitter release apparatus (Table 1); 2) a translocation domain
contained within the amino-
terminal half of the heavy chain (HN domain) that facilitates release of the
LC from intracellular vesicles
into the cytoplasm of the target cell (Table 1); and 3) a binding domain found
within the carboxyl-terminal
half of the heavy chain (Hc domain) that determines the binding activity and
binding specificity of the toxin
to the receptor complex located at the surface of the target cell (Table 1),
see, e.g., Kathryn Turton et al.,
Botulinum and Tetanus Neurotoxins: Structure, Function and Therapeutic
Utility, 27(11) Trends Biochem.
Sci. 552-558. (2002); John A. Chaddock and P. M. H. Marks, Clostridial
Neurotoxins: Structure-Function
Led Design of New Therapeutics, 63(5) Cell. Mol. Life Sci. 540-551 (2006); and
Keith Foster et al., Re-
engineering the Target Specificity of Clostridial Neurotoxins - A Route To
Novel Therapeutics, 9(2-3)
Neurotox Res. 101-107 (2006).
Table 1. Clostridial Toxin Reference Sequences and Regions
Hc
Toxin SEQ ID NO: LC HN
HCN Hcc
BoNT/A 1 M1-K448 A449-1873 1874-P1110 Y1111-L1296
BoNT/B 2 M1-K441 A442-1860 L861-E1097 Y1098-E1291
BoNT/C1 3 M1-K449 T450-1868 N869-E1111 Y1112-E1291
BoNT/D 4 M1-R445 D446-1864 N865-E1098 Y1099-E1276
BoNT/E 5 M1-R422 K423-1847 K848-E1085 Y1086-K1252
BoNT/F 6 M1-K439 A440-1866 K867-K1105 Y1106-E1274
BoNT/G 7 M1-K446 S447-1865 S866-Q1105 Y1106-E1297
TeNT 8 M1-A457 S458-L881 K882-E1127 Y1128-D1315
[016] The binding, translocation and enzymatic activities of a Clostridial
toxin are all necessary to
execute the overall cellular intoxication mechanism whereby Clostridial toxins
enter a neuron and inhibit
neurotransmitter release is similar, regardless of serotype or subtype. The
current paradigm describes
the intoxication mechanism as comprising at least four steps: 1) receptor
binding, 2) complex
internalization, 3) light chain translocation, and 4) enzymatic target
modification (see FIG. 1). The
process is initiated when the Hc domain of a Clostridial toxin binds to a
toxin-specific receptor located on
the plasma membrane surface of a target cell. The binding specificity of a
receptor complex is thought to
be achieved, in part, by specific combinations of gangliosides and protein
receptors that appear to
distinctly comprise each Clostridial toxin receptor complex. Once bound, the
toxin/receptor complexes
are internalized by endocytosis and the internalized vesicles are sorted to
specific intracellular routes.
The translocation step, now thought to be mediated by the HN domain and
further facilitated by the HCN
domain, appears to be triggered by the acidification of the vesicle
compartment. This process seems to
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initiate two important pH-dependent structural rearrangements that increase
hydrophobicity and promote
separation of the light chain from the heavy chain of the toxin. Once
activated, light chain endopeptidase
of the toxin is released from the intracellular vesicle into the cytosol where
it appears to specifically target
one of three known core components of the neurotransmitter release apparatus.
These core proteins,
vesicle-associated membrane protein (VAMP)/synaptobrevin, synaptosomal-
associated protein of 25 kDa
(SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking and fusion
at the nerve terminal and
constitute members of the soluble N-ethylmaleimide-sensitive factor-attachment
protein-receptor
(SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl-terminal
region, releasing a nine
or twenty-six amino acid segment, respectively, and BoNT/C1 also cleaves SNAP-
25 near the
carboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G,
and tetanus toxin,
act on the conserved central portion of VAMP, and release the amino-terminal
portion of VAMP into the
cytosol. BoNT/C1 cleaves syntaxin at a single site near the cytosolic membrane
surface. The selective
proteolysis of synaptic SNAREs accounts for the block of neurotransmitter
release caused by Clostridial
toxins in vivo. The SNARE protein targets of Clostridial toxins are common to
exocytosis in a variety of
non-neuronal types; in these cells, as in neurons, light chain peptidase
activity inhibits exocytosis, see,
e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins Block
Neurotransmitter Release,
82(5) Biochimie. 427-446 (2000); and Giovanna Lalli et al., The Journey of
Tetanus and Botulinum
Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437, (2003).
[017] The three-dimensional crystal structures of BoNT/A, BoNT/B and the Hc
domain of TeNT indicate
that the three functional domains of Clostridial neurotoxins are structurally
distinct (see FIG. 3). The
HEXXH consensus motif of the light chain forms the tetrahedral zinc binding
pocket of the catalytic site
located in a deep cleft on the protein surface that is accessible by a
channel. The structure of the HN and
Hc domains consists primarily of (3-sheet topologies that are linked by a
single a-helix. The cylindrical-
shaped HN domain comprises two long amphipathic a-helices that resemble the
coiled-coil motif found in
some viral proteins. The HN domain also forms a long unstructured loop called
the `translocation belt,'
which wraps around a large negatively charged cleft of the light chain that
blocks access of the zinc atom
to the catalytic-binding pocket of active site. The Hc domain comprises two
distinct structural features of
roughly equal size that indicate function. The first, designated the HCN
domain, is located in the amino
half of the Hc domain. The HCN domain forms a(3-barrel, jelly-roll fold. The
Hcc domain is the second
domain that comprises the Hc domain. This carboxyl-terminal domain comprises a
modified (3-trefoil
domain which forms three distinct carbohydrate binding regions that resembles
the carbohydrate binding
moiety found in many sugar-binding proteins, such as, e.g., serum amyloid P,
sialidase, cryia, insecticidal
a-endotoxin and lectins. Biochemical studies indicate that the (3-trefoil
domain structure of the Hcc
domain appears to mediate the binding to specific carbohydrate containing
components of the Clostridial
toxin receptor on the cell surface, see, e.g., Krzysztof Ginalski et al.,
Structure-based Sequence
Alignment for the Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family
Provides Residue Level
Information About the Putative Ganglioside Binding Site, 482(1-2) FEBS Lett.
119-124 (2000). The Hc
domain tilts away from the HN domain exposing the surface loops and making
them accessible for
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binding. No contacts occur between the light chain and the Hc domain.
[018] We know that only the Hcc domain participates in receptor binding
because the (3-trefoil domains
are restricted to this domain. Proteins containing the structural (3-trefoil
domain represents a diverse
group of proteins organized into at least eight superfamilies including the
cytokines, MIR domain proteins,
Ricin B-like lectins, agglutinins, Soybean trypsin inhibitor like proteins,
Actin-crosslinking proteins, LAG-1
proteins and AbfB domain proteins, see, e.g., C. A. Orengo et al., Protein
Superfamilies and Domain
Superfolds, 372 Nature 631-634 (1994); and Alexey G. Murzin et al., SCOP: A
Structural Classification of
Proteins Database for the Investigation of Sequences and Structures, 247(4) J.
Mol. Biol. 536-540 (1995).
While having diverse cellular roles, members of these superfamilies
mechanistically function via protein-
protein associations through the (3-trefoil domain. Of particular interest is
the fact that many of these
members are specifically involved in receptor interactions, including, e.g.,
the cytokine superfamily
members Fibroblast Growth Factors (FGFs) and the Interleukin-1s (IL-1s); the
Ricin B-like lectins; the
agglutinins; and STI-like members the Kunitz inhibitors and Clostridium
neurotoxins. That only the Hcc
domain alone mediates the cell binding step of intoxication is further
supported by the finding that
mutations that disrupt the receptor binding activity of Clostridial toxins
have been confined to the Hcc
domain, see, e.g., Andreas Rummel et al., The Hcc-Domain of Botulinum
Neurotoxins A and B Exhibits a
Singular Ganglioside Binding Site Displaying Serotype Specific Carbohydrate
Interaction, 51(3) Mol.
Microbiol. 631-643 (2004).
[019] Because the Hcc domain appears not only necessary, but sufficient for
selective binding of a
Clostridial toxin to its receptor, we have deduced that the primary function
of the HCN domain of Clostridial
toxins is involved in the translocation step of the intoxication process, and
not in the cell binding step,
because the lack of HCN domain appears to reduce intoxication efficiency. For
example, a modified
BoNT/A comprising a Substance P targeting domain was inefficient in
intoxicating its corresponding target
cells. In this modified toxin, the entire BoNT/A Hc domain, comprising both
the BoNT/A Hcc domain and
the BoNT/A HCN domain, was replaced by the Substance P targeting domain.
Likewise, we have
determined that several other modified Clostridial toxins that have replaced
the entire BoNT/A Hc domain
with an exogenous targeting domain have exhibited reduced intoxication
capabilities. Thus, the HCN
domain possess a translocation facilitating function because 1) Hcc domain
primarily mediates the
receptor binding step of the intoxication process; 2) modified Clostridial
toxins lacking the HCN domain
exhibit a reduced ability to translocate into the cytoplasm as evident by such
modified toxins exhibiting
decreased proteolysis of their SNARE substrates; and 3) the LC domain mediates
the enzymatic activity
of the toxin. While the exact translocation facilitating mechanism of the HCN
domain is currently not
understood, the HCN domain may 1) participate in the formation of an endosomal
pore; 2) mediate the
insertion of the pore into a vesicle membrane; 3) assist in the delivery of LC
across the endosomal
membrane and/or 4) serve as a structural scaffold or spacer that facilitates
the appropriate orientation a
the targeting domain in relationship to the translocation domain. In this last
point, the HCN domain would
serve to orient the translocation domain to facilitate the proper presentation
of the translocation domain
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for insertion into the membrane following binding of the ligand by the
receptor. This novel role of teh HCN
domain in the translocation step is contrary to the widely accepted view that
the Clostridial toxin HCN
domain played an integral role in the cell binding step of the intoxication
process.
[020] Thus, the present invention discloses modified Clostridial toxins that
exhibit 1) an enhanced
translocation capability; and 2) an altered targeting capability for a
naturally-occurring Clostridial toxin
target cell. The enhanced translocation capability is mediated by a
translocation facilitating domain
comprising, e.g., a HCN region of Clostridial toxins. The HCN domain enhances
the process by which the
HN domain mediates the release of the light chain from internalized
intracellular vesicles into the
cytoplasm of the target cell during the translocation step. Enhanced
translocation capability is obtained
by including or maintaining a Clostridial toxin HCN domain in a modified
Clostridial toxin disclosed in the
present specification. The altered targeting capability for a naturally-
occurring Clostridial toxin target cell
is mediated by an altered targeting domain comprising a modified Hcc targeting
domain of Clostridial
toxins. The Hcc domain primarily determines the binding activity and binding
specificity of the toxin to the
receptor complex located at the surface of the target cell. Altered targeting
activity is achieved by
replacing a naturally-occurring Hcc targeting domain of a Clostridial toxin
with a binding domain for a non-
Clostridial toxin receptor present on a Clostridial toxin target cell
[021] Thus modified Clostridial toxins with an enhanced translocation
capability will reduce the
undesirable dispersal of the toxin to areas not targeted for treatment, due to
the lower dose requirement,
thereby reducing or preventing the undesirable side-effects associated with
diffusion of a Clostridial toxin
to an unwanted location. Furthermore, an altered binding activity will provide
novel Clostridial toxins that
greatly extended the number of therapeutic applications that can exploit the
advantages offered by
current Clostridial toxin therapies.
[022] Thus, aspects of the present invention provide modified Clostridial
toxins comprising a Clostridial
toxin enzymatic domain, a Clostridial toxin translocation domain, a
translocation facilitating domain and
an altered targeting domain, wherein the modified Clostridial toxin exhibits a
binding activity for a non-
Clostridial toxin receptor present on a non-Clostridial toxin target cell. It
is envisioned that any
translocation facilitating domain capable of further facilitating the
translocation step of the intoxication
process where the light chain is released from intracellular vesicles into the
cytoplasm of the target cell
will be useful to practice aspects of the present invention, including,
without limitation, a Clostridial toxin
translocation facilitating domain and an enveloped virus fusogenic peptide
domain. Likewise, a multitude
of altered targeting domains are envisioned, including, without limitation,
opioids, melanocortin peptides,
galanins, granins, tachykinin peptides, cholecystokinins, Neuropeptide Y
related peptides, kinin peptides,
PAR peptides, corticotropin-releasing hormones, thyrotropin-releasing hormones
and somatostatins. It is
also envisioned that the location of the altered targeting domain in the
modified Clostridial toxins of the
present specification can be located at the amino terminus of the toxin,
between the enzymatic and
translocation domains or at the carboxyl terminus of the toxin. Thus, a
modified Clostridial toxins
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disclosed in the present specification can comprise an amino to carboxyl
domain arrangement of, e.g., an
altered targeting domain, a Clostridial toxin translocation domain, a
translocation facilitating domain and a
Clostridial toxin enzymatic domain; an altered targeting domain, a Clostridial
toxin enzymatic domain, a
Clostridial toxin translocation domain and a translocation facilitating
domain; a Clostridial toxin enzymatic
domain, an altered targeting domain, a Clostridial toxin translocation domain
and a translocation
facilitating domain; a Clostridial toxin translocation domain, a translocation
facilitating domain, an altered
targeting domain and a Clostridial toxin enzymatic domain; a Clostridial toxin
enzymatic domain, a
Clostridial toxin translocation domain, a translocation facilitating domain
and an altered targeting domain;
and a Clostridial toxin translocation domain, a translocation facilitating
domain, a Clostridial toxin
enzymatic domain and an altered targeting domain.
[023] Other aspects of the present invention provide polynucleotide molecules
encoding modified
Clostridial toxins comprising a Clostridial toxin enzymatic domain, a
Clostridial toxin translocation domain,
a translocation facilitating domain and an altered targeting domain, wherein
the modified Clostridial toxin
exhibits a binding activity for a non-Clostridial toxin receptor present on a
non-Clostridial toxin target cell.
It is envisioned that any of the modified Clostridial toxins disclosed in the
present specification can be
encoded by a polynucleotide molecule.
[024] Other aspects of the present invention provide methods of producing a
modified Clostridial toxin
disclosed in the present specification, the method comprising the step of
expressing in a cell a
polynucleotide molecule encoding a modified Clostridial toxin comprising a
Clostridial toxin enzymatic
domain, a Clostridial toxin translocation domain, a translocation facilitating
domain and an altered
targeting domain, wherein the modified Clostridial toxin exhibits a binding
activity for a non-Clostridial
toxin receptor present on a non-Clostridial toxin target cell. Other aspects
of the present invention
provide methods of producing a modified Clostridial toxin disclosed in the
present specification, the
method comprising the steps of introducing in a cell an expression construct
comprising a polynucleotide
molecule encoding a modified Clostridial toxin comprising a Clostridial toxin
enzymatic domain, a
Clostridial toxin translocation domain, a translocation facilitating domain
and an altered targeting domain,
wherein the modified Clostridial toxin exhibits a binding activity for a non-
Clostridial toxin receptor present
on a non-Clostridial toxin target cell and expressing the expression construct
in the cell.
[025] Aspects of the present invention provide, in part, a modified
Clostridial toxin. As used herein, the
term "modified Clostridial toxin" means any polypeptide that can execute the
overall cellular mechanism
whereby a Clostridial toxin enters a neuron and inhibits neurotransmitter
release and encompasses the
binding of a Clostridial toxin to a low or high affinity receptor complex, the
internalization of the toxin, the
translocation of the Clostridial toxin light chain into the cytoplasm and the
enzymatic modification of a
Clostridial toxin substrate. A modified Clostridial toxin disclosed in the
present specification is
distinguished from a naturally-occurring Clostridial toxin by the fact that a
modified Clostridial toxin
comprises a translocation facilitating domain that enhances the process by
which a light chain from a
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modified toxin translocates into the cytoplasm of a target cell and a modified
Clostridial toxin lacks the cell
binding activity of a naturally-occurring binding domain found in a
Clostridial toxin. Instead, a modified
Clostridial toxin disclosed in the present specification comprises an altered
targeting domain that
determines the binding activity of the modified Clostridial toxin to a non-
Clostridial toxin receptor located
at the surface of the target cell. By definition, a naturally-occurring
Clostridial toxin lacks an altered
targeting domain. Examples of modified Clostridial toxin are described in,
e.g., Keith A. Foster et al.,
Clostridial Toxin Derivatives Able To Modify Peripheral Sensory Afferent
Functions, U.S. Patent
5,989,545 (Nov. 23, 1999); Clifford C. Shone et al., Recombinant Toxin
Fragments, U.S. Patent
6,461,617 (Oct. 8, 2002); Conrad P. Quinn et al., Methods and Compounds for
the Treatment of Mucus
Hypersecretion, U.S. Patent 6,632,440 (Oct. 14, 2003); Lance E. Steward et
al., Methods And
Compositions For The Treatment Of Pancreatitis, U.S. Patent 6,843,998 (Jan.
18, 2005); Stephan
Donovan, Clostridial Toxin Derivatives and Methods For Treating Pain, U.S.
Patent Publication
2002/0037833 (Mar. 28, 2002); Keith A. Foster et al., Inhibition of Secretion
from Non-neural Cells, U.S.
Patent Publication 2003/0180289 (Sep. 25, 2003); J. Oliver Dolly et al.,
Activatable Recombinant
Neurotoxins, WO 2001/014570 (Mar. 1, 2001); Keith A. Foster et al., Re-
targeted Toxin Conjugates,
International Patent Publication WO 2005/023309 (Mar. 17, 2005); and Lance E.
Steward et al.,
Multivalent Clostridial Toxin Derivatives and Methods of Their Use, U.S.
Patent Application No.
11/376,696 (Mar. 15, 2006). Any of the modified Clostridial toxins described
in, e.g., Foster, supra,
(1999), Dolly, supra, (2001), Donovan, supra, (2002), Shone, supra, (2002),
Foster, supar, (2003), Quinn,
supra, (2003), Foster, supra, (2005), Steward, supra, (2005) and Steward,
supra, (2006), can be further
modified to include a translocation facilitating domain as disclosed in the
present specification.
[026] Aspects of the present invention provide, in part, a Clostridial toxin
enzymatic domain. As used
herein, the term "Clostridial toxin enzymatic domain" means any Clostridial
toxin polypeptide that can
execute the enzymatic target modification step of the intoxication process.
Thus, a Clostridial toxin
enzymatic domain specifically targets a Clostridial toxin substrate and
encompasses the proteolytic
cleavage of a Clostridial toxin substrate, such as, e.g., SNARE proteins like
a SNAP-25 substrate, a
VAMP substrate and a Syntaxin substrate. Non-limiting examples of a
Clostridial toxin enzymatic domain
include, e.g., a Clostridial toxin light chain region such as, e.g., a BoNT/A
light chain region, a BoNT/B
light chain region, a BoNT/C1 light chain region, a BoNT/D light chain region,
a BoNT/E light chain region,
a BoNT/F light chain region, a BoNT/G light chain region, and a TeNT light
chain region.
[027] A Clostridial toxin enzymatic domain includes, without limitation,
naturally occurring Clostridial
toxin light chain variants, such as, e.g., Clostridial toxin light chain
isoforms and Clostridial toxin light
chain subtypes; non-naturally occurring Clostridial toxin light chain
variants, such as, e.g., conservative
Clostridial toxin light chain variants, non-conservative Clostridial toxin
light chain variants, Clostridial toxin
light chain chimerics, active Clostridial toxin light chain fragments thereof,
or any combination thereof.
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[028] As used herein, the term "Clostridial toxin light chain variant,"
whether naturally-occurring or non-
naturally-occurring, means a Clostridial toxin light chain that has at least
one amino acid change from the
corresponding region of the disclosed reference sequences (see Table 1) and
can be described in
percent identity to the corresponding region of that reference sequence.
Unless expressly indicated, all
Clostridial toxin light chain variants disclosed in the present specification
are capable of executing the
enzymatic target modification step of the intoxication process. As non-
limiting examples, a BoNT/A light
chain variant comprising amino acids 1-448 of SEQ ID NO: 1 will have at least
one amino acid difference,
such as, e.g., an amino acid substitution, deletion or addition, as compared
to the amino acid region 1-
448 of SEQ ID NO: 1; a BoNT/B light chain variant comprising amino acids 1-441
of SEQ ID NO: 2 will
have at least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as
compared to the amino acid region 1-441 of SEQ ID NO: 2; a BoNT/C1 light chain
variant comprising
amino acids 1-449 of SEQ ID NO: 3 will have at least one amino acid
difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to the amino acid region
1-449 of SEQ ID NO: 3; a
BoNT/D light chain variant comprising amino acids 1-445 of SEQ ID NO: 4 will
have at least one amino
acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the amino
acid region 1-445 of SEQ ID NO: 4; a BoNT/E light chain variant comprising
amino acids 1-422 of SEQ ID
NO: 5 will have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or
addition, as compared to the amino acid region 1-422 of SEQ ID NO: 5; a BoNT/F
light chain variant
comprising amino acids 1-439 of SEQ ID NO: 6 will have at least one amino acid
difference, such as,
e.g., an amino acid substitution, deletion or addition, as compared to the
amino acid region 1-439 of SEQ
ID NO: 6; a BoNT/G light chain variant comprising amino acids 1-446 of SEQ ID
NO: 7 will have at least
one amino acid difference, such as, e.g., an amino acid substitution, deletion
or addition, as compared to
the amino acid region 1-446 of SEQ ID NO: 7; and a TeNT light chain variant
comprising amino acids 1-
457 of SEQ ID NO: 8 will have at least one amino acid difference, such as,
e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid region 1-457
of SEQ ID NO: 8.
[029] It is recognized by those of skill in the art that within each serotype
of Clostridial toxin there can
be naturally occurring Clostridial toxin light chain variants that differ
somewhat in their amino acid
sequence, and also in the nucleic acids encoding these proteins. For example,
there are presently four
BoNT/A subtypes, BoNT/Al, BoNT/A2, BoNT/A3 and BoNT/A4, with specific light
chain subtypes
showing approximately 95% amino acid identity when compared to another BoNT/A
light chain subtype.
As used herein, the term "naturally occurring Clostridial toxin light chain
variant" means any Clostridial
toxin light chain produced by a naturally-occurring process, including,
without limitation, Clostridial toxin
light chain isoforms produced from alternatively-spliced transcripts,
Clostridial toxin light chain isoforms
produced by spontaneous mutation and Clostridial toxin light chain subtypes. A
naturally occurring
Clostridial toxin light chain variant can function in substantially the same
manner as the reference
Clostridial toxin light chain on which the naturally occurring Clostridial
toxin light chain variant is based,
and can be substituted for the reference Clostridial toxin light chain in any
aspect of the present invention.
A naturally occurring Clostridial toxin light chain variant may substitute one
or more amino acids, two or
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more amino acids, three or more amino acids, four or more amino acids, five or
more amino acids, ten or
more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more
amino acids, 50 or more
amino acids or 100 or more amino acids from the reference Clostridial toxin
light chain on which the
naturally occurring Clostridial toxin light chain variant is based. A
naturally occurring Clostridial toxin light
chain variant can also substitute at least 10 contiguous amino acids, at least
15 contiguous amino acids,
at least 20 contiguous amino acids, or at least 25 contiguous amino acids from
the reference Clostridial
toxin light chain on which the naturally occurring Clostridial toxin light
chain variant is based, that possess
at least 50% amino acid identity, 65% amino acid identity, 75% amino acid
identity, 85% amino acid
identity or 95% amino acid identity to the reference Clostridial toxin light
chain on which the naturally
occurring Clostridial toxin light chain variant is based.
[030] A non-limiting examples of a naturally occurring Clostridial toxin light
chain variant is a Clostridial
toxin light chain isoform such as, e.g., a BoNT/A light chain isoform, a
BoNT/B light chain isoform, a
BoNT/C1 light chain isoform, a BoNT/D light chain isoform, a BoNT/E light
chain isoform, a BoNT/F light
chain isoform, a BoNT/G light chain isoform, and a TeNT light chain isoform. A
Clostridial toxin light chain
isoform can function in substantially the same manner as the reference
Clostridial toxin light chain on
which the Clostridial toxin light chain isoform is based, and can be
substituted for the reference Clostridial
toxin light chain in any aspect of the present invention.
[031] Another non-limiting examples of a naturally occurring Clostridial toxin
light chain variant is a
Clostridial toxin light chain subtype such as, e.g., a light chain from
subtype BoNT/Al, BoNT/A2,
BoNT/A3 and BoNT/A4; a light chain from subtype BoNT/B1, BoNT/B2, BoNT/B
bivalent and BoNT/B
nonproteolytic; a light chain from subtype BoNT/C1-1 and BoNT/C1-2; a light
chain from subtype
BoNT/El, BoNT/E2 and BoNT/E3; and a light chain from subtype BoNT/F1, BoNT/F2,
BoNT/F3 and
BoNT/F4.. A Clostridial toxin light chain subtype can function in
substantially the same manner as the
reference Clostridial toxin light chain on which the Clostridial toxin light
chain subtype is based, and can
be substituted for the reference Clostridial toxin light chain in any aspect
of the present invention.
[032] As used herein, the term "non-naturally occurring Clostridial toxin
light chain variant" means any
Clostridial toxin light chain produced with the aid of human manipulation,
including, without limitation,
Clostridial toxin light chains produced by genetic engineering using random
mutagenesis or rational
design and Clostridial toxin light chains produced by chemical synthesis. Non-
limiting examples of non-
naturally occurring Clostridial toxin light chain variants include, e.g.,
conservative Clostridial toxin light
chain variants, non-conservative Clostridial toxin light chain variants,
Clostridial toxin light chain chimeric
variants and active Clostridial toxin light chain fragments.
[033] As used herein, the term "conservative Clostridial toxin light chain
variant" means a Clostridial
toxin light chain that has at least one amino acid substituted by another
amino acid or an amino acid
analog that has at least one property similar to that of the original amino
acid from the reference
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Clostridial toxin light chain sequence (Table 1). Examples of properties
include, without limitation, similar
size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity,
hydrogen-bonding capacity, a physicochemical property, of the like, or any
combination thereof. A
conservative Clostridial toxin light chain variant can function in
substantially the same manner as the
reference Clostridial toxin light chain on which the conservative Clostridial
toxin light chain variant is
based, and can be substituted for the reference Clostridial toxin light chain
in any aspect of the present
invention. A conservative Clostridial toxin light chain variant may substitute
one or more amino acids, two
or more amino acids, three or more amino acids, four or more amino acids, five
or more amino acids, ten
or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or
more amino acids, 50 or
more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or
more amino acids, 400 or
more amino acids, or 500 or more amino acids from the reference Clostridial
toxin light chain on which the
conservative Clostridial toxin light chain variant is based. A conservative
Clostridial toxin light chain
variant can also substitute at least 10 contiguous amino acids, at least 15
contiguous amino acids, at
least 20 contiguous amino acids, or at least 25 contiguous amino acids from
the reference Clostridial
toxin light chain on which the conservative Clostridial toxin light chain
variant is based, that possess at
least 50% amino acid identity, 65% amino acid identity, 75% amino acid
identity, 85% amino acid identity
or 95% amino acid identity to the reference Clostridial toxin light chain on
which the conservative
Clostridial toxin light chain variant is based. Non-limiting examples of a
conservative Clostridial toxin light
chain variant include, e.g., conservative BoNT/A light chain variants,
conservative BoNT/B light chain
variants, conservative BoNT/C1 light chain variants, conservative BoNT/D light
chain variants,
conservative BoNT/E light chain variants, conservative BoNT/F light chain
variants, conservative BoNT/G
light chain variants, and conservative TeNT light chain variants.
[034] As used herein, the term "non-conservative Clostridial toxin light chain
variant" means a
Clostridial toxin light chain in which 1) at least one amino acid is deleted
from the reference Clostridial
toxin light chain on which the non-conservative Clostridial toxin light chain
variant is based; 2) at least one
amino acid added to the reference Clostridial toxin light chain on which the
non-conservative Clostridial
toxin light chain is based; or 3) at least one amino acid is substituted by
another amino acid or an amino
acid analog that does not share any property similar to that of the original
amino acid from the reference
Clostridial toxin light chain sequence (Table 1). A non-conservative
Clostridial toxin light chain variant
can function in substantially the same manner as the reference Clostridial
toxin light chain on which the
non-conservative Clostridial toxin light chain variant is based, and can be
substituted for the reference
Clostridial toxin light chain in any aspect of the present invention. A non-
conservative Clostridial toxin
light chain variant can delete one or more amino acids, two or more amino
acids, three or more amino
acids, four or more amino acids, five or more amino acids, and ten or more
amino acids from the
reference Clostridial toxin light chain on which the non-conservative
Clostridial toxin light chain variant is
based. A non-conservative Clostridial toxin light chain variant can add one or
more amino acids, two or
more amino acids, three or more amino acids, four or more amino acids, five or
more amino acids, and
ten or more amino acids to the reference Clostridial toxin light chain on
which the non-conservative
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WO 2008/008805 PCT/US2007/073202
Clostridial toxin light chain variant is based. A non-conservative Clostridial
toxin light chain variant may
substitute one or more amino acids, two or more amino acids, three or more
amino acids, four or more
amino acids, five or more amino acids, ten or more amino acids, 20 or more
amino acids, 30 or more
amino acids, 40 or more amino acids, 50 or more amino acids, 100 or more amino
acids, 200 or more
amino acids, 300 or more amino acids, 400 or more amino acids, or 500 or more
amino acids from the
reference Clostridial toxin light chain on which the non-conservative
Clostridial toxin light chain variant is
based. A non-conservative Clostridial toxin light chain variant can also
substitute at least 10 contiguous
amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino
acids, or at least 25
contiguous amino acids from the reference Clostridial toxin light chain on
which the non-conservative
Clostridial toxin light chain variant is based, that possess at least 50%
amino acid identity, 65% amino
acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino
acid identity to the
reference Clostridial toxin light chain on which the non-conservative
Clostridial toxin light chain variant is
based. Non-limiting examples of a non-conservative Clostridial toxin light
chain variant include, e.g., non-
conservative BoNT/A light chain variants, non-conservative BoNT/B light chain
variants, non-conservative
BoNT/C1 light chain variants, non-conservative BoNT/D light chain variants,
non-conservative BoNT/E
light chain variants, non-conservative BoNT/F light chain variants, non-
conservative BoNT/G light chain
variants, and non-conservative TeNT light chain variants.
[035] As used herein, the term "Clostridial toxin light chain chimeric" means
a polypeptide comprising at
least a portion of a Clostridial toxin light chain and at least a portion of
at least one other polypeptide to
form a toxin light chain with at least one property different from the
reference Clostridial toxin light chains
of Table 1, with the proviso that this Clostridial toxin light chain chimeric
is still capable of specifically
targeting the core components of the neurotransmitter release apparatus and
thus participate in executing
the overall cellular mechanism whereby a Clostridial toxin proteolytically
cleaves a substrate. Such
Clostridial toxin light chain chimerics are described in, e.g., Lance E.
Steward et al., Leucine-based Motif
and Clostridial Toxins, U.S. Patent Publication 2003/0027752 (Feb. 6, 2003);
Lance E. Steward et al.,
Clostridial Neurotoxin Compositions and Modified Clostridial Neurotoxins, U.S.
Patent Publication
2003/0219462 (Nov. 27, 2003); and Lance E. Steward et al., Clostridial
Neurotoxin Compositions and
Modified Clostridial Neurotoxins, U.S. Patent Publication 2004/0220386 (Nov.
4, 2004).
[036] As used herein, the term "active Clostridial toxin light chain fragment"
means any of a variety of
Clostridial toxin fragments comprising the light chain can be useful in
aspects of the present invention
with the proviso that these light chain fragments can specifically target the
core components of the
neurotransmitter release apparatus and thus participate in executing the
overall cellular mechanism
whereby a Clostridial toxin proteolytically cleaves a substrate. The light
chains of Clostridial toxins are
approximately 420-460 amino acids in length and comprise an enzymatic domain
(Table 1). Research
has shown that the entire length of a Clostridial toxin light chain is not
necessary for the enzymatic activity
of the enzymatic domain. As a non-limiting example, the first eight amino
acids of the BoNT/A light chain
(residues 1-8 of SEQ ID NO: 1) are not required for enzymatic activity. As
another non-limiting example,
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the first eight amino acids of the TeNT light chain (residues 1-8 of SEQ ID
NO: 8) are not required for
enzymatic activity. Likewise, the carboxyl-terminus of the light chain is not
necessary for activity. As a
non-limiting example, the last 32 amino acids of the BoNT/A light chain
(residues 417-448 of SEQ ID NO:
1) are not required for enzymatic activity. As another non-limiting example,
the last 31 amino acids of the
TeNT light chain (residues 427-457 of SEQ ID NO: 8) are not required for
enzymatic activity. Thus,
aspects of this embodiment can include Clostridial toxin light chains
comprising an enzymatic domain
having a length of, e.g., at least 350 amino acids, at least 375 amino acids,
at least 400 amino acids, at
least 425 amino acids and at least 450 amino acids. Other aspects of this
embodiment can include
Clostridial toxin light chains comprising an enzymatic domain having a length
of, e.g., at most 350 amino
acids, at most 375 amino acids, at most 400 amino acids, at most 425 amino
acids and at most 450
amino acids.
[037] Any of a variety of sequence alignment methods can be used to determine
percent identity of
naturally-occurring Clostridial toxin light chain variants and non-naturally-
occurring Clostridial toxin light
chain variants, including, without limitation, global methods, local methods
and hybrid methods, such as,
e.g., segment approach methods. Protocols to determine percent identity are
routine procedures within
the scope of one skilled in the art and from the teaching herein.
[038] Global methods align sequences from the beginning to the end of the
molecule and determine the
best alignment by adding up scores of individual residue pairs and by imposing
gap penalties. Non-
limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et
al., CLUSTAL W: Improving
the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence
Weighting, Position-
Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research
4673-4680 (1994); and
iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in
Accuracy of Multiple Protein
Sequence Alignments by Iterative Refinement as Assessed by Reference to
Structural Alignments,
264(4) J. Mol. Biol. 823-838 (1996).
[039] Local methods align sequences by identifying one or more conserved
motifs shared by all of the
input sequences. Non-limiting methods include, e.g., Match-box, see, e.g.,
Eric Depiereux and Ernest
Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous
Alignment of Several
Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C.
E. Lawrence et al.,
Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple
Alignment, 262(5131)
Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M - A
New Algorithm for Multiple
Alignment of Highly Divergent Sequences, 20(9) Bioinformatics,:1428-1435
(2004).
[040] Hybrid methods combine functional aspects of both global and local
alignment methods. Non-
limiting methods include, e.g., segment-to-segment comparison, see, e.g.,
Burkhard Morgenstern et al.,
Multiple DNA and Protein Sequence Alignment Based On Segment-To-Segment
Comparison, 93(22)
Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996); T-Coffee, see, e.g., Cedric
Notredame et al., T-Coffee:
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A Novel Algorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-
217 (2000); MUSCLE, see,
e.g., Robert C. Edgar, MUSCLE: Multiple Sequence Alignment With High Score
Accuracy and High
Throughput, 32(5) Nucleic Acids Res. 1792-1797 (2004); and DIALIGN-T, see,
e.g., Amarendran R
Subramanian et al., DIALIGN-T.- An Improved Algorithm for Segment-Based
Multiple Sequence
Alignment, 6(1) BMC Bioinformatics 66 (2005).
[041] Thus, in an embodiment, a modified Clostridial toxin disclosed in the
present specification
comprises a Clostridial toxin enzymatic domain. In an aspect of this
embodiment, a Clostridial toxin
enzymatic domain comprises a naturally occurring Clostridial toxin light chain
variant, such as, e.g., a
Clostridial toxin light chain isoform or a Clostridial toxin light chain
subtype. In another aspect of this
embodiment, a Clostridial toxin enzymatic domain comprises a non-naturally
occurring Clostridial toxin
light chain variant, such as, e.g., a conservative Clostridial toxin light
chain variant, a non-conservative
Clostridial toxin light chain variant, a Clostridial toxin chimeric light
chain, an active Clostridial toxin light
chain fragment, or any combination thereof.
[042] In another embodiment, a Clostridial toxin enzymatic domain comprises a
BoNT/A light chain. In
an aspect of this embodiment, a BoNT/A light chain comprises amino acids 1-448
of SEQ ID NO: 1. In
another aspect of this embodiment, a BoNT/A light chain comprises a naturally
occurring BoNT/A light
chain variant, such as, e.g., a light chain from a BoNT/A isoform or a light
chain from a BoNT/A subtype.
In another aspect of this embodiment, a BoNT/A light chain comprises amino
acids 1-448 of a naturally
occurring BoNT/A light chain variant of SEQ ID NO: 1, such as, e.g., amino
acids 1-448 of a BoNT/A
isoform of SEQ ID NO: 1 or amino acids 1-448 of a BoNT/A subtype of SEQ ID NO:
1. In still another
aspect of this embodiment, a BoNT/A light chain comprises a non-naturally
occurring BoNT/A light chain
variant, such as, e.g., a conservative BoNT/A light chain variant, a non-
conservative BoNT/A light chain
variant, a BoNT/A chimeric light chain, an active BoNT/A light chain fragment,
or any combination thereof.
In still another aspect of this embodiment, a BoNT/A light chain comprises
amino acids 1-448 of a non-
naturally occurring BoNT/A light chain variant of SEQ ID NO: 1, such as, e.g.,
amino acids 1-448 of a
conservative BoNT/A light chain variant of SEQ ID NO: 1, amino acids 1-448 of
a non-conservative
BoNT/A light chain variant of SEQ ID NO: 1, amino acids 1-448 of an active
BoNT/A light chain fragment
of SEQ ID NO: 1, or any combination thereof.
[043] In other aspects of this embodiment, a BoNT/A light chain comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 1-448 of SEQ ID NO: 1, at
least 75% amino acid
identity with amino acids 1-448 of SEQ ID NO: 1, at least 80% amino acid
identity with amino acids 1-448
of SEQ ID NO: 1, at least 85% amino acid identity with amino acids 1-448 of
SEQ ID NO: 1, at least 90%
amino acid identity with amino acids 1-448 of SEQ ID NO: 1 or at least 95%
amino acid identity with
amino acids 1-448 of SEQ ID NO: 1. In yet other aspects of this embodiment, a
BoNT/A light chain
comprises a polypeptide having, e.g., at most 70% amino acid identity with
amino acids 1-448 of SEQ ID
NO: 1, at most 75% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,
at most 80% amino acid
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identity with amino acids 1-448 of SEQ ID NO: 1, at most 85% amino acid
identity with amino acids 1-448
of SEQ ID NO: 1, at most 90% amino acid identity with amino acids 1-448 of SEQ
ID NO: 1 or at most
95% amino acid identity with amino acids 1-448 of SEQ ID NO: 1.
[044] In other aspects of this embodiment, a BoNT/A light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 1-448 of SEQ ID NO: 1. In
other aspects of this
embodiment, a BoNT/A light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 1-448 of SEQ ID NO: 1. In yet other aspects of this
embodiment, a BoNT/A light
chain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 1-448 of SEQ ID
NO: 1. In other aspects of this embodiment, a BoNT/A light chain comprises a
polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40
, 50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 1-448 of SEQ ID NO: 1. In still
other aspects of this
embodiment, a BoNT/A light chain comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 1-448 of SEQ ID NO: 1. In other aspects of this embodiment, a
BoNT/A light chain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 1-448 of SEQ ID NO: 1.
[045] In other aspects of this embodiment, a BoNT/A light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 1-448 of SEQ ID NO: 1. In
other aspects of this
embodiment, a BoNT/A light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
amino acids 1-448 of SEQ ID NO: 1. In yet other aspects of this embodiment, a
BoNT/A light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 contiguous amino acid deletions relative to amino
acids 1-448 of SEQ ID NO: 1.
In other aspects of this embodiment, a BoNT/A light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
deletions relative to amino acids 1-448 of SEQ ID NO: 1. In still other
aspects of this embodiment, a
BoNT/A light chain comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 1-448
of SEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A light chain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to amino acids 1-448 of SEQ ID NO: 1.
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[046] In another embodiment, a Clostridial toxin enzymatic domain comprises a
BoNT/B light chain. In
an aspect of this embodiment, a BoNT/B light chain comprises amino acids 1-441
of SEQ ID NO: 2. In
another aspect of this embodiment, a BoNT/B light chain comprises a naturally
occurring BoNT/B light
chain variant, such as, e.g., a light chain from a BoNT/B isoform or a light
chain from a BoNT/B subtype.
In another aspect of this embodiment, a BoNT/B light chain comprises amino
acids 1-441 of a naturally
occurring BoNT/B light chain variant of SEQ ID NO: 2, such as, e.g., amino
acids 1-441 of a BoNT/B
isoform of SEQ ID NO: 2 or amino acids 1-441 of a BoNT/B subtype of SEQ ID NO:
2. In still another
aspect of this embodiment, a BoNT/B light chain comprises a non-naturally
occurring BoNT/B light chain
variant, such as, e.g., a conservative BoNT/B light chain variant, a non-
conservative BoNT/B light chain
variant, a BoNT/B chimeric light chain, an active BoNT/B light chain fragment,
or any combination thereof.
In still another aspect of this embodiment, a BoNT/B light chain comprises
amino acids 1-441 of a non-
naturally occurring BoNT/B light chain variant of SEQ ID NO: 2, such as, e.g.,
amino acids 1-441 of a
conservative BoNT/B light chain variant of SEQ ID NO: 2, amino acids 1-441 of
a non-conservative
BoNT/B light chain variant of SEQ ID NO: 2, amino acids 1-441 of an active
BoNT/B light chain fragment
of SEQ ID NO: 2, or any combination thereof.
[047] In other aspects of this embodiment, a BoNT/B light chain comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 1-441 of SEQ ID NO: 2, at
least 75% amino acid
identity with amino acids 1-441 of SEQ ID NO: 2, at least 80% amino acid
identity with amino acids 1-441
of SEQ ID NO: 2, at least 85% amino acid identity with amino acids 1-441 of
SEQ ID NO: 2, at least 90%
amino acid identity with amino acids 1-441 of SEQ ID NO: 2 or at least 95%
amino acid identity with
amino acids 1-441 of SEQ ID NO: 2. In yet other aspects of this embodiment, a
BoNT/B light chain
comprises a polypeptide having, e.g., at most 70% amino acid identity with
amino acids 1-441 of SEQ ID
NO: 2, at most 75% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,
at most 80% amino acid
identity with amino acids 1-441 of SEQ ID NO: 2, at most 85% amino acid
identity with amino acids 1-441
of SEQ ID NO: 2, at most 90% amino acid identity with amino acids 1-441 of SEQ
ID NO: 2 or at most
95% amino acid identity with amino acids 1-441 of SEQ ID NO: 2.
[048] In other aspects of this embodiment, a BoNT/B light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 1-441 of SEQ ID NO: 2. In
other aspects of this
embodiment, a BoNT/B light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 1-441 of SEQ ID NO: 2. In yet other aspects of this
embodiment, a BoNT/B light
chain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 1-441 of SEQ ID
NO: 2. In other aspects of this embodiment, a BoNT/B light chain comprises a
polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40
, 50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 1-441 of SEQ ID NO: 2. In still
other aspects of this
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embodiment, a BoNT/B light chain comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 1-441 of SEQ ID NO: 2. In other aspects of this embodiment, a
BoNT/B light chain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 1-441 of SEQ ID NO: 2.
[049] In other aspects of this embodiment, a BoNT/B light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 1-441 of SEQ ID NO: 2. In
other aspects of this
embodiment, a BoNT/B light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
amino acids 1-441 of SEQ ID NO: 2. In yet other aspects of this embodiment, a
BoNT/B light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 contiguous amino acid deletions relative to amino
acids 1-441 of SEQ ID NO: 2.
In other aspects of this embodiment, a BoNT/B light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
deletions relative to amino acids 1-441 of SEQ ID NO: 2. In still other
aspects of this embodiment, a
BoNT/B light chain comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 1-441
of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B light chain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to amino acids 1-441 of SEQ ID NO: 2.
[050] In another embodiment, a Clostridial toxin enzymatic domain comprises a
BoNT/C1 light chain.
In an aspect of this embodiment, a BoNT/C1 light chain comprises amino acids 1-
449 of SEQ ID NO: 3.
In another aspect of this embodiment, a BoNT/C1 light chain comprises a
naturally occurring BoNT/C1
light chain variant, such as, e.g., a light chain from a BoNT/C1 isoform or a
light chain from a BoNT/C1
subtype. In another aspect of this embodiment, a BoNT/C1 light chain comprises
amino acids 1-449 of a
naturally occurring BoNT/C1 light chain variant of SEQ ID NO: 3, such as,
e.g., amino acids 1-449 of a
BoNT/C1 isoform of SEQ ID NO: 3 or amino acids 1-449 of a BoNT/C1 subtype of
SEQ ID NO: 3. In still
another aspect of this embodiment, a BoNT/C1 light chain comprises a non-
naturally occurring BoNT/C1
light chain variant, such as, e.g., a conservative BoNT/C1 light chain
variant, a non-conservative
BoNT/C1 light chain variant, a BoNT/C1 chimeric light chain, an active BoNT/C1
light chain fragment, or
any combination thereof. In still another aspect of this embodiment, a BoNT/C1
light chain comprises
amino acids 1-449 of a non-naturally occurring BoNT/C1 light chain variant of
SEQ ID NO: 3, such as,
e.g., amino acids 1-449 of a conservative BoNT/C1 light chain variant of SEQ
ID NO: 3, amino acids 1-
449 of a non-conservative BoNT/C1 light chain variant of SEQ ID NO: 3, amino
acids 1-449 of an active
BoNT/C1 light chain fragment of SEQ ID NO: 3, or any combination thereof.
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[051] In other aspects of this embodiment, a BoNT/C1 light chain comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 1-449 of SEQ ID NO: 3, at
least 75% amino acid
identity with amino acids 1-449 of SEQ ID NO: 3, at least 80% amino acid
identity with amino acids 1-449
of SEQ ID NO: 3, at least 85% amino acid identity with amino acids 1-449 of
SEQ ID NO: 3, at least 90%
amino acid identity with amino acids 1-449 of SEQ ID NO: 3 or at least 95%
amino acid identity with
amino acids 1-449 of SEQ ID NO: 3. In yet other aspects of this embodiment, a
BoNT/C1 light chain
comprises a polypeptide having, e.g., at most 70% amino acid identity with
amino acids 1-449 of SEQ ID
NO: 3, at most 75% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,
at most 80% amino acid
identity with amino acids 1-449 of SEQ ID NO: 3, at most 85% amino acid
identity with amino acids 1-449
of SEQ ID NO: 3, at most 90% amino acid identity with amino acids 1-449 of SEQ
ID NO: 3 or at most
95% amino acid identity with amino acids 1-449 of SEQ ID NO: 3.
[052] In other aspects of this embodiment, a BoNT/C1 light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 1-449 of SEQ ID NO: 3. In
other aspects of this
embodiment, a BoNT/C1 light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 1-449 of SEQ ID NO: 3. In yet other aspects of this
embodiment, a BoNT/C1 light
chain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 1-449 of SEQ ID
NO: 3. In other aspects of this embodiment, a BoNT/C1 light chain comprises a
polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40
, 50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 1-449 of SEQ ID NO: 3. In still
other aspects of this
embodiment, a BoNT/C1 light chain comprises a polypeptide having, e.g., at
most one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 1-449 of SEQ ID NO: 3. In other aspects of this embodiment, a
BoNT/C1 light chain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 1-449 of SEQ ID NO: 3.
[053] In other aspects of this embodiment, a BoNT/C1 light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 1-449 of SEQ ID NO: 3. In
other aspects of this
embodiment, a BoNT/C1 light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
amino acids 1-449 of SEQ ID NO: 3. In yet other aspects of this embodiment, a
BoNT/C1 light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 contiguous amino acid deletions relative to amino
acids 1-449 of SEQ ID NO: 3.
In other aspects of this embodiment, a BoNT/C1 light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
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deletions relative to amino acids 1-449 of SEQ ID NO: 3. In still other
aspects of this embodiment, a
BoNT/C1 light chain comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 1-449
of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 light chain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to amino acids 1-449 of SEQ ID NO: 3.
[054] In another embodiment, a Clostridial toxin enzymatic domain comprises a
BoNT/D light chain. In
an aspect of this embodiment, a BoNT/D light chain comprises amino acids 1-445
of SEQ ID NO: 4. In
another aspect of this embodiment, a BoNT/D light chain comprises a naturally
occurring BoNT/D light
chain variant, such as, e.g., a light chain from a BoNT/D isoform or a light
chain from a BoNT/D subtype.
In another aspect of this embodiment, a BoNT/D light chain comprises amino
acids 1-445 of a naturally
occurring BoNT/D light chain variant of SEQ ID NO: 4, such as, e.g., amino
acids 1-445 of a BoNT/D
isoform of SEQ ID NO: 4 or amino acids 1-445 of a BoNT/D subtype of SEQ ID NO:
4. In still another
aspect of this embodiment, a BoNT/D light chain comprises a non-naturally
occurring BoNT/D light chain
variant, such as, e.g., a conservative BoNT/D light chain variant, a non-
conservative BoNT/D light chain
variant, a BoNT/D chimeric light chain, an active BoNT/D light chain fragment,
or any combination thereof.
In still another aspect of this embodiment, a BoNT/D light chain comprises
amino acids 1-445 of a non-
naturally occurring BoNT/D light chain variant of SEQ ID NO: 4, such as, e.g.,
amino acids 1-445 of a
conservative BoNT/D light chain variant of SEQ ID NO: 4, amino acids 1-445 of
a non-conservative
BoNT/D light chain variant of SEQ ID NO: 4, amino acids 1-445 of an active
BoNT/D light chain fragment
of SEQ ID NO: 4, or any combination thereof.
[055] In other aspects of this embodiment, a BoNT/D light chain comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 1-445 of SEQ ID NO: 4, at
least 75% amino acid
identity with amino acids 1-445 of SEQ ID NO: 4, at least 80% amino acid
identity with amino acids 1-445
of SEQ ID NO: 4, at least 85% amino acid identity with amino acids 1-445 of
SEQ ID NO: 4, at least 90%
amino acid identity with amino acids 1-445 of SEQ ID NO: 4 or at least 95%
amino acid identity with
amino acids 1-445 of SEQ ID NO: 4. In yet other aspects of this embodiment, a
BoNT/D light chain
comprises a polypeptide having, e.g., at most 70% amino acid identity with
amino acids 1-445 of SEQ ID
NO: 4, at most 75% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,
at most 80% amino acid
identity with amino acids 1-445 of SEQ ID NO: 4, at most 85% amino acid
identity with amino acids 1-445
of SEQ ID NO: 4, at most 90% amino acid identity with amino acids 1-445 of SEQ
ID NO: 4 or at most
95% amino acid identity with amino acids 1-445 of SEQ ID NO: 4.
[056] In other aspects of this embodiment, a BoNT/D light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 1-445 of SEQ ID NO: 4. In
other aspects of this
embodiment, a BoNT/D light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
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five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 1-445 of SEQ ID NO: 4. In yet other aspects of this
embodiment, a BoNT/D light
chain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 1-445 of SEQ ID
NO: 4. In other aspects of this embodiment, a BoNT/D light chain comprises a
polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40
, 50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 1-445 of SEQ ID NO: 4. In still
other aspects of this
embodiment, a BoNT/D light chain comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 1-445 of SEQ ID NO: 4. In other aspects of this embodiment, a
BoNT/D light chain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 1-445 of SEQ ID NO: 4.
[057] In other aspects of this embodiment, a BoNT/D light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 1-445 of SEQ ID NO: 4. In
other aspects of this
embodiment, a BoNT/D light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
amino acids 1-445 of SEQ ID NO: 4. In yet other aspects of this embodiment, a
BoNT/D light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 contiguous amino acid deletions relative to amino
acids 1-445 of SEQ ID NO: 4.
In other aspects of this embodiment, a BoNT/D light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
deletions relative to amino acids 1-445 of SEQ ID NO: 4. In still other
aspects of this embodiment, a
BoNT/D light chain comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 1-445
of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D light chain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to amino acids 1-445 of SEQ ID NO: 4.
[058] In another embodiment, a Clostridial toxin enzymatic domain comprises a
BoNT/E light chain. In
an aspect of this embodiment, a BoNT/E light chain comprises amino acids 1-422
of SEQ ID NO: 5. In
another aspect of this embodiment, a BoNT/E light chain comprises a naturally
occurring BoNT/E light
chain variant, such as, e.g., a light chain from a BoNT/E isoform or a light
chain from a BoNT/E subtype.
In another aspect of this embodiment, a BoNT/E light chain comprises amino
acids 1-422 of a naturally
occurring BoNT/E light chain variant of SEQ ID NO: 5, such as, e.g., amino
acids 1-422 of a BoNT/E
isoform of SEQ ID NO: 5 or amino acids 1-422 of a BoNT/E subtype of SEQ ID NO:
5. In still another
aspect of this embodiment, a BoNT/E light chain comprises a non-naturally
occurring BoNT/E light chain
variant, such as, e.g., a conservative BoNT/E light chain variant, a non-
conservative BoNT/E light chain
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variant, a BoNT/E chimeric light chain, an active BoNT/E light chain fragment,
or any combination thereof.
In still another aspect of this embodiment, a BoNT/E light chain comprises
amino acids 1-422 of a non-
naturally occurring BoNT/E light chain variant of SEQ ID NO: 5, such as, e.g.,
amino acids 1-422 of a
conservative BoNT/E light chain variant of SEQ ID NO: 5, amino acids 1-422 of
a non-conservative
BoNT/E light chain variant of SEQ ID NO: 5, amino acids 1-422 of an active
BoNT/E light chain fragment
of SEQ ID NO: 5, or any combination thereof.
[059] In other aspects of this embodiment, a BoNT/E light chain comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 1-422 of SEQ ID NO: 5, at
least 75% amino acid
identity with amino acids 1-422 of SEQ ID NO: 5, at least 80% amino acid
identity with amino acids 1-422
of SEQ ID NO: 5, at least 85% amino acid identity with amino acids 1-422 of
SEQ ID NO: 5, at least 90%
amino acid identity with amino acids 1-422 of SEQ ID NO: 5 or at least 95%
amino acid identity with
amino acids 1-422 of SEQ ID NO: 5. In yet other aspects of this embodiment, a
BoNT/E light chain
comprises a polypeptide having, e.g., at most 70% amino acid identity with
amino acids 1-422 of SEQ ID
NO: 5, at most 75% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,
at most 80% amino acid
identity with amino acids 1-422 of SEQ ID NO: 5, at most 85% amino acid
identity with amino acids 1-422
of SEQ ID NO: 5, at most 90% amino acid identity with amino acids 1-422 of SEQ
ID NO: 5 or at most
95% amino acid identity with amino acids 1-422 of SEQ ID NO: 5.
[060] In other aspects of this embodiment, a BoNT/E light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 1-422 of SEQ ID NO: 5. In
other aspects of this
embodiment, a BoNT/E light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 1-422 of SEQ ID NO: 5. In yet other aspects of this
embodiment, a BoNT/E light
chain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 1-422 of SEQ ID
NO: 5. In other aspects of this embodiment, a BoNT/E light chain comprises a
polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40
, 50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 1-422 of SEQ ID NO: 5. In still
other aspects of this
embodiment, a BoNT/E light chain comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 1-422 of SEQ ID NO: 5. In other aspects of this embodiment, a
BoNT/E light chain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 1-422 of SEQ ID NO: 5.
[061] In other aspects of this embodiment, a BoNT/E light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 1-422 of SEQ ID NO: 5. In
other aspects of this
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embodiment, a BoNT/E light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
amino acids 1-422 of SEQ ID NO: 5. In yet other aspects of this embodiment, a
BoNT/E light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 contiguous amino acid deletions relative to amino
acids 1-422 of SEQ ID NO: 5.
In other aspects of this embodiment, a BoNT/E light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
deletions relative to amino acids 1-422 of SEQ ID NO: 5. In still other
aspects of this embodiment, a
BoNT/E light chain comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 1-422
of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E light chain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to amino acids 1-422 of SEQ ID NO: 5.
[062] In another embodiment, a Clostridial toxin enzymatic domain comprises a
BoNT/F light chain. In
an aspect of this embodiment, a BoNT/F light chain comprises amino acids 1-439
of SEQ ID NO: 6. In
another aspect of this embodiment, a BoNT/F light chain comprises a naturally
occurring BoNT/F light
chain variant, such as, e.g., a light chain from a BoNT/F isoform or a light
chain from a BoNT/F subtype.
In another aspect of this embodiment, a BoNT/F light chain comprises amino
acids 1-439 of a naturally
occurring BoNT/F light chain variant of SEQ ID NO: 6, such as, e.g., amino
acids 1-439 of a BoNT/F
isoform of SEQ ID NO: 6 or amino acids 1-439 of a BoNT/F subtype of SEQ ID NO:
6. In still another
aspect of this embodiment, a BoNT/F light chain comprises a non-naturally
occurring BoNT/F light chain
variant, such as, e.g., a conservative BoNT/F light chain variant, a non-
conservative BoNT/F light chain
variant, a BoNT/F chimeric light chain, an active BoNT/F light chain fragment,
or any combination thereof.
In still another aspect of this embodiment, a BoNT/F light chain comprises
amino acids 1-439 of a non-
naturally occurring BoNT/F light chain variant of SEQ ID NO: 6, such as, e.g.,
amino acids 1-439 of a
conservative BoNT/F light chain variant of SEQ ID NO: 6, amino acids 1-439 of
a non-conservative
BoNT/F light chain variant of SEQ ID NO: 6, amino acids 1-439 of an active
BoNT/F light chain fragment
of SEQ ID NO: 6, or any combination thereof.
[063] In other aspects of this embodiment, a BoNT/F light chain comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 1-439 of SEQ ID NO: 6, at
least 75% amino acid
identity with amino acids 1-439 of SEQ ID NO: 6, at least 80% amino acid
identity with amino acids 1-439
of SEQ ID NO: 6, at least 85% amino acid identity with amino acids 1-439 of
SEQ ID NO: 6, at least 90%
amino acid identity with amino acids 1-439 of SEQ ID NO: 6 or at least 95%
amino acid identity with
amino acids 1-439 of SEQ ID NO: 6. In yet other aspects of this embodiment, a
BoNT/F light chain
comprises a polypeptide having, e.g., at most 70% amino acid identity with
amino acids 1-439 of SEQ ID
NO: 6, at most 75% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,
at most 80% amino acid
identity with amino acids 1-439 of SEQ ID NO: 6, at most 85% amino acid
identity with amino acids 1-439
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of SEQ ID NO: 6, at most 90% amino acid identity with amino acids 1-439 of SEQ
ID NO: 6 or at most
95% amino acid identity with amino acids 1-439 of SEQ ID NO: 6.
[064] In other aspects of this embodiment, a BoNT/F light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 1-439 of SEQ ID NO: 6. In
other aspects of this
embodiment, a BoNT/F light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 1-439 of SEQ ID NO: 6. In yet other aspects of this
embodiment, a BoNT/F light
chain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 1-439 of SEQ ID
NO: 6. In other aspects of this embodiment, a BoNT/F light chain comprises a
polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40
, 50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 1-439 of SEQ ID NO: 6. In still
other aspects of this
embodiment, a BoNT/F light chain comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 1-439 of SEQ ID NO: 6. In other aspects of this embodiment, a
BoNT/F light chain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 1-439 of SEQ ID NO: 6.
[065] In other aspects of this embodiment, a BoNT/F light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 1-439 of SEQ ID NO: 6. In
other aspects of this
embodiment, a BoNT/F light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
amino acids 1-439 of SEQ ID NO: 6. In yet other aspects of this embodiment, a
BoNT/F light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 contiguous amino acid deletions relative to amino
acids 1-439 of SEQ ID NO: 6.
In other aspects of this embodiment, a BoNT/F light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
deletions relative to amino acids 1-439 of SEQ ID NO: 6. In still other
aspects of this embodiment, a
BoNT/F light chain comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 1-439
of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F light chain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to amino acids 1-439 of SEQ ID NO: 6.
[066] In another embodiment, a Clostridial toxin enzymatic domain comprises a
BoNT/G light chain. In
an aspect of this embodiment, a BoNT/G light chain comprises amino acids 1-446
of SEQ ID NO: 7. In
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another aspect of this embodiment, a BoNT/G light chain comprises a naturally
occurring BoNT/G light
chain variant, such as, e.g., a light chain from a BoNT/G isoform or a light
chain from a BoNT/G subtype.
In another aspect of this embodiment, a BoNT/G light chain comprises amino
acids 1-446 of a naturally
occurring BoNT/G light chain variant of SEQ ID NO: 7, such as, e.g., amino
acids 1-446 of a BoNT/G
isoform of SEQ ID NO: 7 or amino acids 1-446 of a BoNT/G subtype of SEQ ID NO:
7. In still another
aspect of this embodiment, a BoNT/G light chain comprises a non-naturally
occurring BoNT/G light chain
variant, such as, e.g., a conservative BoNT/G light chain variant, a non-
conservative BoNT/G light chain
variant, a BoNT/G chimeric light chain, an active BoNT/G light chain fragment,
or any combination
thereof. In still another aspect of this embodiment, a BoNT/G light chain
comprises amino acids 1-446 of
a non-naturally occurring BoNT/G light chain variant of SEQ ID NO: 7, such as,
e.g., amino acids 1-446 of
a conservative BoNT/G light chain variant of SEQ ID NO: 7, amino acids 1-446
of a non-conservative
BoNT/G light chain variant of SEQ ID NO: 7, amino acids 1-446 of an active
BoNT/G light chain fragment
of SEQ ID NO: 7, or any combination thereof.
[067] In other aspects of this embodiment, a BoNT/G light chain comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 1-446 of SEQ ID NO: 7, at
least 75% amino acid
identity with amino acids 1-446 of SEQ ID NO: 7, at least 80% amino acid
identity with amino acids 1-446
of SEQ ID NO: 7, at least 85% amino acid identity with amino acids 1-446 of
SEQ ID NO: 7, at least 90%
amino acid identity with amino acids 1-446 of SEQ ID NO: 7 or at least 95%
amino acid identity with
amino acids 1-446 of SEQ ID NO: 7. In yet other aspects of this embodiment, a
BoNT/G light chain
comprises a polypeptide having, e.g., at most 70% amino acid identity with
amino acids 1-446 of SEQ ID
NO: 7, at most 75% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,
at most 80% amino acid
identity with amino acids 1-446 of SEQ ID NO: 7, at most 85% amino acid
identity with amino acids 1-446
of SEQ ID NO: 7, at most 90% amino acid identity with amino acids 1-446 of SEQ
ID NO: 7 or at most
95% amino acid identity with amino acids 1-446 of SEQ ID NO: 7.
[068] In other aspects of this embodiment, a BoNT/G light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 1-446 of SEQ ID NO: 7. In
other aspects of this
embodiment, a BoNT/G light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 1-446 of SEQ ID NO: 7. In yet other aspects of this
embodiment, a BoNT/G light
chain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 1-446 of SEQ ID
NO: 7. In other aspects of this embodiment, a BoNT/G light chain comprises a
polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40
, 50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 1-446 of SEQ ID NO: 7. In still
other aspects of this
embodiment, a BoNT/G light chain comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
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to amino acids 1-446 of SEQ ID NO: 7. In other aspects of this embodiment, a
BoNT/G light chain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 1-446 of SEQ ID NO: 7.
[069] In other aspects of this embodiment, a BoNT/G light chain comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 1-446 of SEQ ID NO: 7. In
other aspects of this
embodiment, a BoNT/G light chain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
amino acids 1-446 of SEQ ID NO: 7. In yet other aspects of this embodiment, a
BoNT/G light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 contiguous amino acid deletions relative to amino
acids 1-446 of SEQ ID NO: 7.
In other aspects of this embodiment, a BoNT/G light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
deletions relative to amino acids 1-446 of SEQ ID NO: 7. In still other
aspects of this embodiment, a
BoNT/G light chain comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 1-446
of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G light chain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to amino acids 1-446 of SEQ ID NO: 7.
[070] In another embodiment, a Clostridial toxin enzymatic domain comprises a
TeNT light chain. In an
aspect of this embodiment, a TeNT light chain comprises amino acids 1-457 of
SEQ ID NO: 8. In another
aspect of this embodiment, a TeNT light chain comprises a naturally occurring
TeNT light chain variant,
such as, e.g., a light chain from a TeNT isoform or a light chain from a TeNT
subtype. In another aspect
of this embodiment, a TeNT light chain comprises amino acids 1-457 of a
naturally occurring TeNT light
chain variant of SEQ ID NO: 8, such as, e.g., amino acids 1-457 of a TeNT
isoform of SEQ ID NO: 8 or
amino acids 1-457 of a TeNT subtype of SEQ ID NO: 8. In still another aspect
of this embodiment, a
TeNT light chain comprises a non-naturally occurring TeNT light chain variant,
such as, e.g., a
conservative TeNT light chain variant, a non-conservative TeNT light chain
variant, a TeNT chimeric light
chain, an active TeNT light chain fragment, or any combination thereof. In
still another aspect of this
embodiment, a TeNT light chain comprises amino acids 1-457 of a non-naturally
occurring TeNT light
chain variant of SEQ ID NO: 8, such as, e.g., amino acids 1-457 of a
conservative TeNT light chain
variant of SEQ ID NO: 8, amino acids 1-457 of a non-conservative TeNT light
chain variant of SEQ ID
NO: 8, amino acids 1-457 of an active TeNT light chain fragment of SEQ ID NO:
8, or any combination
thereof.
[071] In other aspects of this embodiment, a TeNT light chain comprises a
polypeptide having, e.g., at
least 70% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, at least
75% amino acid identity
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with amino acids 1-457 of SEQ ID NO: 8, at least 80% amino acid identity with
amino acids 1-457 of SEQ
ID NO: 8, at least 85% amino acid identity with amino acids 1-457 of SEQ ID
NO: 8, at least 90% amino
acid identity with amino acids 1-457 of SEQ ID NO: 8 or at least 95% amino
acid identity with amino acids
1-457 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT light
chain comprises a
polypeptide having, e.g., at most 70% amino acid identity with amino acids 1-
457 of SEQ ID NO: 8, at
most 75% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, at most
80% amino acid identity
with amino acids 1-457 of SEQ ID NO: 8, at most 85% amino acid identity with
amino acids 1-457 of SEQ
ID NO: 8, at most 90% amino acid identity with amino acids 1-457 of SEQ ID NO:
8 or at most 95% amino
acid identity with amino acids 1-457 of SEQ ID NO: 8.
[072] In other aspects of this embodiment, a TeNT light chain comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 1-457 of SEQ ID NO: 8. In
other aspects of this
embodiment, a TeNT light chain comprises a polypeptide having, e.g., at least
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino
acid substitutions relative to
amino acids 1-457 of SEQ ID NO: 8. In yet other aspects of this embodiment, a
TeNT light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to amino
acids 1-457 of SEQ ID NO:
8. In other aspects of this embodiment, a TeNT light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 non-contiguous amino
acid deletions relative to amino acids 1-457 of SEQ ID NO: 8. In still other
aspects of this embodiment, a
TeNT light chain comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino acid
additions relative to amino acids 1-
457 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT light chain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200 non-
contiguous amino acid additions relative to amino acids 1-457 of SEQ ID NO: 8.
[073] In other aspects of this embodiment, a TeNT light chain comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid substitutions relative to amino acids 1-457 of SEQ ID NO: 8. In other
aspects of this embodiment, a
TeNT light chain comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid
substitutions relative to amino acids 1-
457 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT light
chain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid deletions relative to amino acids 1-457 of SEQ ID
NO: 8. In other aspects
of this embodiment, a TeNT light chain comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid deletions relative
to amino acids 1-457 of SEQ ID NO: 8. In still other aspects of this
embodiment, a TeNT light chain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
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30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino
acids 1-457 of SEQ ID NO: 8.
In other aspects of this embodiment, a TeNT light chain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
additions relative to amino acids 1-457 of SEQ ID NO: 8.
[074] Aspects of the present invention provide, in part, a Clostridial toxin
translocation domain. As
used herein, the term "Clostridial toxin translocation domain" means any
Clostridial toxin polypeptide that
can execute the translocation step of the intoxication process that mediates
Clostridial toxin light chain
translocation. Thus, a Clostridial toxin translocation domain facilitates the
movement of a Clostridial toxin
light chain across a membrane and encompasses the movement of a Clostridial
toxin light chain through
the membrane an intracellular vesicle into the cytoplasm of a cell. Non-
limiting examples of a Clostridial
toxin translocation domain include, e.g., a Clostridial toxin HN region such
as, e.g., a BoNT/A HN region, a
BoNT/B HN region, a BoNT/C1 HN region, a BoNT/D HN region, a BoNT/E HN region,
a BoNT/F HN region,
a BoNT/G HN region, and a TeNT HN region.
[075] A Clostridial toxin translocation domain includes, without limitation,
naturally occurring Clostridial
toxin HN region variants, such as, e.g., Clostridial toxin HN region isoforms
and Clostridial toxin HN region
subtypes; non-naturally occurring Clostridial toxin HN region variants, such
as, e.g., conservative
Clostridial toxin HN region variants, non-conservative Clostridial toxin HN
region variants, Clostridial toxin
HN region chimerics, active Clostridial toxin HN region fragments thereof, or
any combination thereof.
[076] As used herein, the term "Clostridial toxin HN region variant," whether
naturally-occurring or non-
naturally-occurring, means a Clostridial toxin HN region that has at least one
amino acid change from the
corresponding region of the disclosed reference sequences (see Table 1) and
can be described in
percent identity to the corresponding region of that reference sequence.
Unless expressly indicated, all
Clostridial toxin HN region variants disclosed in the present specification
are capable of executing the
translocation step of the intoxication process that mediates Clostridial toxin
light chain translocation. As
non-limiting examples, a BoNT/A HN region variant comprising amino acids 449-
873 of SEQ ID NO: 1 will
have at least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as
compared to the amino acid region 449-873 of SEQ ID NO: 1; a BoNT/B HN region
variant comprising
amino acids 442-860of SEQ ID NO: 2 will have at least one amino acid
difference, such as, e.g., an
amino acid substitution, deletion or addition, as compared to the amino acid
region 442-860of SEQ ID
NO: 2; a BoNT/C1 HN region variant comprising amino acids 450-868 of SEQ ID
NO: 3 will have at least
one amino acid difference, such as, e.g., an amino acid substitution, deletion
or addition, as compared to
the amino acid region 450-868 of SEQ ID NO: 3; a BoNT/D HN region variant
comprising amino acids
446-864 of SEQ ID NO: 4 will have at least one amino acid difference, such as,
e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid region 446-
864 of SEQ ID NO: 4; a
BoNT/E HN region variant comprising amino acids 423-847 of SEQ ID NO: 5 will
have at least one amino
acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the amino
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acid region 423-847 of SEQ ID NO: 5; a BoNT/F HN region variant comprising
amino acids 440-866 of
SEQ ID NO: 6 will have at least one amino acid difference, such as, e.g., an
amino acid substitution,
deletion or addition, as compared to the amino acid region 440-866 of SEQ ID
NO: 6; a BoNT/G HN
region variant comprising amino acids 447-865 of SEQ ID NO: 7 will have at
least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or addition,
as compared to the amino acid
region 447-865 of SEQ ID NO: 7; and a TeNT HN region variant comprising amino
acids 458-881 of SEQ
ID NO: 8 will have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion
or addition, as compared to the amino acid region 458-881 of SEQ ID NO: 8.
[077] It is recognized by those of skill in the art that within each serotype
of Clostridial toxin there can
be naturally occurring Clostridial toxin HN region variants that differ
somewhat in their amino acid
sequence, and also in the nucleic acids encoding these proteins. For example,
there are presently four
BoNT/A subtypes, BoNT/Al, BoNT/A2, BoNT/A3 and BoNT/A4, with specific HN
region subtypes showing
approximately 87% amino acid identity when compared to another BoNT/A HN
region subtype. As used
herein, the term "naturally occurring Clostridial toxin HN region variant"
means any Clostridial toxin HN
region produced by a naturally-occurring process, including, without
limitation, Clostridial toxin HN region
isoforms produced from alternatively-spliced transcripts, Clostridial toxin HN
region isoforms produced by
spontaneous mutation and Clostridial toxin HN region subtypes. A naturally
occurring Clostridial toxin HN
region variant can function in substantially the same manner as the reference
Clostridial toxin HN region
on which the naturally occurring Clostridial toxin HN region variant is based,
and can be substituted for the
reference Clostridial toxin HN region in any aspect of the present invention.
A naturally occurring
Clostridial toxin HN region variant may substitute one or more amino acids,
two or more amino acids,
three or more amino acids, four or more amino acids, five or more amino acids,
ten or more amino acids,
20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or
more amino acids or 100
or more amino acids from the reference Clostridial toxin HN region on which
the naturally occurring
Clostridial toxin HN region variant is based. A naturally occurring
Clostridial toxin HN region variant can
also substitute at least 10 contiguous amino acids, at least 15 contiguous
amino acids, at least 20
contiguous amino acids, or at least 25 contiguous amino acids from the
reference Clostridial toxin HN
region on which the naturally occurring Clostridial toxin HN region variant is
based, that possess at least
50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85%
amino acid identity or
95% amino acid identity to the reference Clostridial toxin HN region on which
the naturally occurring
Clostridial toxin HN region variant is based.
[078] A non-limiting examples of a naturally occurring Clostridial toxin HN
region variant is a Clostridial
toxin HN region isoform such as, e.g., a BoNT/A HN region isoform, a BoNT/B HN
region isoform, a
BoNT/C1 HN region isoform, a BoNT/D HN region isoform, a BoNT/E HN region
isoform, a BoNT/F HN
region isoform, a BoNT/G HN region isoform, and a TeNT HN region isoform. A
Clostridial toxin HN region
isoform can function in substantially the same manner as the reference
Clostridial toxin HN region on
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which the Clostridial toxin HN region isoform is based, and can be substituted
for the reference Clostridial
toxin HN region in any aspect of the present invention.
[079] Another non-limiting examples of a naturally occurring Clostridial toxin
HN region variant is a
Clostridial toxin HN region subtype such as, e.g., a HN region from subtype
BoNT/Al, BoNT/A2, BoNT/A3
and BoNT/A4; a HN region from subtype BoNT/B1, BoNT/B2, BoNT/B bivalent and
BoNT/B
nonproteolytic; a HN region from subtype BoNT/C1-1 and BoNT/C1-2; a HN region
from subtype BoNT/El,
BoNT/E2 and BoNT/E3; and a HN region from subtype BoNT/F1, BoNT/F2, BoNT/F3
and BoNT/F4. A
Clostridial toxin HN region subtype can function in substantially the same
manner as the reference
Clostridial toxin HN region on which the Clostridial toxin HN region subtype
is based, and can be
substituted for the reference Clostridial toxin HN region in any aspect of the
present invention.
[080] As used herein, the term "non-naturally occurring Clostridial toxin HN
region variant" means any
Clostridial toxin HN region produced with the aid of human manipulation,
including, without limitation,
Clostridial toxin HN regions produced by genetic engineering using random
mutagenesis or rational
design and Clostridial toxin HN regions produced by chemical synthesis. Non-
limiting examples of non-
naturally occurring Clostridial toxin HN region variants include, e.g.,
conservative Clostridial toxin HN
region variants, non-conservative Clostridial toxin HN region variants,
Clostridial toxin HN region chimeric
variants and active Clostridial toxin HN region fragments.
[081] As used herein, the term "conservative Clostridial toxin HN region
variant" means a Clostridial
toxin HN region that has at least one amino acid substituted by another amino
acid or an amino acid
analog that has at least one property similar to that of the original amino
acid from the reference
Clostridial toxin HN region sequence (Table 1). Examples of properties
include, without limitation, similar
size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity,
hydrogen-bonding capacity, a physicochemical property, of the like, or any
combination thereof. A
conservative Clostridial toxin HN region variant can function in substantially
the same manner as the
reference Clostridial toxin HN region on which the conservative Clostridial
toxin HN region variant is based,
and can be substituted for the reference Clostridial toxin HN region in any
aspect of the present invention.
A conservative Clostridial toxin HN region variant may substitute one or more
amino acids, two or more
amino acids, three or more amino acids, four or more amino acids, five or more
amino acids, ten or more
amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino
acids, 50 or more amino
acids, 100 or more amino acids, 200 or more amino acids, 300 or more amino
acids, 400 or more amino
acids, or 500 or more amino acids from the reference Clostridial toxin HN
region on which the
conservative Clostridial toxin HN region variant is based. A conservative
Clostridial toxin HN region variant
can also substitute at least 10 contiguous amino acids, at least 15 contiguous
amino acids, at least 20
contiguous amino acids, or at least 25 contiguous amino acids from the
reference Clostridial toxin HN
region on which the conservative Clostridial toxin HN region variant is based,
that possess at least 50%
amino acid identity, 65% amino acid identity, 75% amino acid identity, 85%
amino acid identity or 95%
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amino acid identity to the reference Clostridial toxin HN region on which the
conservative Clostridial toxin
HN region variant is based. Non-limiting examples of a conservative
Clostridial toxin HN region variant
include, e.g., conservative BoNT/A HN region variants, conservative BoNT/B HN
region variants,
conservative BoNT/C1 HN region variants, conservative BoNT/D HN region
variants, conservative BoNT/E
HN region variants, conservative BoNT/F HN region variants, conservative
BoNT/G HN region variants,
and conservative TeNT HN region variants.
[082] As used herein, the term "non-conservative Clostridial toxin HN region
variant" means a Clostridial
toxin HN region in which 1) at least one amino acid is deleted from the
reference Clostridial toxin HN
region on which the non-conservative Clostridial toxin HN region variant is
based; 2) at least one amino
acid added to the reference Clostridial toxin HN region on which the non-
conservative Clostridial toxin HN
region is based; or 3) at least one amino acid is substituted by another amino
acid or an amino acid
analog that does not share any property similar to that of the original amino
acid from the reference
Clostridial toxin HN region sequence (Table 1). A non-conservative Clostridial
toxin HN region variant can
function in substantially the same manner as the reference Clostridial toxin
HN region on which the non-
conservative Clostridial toxin HN region variant is based, and can be
substituted for the reference
Clostridial toxin HN region in any aspect of the present invention. A non-
conservative Clostridial toxin HN
region variant can delete one or more amino acids, two or more amino acids,
three or more amino acids,
four or more amino acids, five or more amino acids, and ten or more amino
acids from the reference
Clostridial toxin HN region on which the non-conservative Clostridial toxin HN
region variant is based. A
non-conservative Clostridial toxin HN region variant can add one or more amino
acids, two or more amino
acids, three or more amino acids, four or more amino acids, five or more amino
acids, and ten or more
amino acids to the reference Clostridial toxin HN region on which the non-
conservative Clostridial toxin HN
region variant is based. A non-conservative Clostridial toxin HN region
variant may substitute one or more
amino acids, two or more amino acids, three or more amino acids, four or more
amino acids, five or more
amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino
acids, 40 or more
amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more
amino acids, 300 or more
amino acids, 400 or more amino acids, or 500 or more amino acids from the
reference Clostridial toxin HN
region on which the non-conservative Clostridial toxin HN region variant is
based. A non-conservative
Clostridial toxin HN region variant can also substitute at least 10 contiguous
amino acids, at least 15
contiguous amino acids, at least 20 contiguous amino acids, or at least 25
contiguous amino acids from
the reference Clostridial toxin HN region on which the non-conservative
Clostridial toxin HN region variant
is based, that possess at least 50% amino acid identity, 65% amino acid
identity, 75% amino acid identity,
85% amino acid identity or 95% amino acid identity to the reference
Clostridial toxin HN region on which
the non-conservative Clostridial toxin HN region variant is based. Non-
limiting examples of a non-
conservative Clostridial toxin HN region variant include, e.g., non-
conservative BoNT/A HN region variants,
non-conservative BoNT/B HN region variants, non-conservative BoNT/C1 HN region
variants, non-
conservative BoNT/D HN region variants, non-conservative BoNT/E HN region
variants, non-conservative
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BoNT/F HN region variants, non-conservative BoNT/G HN region variants, and non-
conservative TeNT HN
region variants.
[083] As used herein, the term "Clostridial toxin HN region chimeric" means a
polypeptide comprising at
least a portion of a Clostridial toxin HN region and at least a portion of at
least one other polypeptide to
form a toxin HN region with at least one property different from the reference
Clostridial toxin HN regions of
Table 1, with the proviso that this Clostridial toxin HN region chimeric is
still capable of specifically
targeting the core components of the neurotransmitter release apparatus and
thus participate in executing
the overall cellular mechanism whereby a Clostridial toxin proteolytically
cleaves a substrate.
[084] As used herein, the term "active Clostridial toxin HN region fragment"
means any of a variety of
Clostridial toxin fragments comprising the HN region can be useful in aspects
of the present invention with
the proviso that these active fragments can facilitate the release of the LC
from intracellular vesicles into
the cytoplasm of the target cell and thus participate in executing the overall
cellular mechanism whereby
a Clostridial toxin proteolytically cleaves a substrate. The HN regions from
the heavy chains of Clostridial
toxins are approximately 410-430 amino acids in length and comprise a
translocation domain (Table 1).
Research has shown that the entire length of a HN region from a Clostridial
toxin heavy chain is not
necessary for the translocating activity of the translocation domain. Thus,
aspects of this embodiment
can include Clostridial toxin HN regions comprising a translocation domain
having a length of, e.g., at least
350 amino acids, at least 375 amino acids, at least 400 amino acids and at
least 425 amino acids. Other
aspects of this embodiment can include Clostridial toxin HN regions comprising
translocation domain
having a length of, e.g., at most 350 amino acids, at most 375 amino acids, at
most 400 amino acids and
at most 425 amino acids.
[085] Any of a variety of sequence alignment methods can be used to determine
percent identity of
naturally-occurring Clostridial toxin HN region variants and non-naturally-
occurring Clostridial toxin HN
region variants, including, without limitation, global methods, local methods
and hybrid methods, such as,
e.g., segment approach methods. Protocols to determine percent identity are
routine procedures within
the scope of one skilled in the art and from the teaching herein.
[086] Thus, in an embodiment, a modified Clostridial toxin disclosed in the
present specification
comprises a Clostridial toxin translocation domain. In an aspect of this
embodiment, a Clostridial toxin
translocation domain comprises a naturally occurring Clostridial toxin HN
region variant, such as, e.g., a
Clostridial toxin HN region isoform or a Clostridial toxin HN region subtype.
In another aspect of this
embodiment, a Clostridial toxin translocation domain comprises a non-naturally
occurring Clostridial toxin
HN region variant, such as, e.g., a conservative Clostridial toxin HN region
variant, a non-conservative
Clostridial toxin HN region variant, a Clostridial toxin chimeric HN region,
an active Clostridial toxin HN
region fragment, or any combination thereof.
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[087] In another embodiment, a Clostridial toxin translocation domain
comprises a BoNT/A HN region.
In an aspect of this embodiment, a BoNT/A HN region comprises amino acids 449-
873 of SEQ ID NO: 1.
In another aspect of this embodiment, a BoNT/A HN region comprises a naturally
occurring BoNT/A HN
region variant, such as, e.g., a HN region from a BoNT/A isoform or a HN
region from a BoNT/A subtype.
In another aspect of this embodiment, a BoNT/A HN region comprises amino acids
449-873 of a naturally
occurring BoNT/A HN region variant of SEQ ID NO: 1, such as, e.g., amino acids
449-873 of a BoNT/A
isoform of SEQ ID NO: 1 or amino acids 449-873 of a BoNT/A subtype of SEQ ID
NO: 1. In still another
aspect of this embodiment, a BoNT/A HN region comprises a non-naturally
occurring BoNT/A HN region
variant, such as, e.g., a conservative BoNT/A HN region variant, a non-
conservative BoNT/A HN region
variant, a BoNT/A chimeric HN region, an active BoNT/A HN region fragment, or
any combination thereof.
In still another aspect of this embodiment, a BoNT/A HN region comprises amino
acids 449-873 of a non-
naturally occurring BoNT/A HN region variant of SEQ ID NO: 1, such as, e.g.,
amino acids 449-873 of a
conservative BoNT/A HN region variant of SEQ ID NO: 1, amino acids 449-873 of
a non-conservative
BoNT/A HN region variant of SEQ ID NO: 1, amino acids 449-873 of an active
BoNT/A HN region fragment
of SEQ ID NO: 1, or any combination thereof.
[088] In other aspects of this embodiment, a BoNT/A HN region comprises a
polypeptide having, e.g., at
least 70% amino acid identity with amino acids 449-873 of SEQ ID NO: 1, at
least 75% amino acid
identity with amino acids 449-873 of SEQ ID NO: 1, at least 80% amino acid
identity with amino acids
449-873 of SEQ ID NO: 1, at least 85% amino acid identity with amino acids 449-
873 of SEQ ID NO: 1, at
least 90% amino acid identity with amino acids 449-873 of SEQ ID NO: 1 or at
least 95% amino acid
identity with amino acids 449-873 of SEQ ID NO: 1. In yet other aspects of
this embodiment, a BoNT/A
HN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 449-
873 of SEQ ID NO: 1, at most 75% amino acid identity with amino acids 449-873
of SEQ ID NO: 1, at
most 80% amino acid identity with amino acids 449-873 of SEQ ID NO: 1, at most
85% amino acid
identity with amino acids 449-873 of SEQ ID NO: 1, at most 90% amino acid
identity with amino acids
449-873 of SEQ ID NO: 1 or at most 95% amino acid identity with amino acids
449-873 of SEQ ID NO: 1.
[089] In other aspects of this embodiment, a BoNT/A HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 449-873 of SEQ ID NO: 1. In
other aspects of this
embodiment, a BoNT/A HN region comprises a polypeptide having, e.g., at least
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino
acid substitutions relative to
amino acids 449-873 of SEQ ID NO: 1. In yet other aspects of this embodiment,
a BoNT/A HN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to amino
acids 449-873 of SEQ ID
NO: 1. In other aspects of this embodiment, a BoNT/A HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 449-873 of SEQ ID NO: 1. In still
other aspects of this
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embodiment, a BoNT/A HN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 449-873 of SEQ ID NO: 1. In other aspects of this embodiment, a
BoNT/A HN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 449-873 of SEQ ID NO:
1.
[090] In other aspects of this embodiment, a BoNT/A HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid substitutions relative to amino acids 449-873 of SEQ ID NO: 1. In other
aspects of this embodiment,
a BoNT/A HN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid
substitutions relative to amino acids
449-873 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A HN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid deletions relative to amino acids 449-873 of SEQ
ID NO: 1. In other
aspects of this embodiment, a BoNT/A HN region comprises a polypeptide having,
e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid deletions
relative to amino acids 449-873 of SEQ ID NO: 1. In still other aspects of
this embodiment, a BoNT/A HN
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino
acids 449-873 of SEQ ID
NO: 1. In other aspects of this embodiment, a BoNT/A HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid additions relative to amino acids 449-873 of SEQ ID NO: 1.
[091] In another embodiment, a Clostridial toxin translocation domain
comprises a BoNT/B HN region.
In an aspect of this embodiment, a BoNT/B HN region comprises amino acids 442-
860of SEQ ID NO: 2.
In another aspect of this embodiment, a BoNT/B HN region comprises a naturally
occurring BoNT/B HN
region variant, such as, e.g., a HN region from a BoNT/B isoform or a HN
region from a BoNT/B subtype.
In another aspect of this embodiment, a BoNT/B HN region comprises amino acids
442-860of a naturally
occurring BoNT/B HN region variant of SEQ ID NO: 2, such as, e.g., amino acids
442-860of a BoNT/B
isoform of SEQ ID NO: 2 or amino acids 442-860of a BoNT/B subtype of SEQ ID
NO: 2. In still another
aspect of this embodiment, a BoNT/B HN region comprises a non-naturally
occurring BoNT/B HN region
variant, such as, e.g., a conservative BoNT/B HN region variant, a non-
conservative BoNT/B HN region
variant, a BoNT/B chimeric HN region, an active BoNT/B HN region fragment, or
any combination thereof.
In still another aspect of this embodiment, a BoNT/B HN region comprises amino
acids 442-860of a non-
naturally occurring BoNT/B HN region variant of SEQ ID NO: 2, such as, e.g.,
amino acids 442-860of a
conservative BoNT/B HN region variant of SEQ ID NO: 2, amino acids 442-860of a
non-conservative
BoNT/B HN region variant of SEQ ID NO: 2, amino acids 442-860of an active
BoNT/B HN region fragment
of SEQ ID NO: 2, or any combination thereof.
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[092] In other aspects of this embodiment, a BoNT/B HN region comprises a
polypeptide having, e.g., at
least 70% amino acid identity with amino acids 442-860of SEQ ID NO: 2, at
least 75% amino acid identity
with amino acids 442-860of SEQ ID NO: 2, at least 80% amino acid identity with
amino acids 442-860of
SEQ ID NO: 2, at least 85% amino acid identity with amino acids 442-860of SEQ
ID NO: 2, at least 90%
amino acid identity with amino acids 442-860of SEQ ID NO: 2 or at least 95%
amino acid identity with
amino acids 442-860of SEQ ID NO: 2. In yet other aspects of this embodiment, a
BoNT/B HN region
comprises a polypeptide having, e.g., at most 70% amino acid identity with
amino acids 442-860of SEQ
ID NO: 2, at most 75% amino acid identity with amino acids 442-860of SEQ ID
NO: 2, at most 80% amino
acid identity with amino acids 442-860of SEQ ID NO: 2, at most 85% amino acid
identity with amino acids
442-860of SEQ ID NO: 2, at most 90% amino acid identity with amino acids 442-
860of SEQ ID NO: 2 or
at most 95% amino acid identity with amino acids 442-860of SEQ ID NO: 2.
[093] In other aspects of this embodiment, a BoNT/B HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 442-860of SEQ ID NO: 2. In
other aspects of this
embodiment, a BoNT/B HN region comprises a polypeptide having, e.g., at least
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino
acid substitutions relative to
amino acids 442-860of SEQ ID NO: 2. In yet other aspects of this embodiment, a
BoNT/B HN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to amino
acids 442-860of SEQ ID
NO: 2. In other aspects of this embodiment, a BoNT/B HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 442-860of SEQ ID NO: 2. In still
other aspects of this
embodiment, a BoNT/B HN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 442-860of SEQ ID NO: 2. In other aspects of this embodiment, a
BoNT/B HN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 442-860of SEQ ID NO: 2.
[094] In other aspects of this embodiment, a BoNT/B HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid substitutions relative to amino acids 442-860of SEQ ID NO: 2. In other
aspects of this embodiment,
a BoNT/B HN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid
substitutions relative to amino acids
442-860of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B HN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid deletions relative to amino acids 442-860of SEQ
ID NO: 2. In other
aspects of this embodiment, a BoNT/B HN region comprises a polypeptide having,
e.g., at least one, two,
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three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid deletions
relative to amino acids 442-860of SEQ ID NO: 2. In still other aspects of this
embodiment, a BoNT/B HN
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino
acids 442-860of SEQ ID
NO: 2. In other aspects of this embodiment, a BoNT/B HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid additions relative to amino acids 442-860of SEQ ID NO: 2.
[095] In another embodiment, a Clostridial toxin translocation domain
comprises a BoNT/C1 HN region.
In an aspect of this embodiment, a BoNT/C1 HN region comprises amino acids 450-
868 of SEQ ID NO: 3.
In another aspect of this embodiment, a BoNT/C1 HN region comprises a
naturally occurring BoNT/C1 HN
region variant, such as, e.g., a HN region from a BoNT/C1 isoform or a HN
region from a BoNT/C1
subtype. In another aspect of this embodiment, a BoNT/C1 HN region comprises
amino acids 450-868 of
a naturally occurring BoNT/C1 HN region variant of SEQ ID NO: 3, such as,
e.g., amino acids 450-868 of
a BoNT/C1 isoform of SEQ ID NO: 3 or amino acids 450-868 of a BoNT/C1 subtype
of SEQ ID NO: 3. In
still another aspect of this embodiment, a BoNT/C1 HN region comprises a non-
naturally occurring
BoNT/C1 HN region variant, such as, e.g., a conservative BoNT/C1 HN region
variant, a non-conservative
BoNT/C1 HN region variant, a BoNT/C1 chimeric HN region, an active BoNT/C1 HN
region fragment, or
any combination thereof. In still another aspect of this embodiment, a BoNT/C1
HN region comprises
amino acids 450-868 of a non-naturally occurring BoNT/C1 HN region variant of
SEQ ID NO: 3, such as,
e.g., amino acids 450-868 of a conservative BoNT/C1 HN region variant of SEQ
ID NO: 3, amino acids
450-868 of a non-conservative BoNT/C1 HN region variant of SEQ ID NO: 3, amino
acids 450-868 of an
active BoNT/C1 HN region fragment of SEQ ID NO: 3, or any combination thereof.
[096] In other aspects of this embodiment, a BoNT/C1 HN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 450-868 of SEQ ID NO: 3, at
least 75% amino acid
identity with amino acids 450-868 of SEQ ID NO: 3, at least 80% amino acid
identity with amino acids
450-868 of SEQ ID NO: 3, at least 85% amino acid identity with amino acids 450-
868 of SEQ ID NO: 3, at
least 90% amino acid identity with amino acids 450-868 of SEQ ID NO: 3 or at
least 95% amino acid
identity with amino acids 450-868 of SEQ ID NO: 3. In yet other aspects of
this embodiment, a BoNT/C1
HN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 450-
868 of SEQ ID NO: 3, at most 75% amino acid identity with amino acids 450-868
of SEQ ID NO: 3, at
most 80% amino acid identity with amino acids 450-868 of SEQ ID NO: 3, at most
85% amino acid
identity with amino acids 450-868 of SEQ ID NO: 3, at most 90% amino acid
identity with amino acids
450-868 of SEQ ID NO: 3 or at most 95% amino acid identity with amino acids
450-868 of SEQ ID NO: 3.
[097] In other aspects of this embodiment, a BoNT/C1 HN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 450-868 of SEQ ID NO: 3. In
other aspects of this
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embodiment, a BoNT/C1 HN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 450-868 of SEQ ID NO: 3. In yet other aspects of this
embodiment, a BoNT/C1
HN region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative
to amino acids 450-868 of
SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 HN region
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200 non-
contiguous amino acid deletions relative to amino acids 450-868 of SEQ ID NO:
3. In still other aspects
of this embodiment, a BoNT/C1 HN region comprises a polypeptide having, e.g.,
at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-
contiguous amino acid additions
relative to amino acids 450-868 of SEQ ID NO: 3. In other aspects of this
embodiment, a BoNT/C1 HN
region comprises a polypeptide having, e.g., at least one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid additions relative to
amino acids 450-868 of SEQ
ID NO: 3.
[098] In other aspects of this embodiment, a BoNT/C1 HN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 450-868 of SEQ ID NO: 3. In
other aspects of this
embodiment, a BoNT/C1 HN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
amino acids 450-868 of SEQ ID NO: 3. In yet other aspects of this embodiment,
a BoNT/C1 HN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids
450-868 of SEQ ID NO: 3.
In other aspects of this embodiment, a BoNT/C1 HN region comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
deletions relative to amino acids 450-868 of SEQ ID NO: 3. In still other
aspects of this embodiment, a
BoNT/C1 HN region comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 450-
868 of SEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 HN region
comprises a
polypeptide having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid additions relative to amino acids 450-868 of SEQ
ID NO: 3.
[099] In another embodiment, a Clostridial toxin translocation domain
comprises a BoNT/D HN region.
In an aspect of this embodiment, a BoNT/D HN region comprises amino acids 446-
864 of SEQ ID NO: 4.
In another aspect of this embodiment, a BoNT/D HN region comprises a naturally
occurring BoNT/D HN
region variant, such as, e.g., a HN region from a BoNT/D isoform or a HN
region from a BoNT/D subtype.
In another aspect of this embodiment, a BoNT/D HN region comprises amino acids
446-864 of a naturally
occurring BoNT/D HN region variant of SEQ ID NO: 4, such as, e.g., amino acids
446-864 of a BoNT/D
isoform of SEQ ID NO: 4 or amino acids 446-864 of a BoNT/D subtype of SEQ ID
NO: 4. In still another
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aspect of this embodiment, a BoNT/D HN region comprises a non-naturally
occurring BoNT/D HN region
variant, such as, e.g., a conservative BoNT/D HN region variant, a non-
conservative BoNT/D HN region
variant, a BoNT/D chimeric HN region, an active BoNT/D HN region fragment, or
any combination thereof.
In still another aspect of this embodiment, a BoNT/D HN region comprises amino
acids 446-864 of a non-
naturally occurring BoNT/D HN region variant of SEQ ID NO: 4, such as, e.g.,
amino acids 446-864 of a
conservative BoNT/D HN region variant of SEQ ID NO: 4, amino acids 446-864 of
a non-conservative
BoNT/D HN region variant of SEQ ID NO: 4, amino acids 446-864 of an active
BoNT/D HN region
fragment of SEQ ID NO: 4, or any combination thereof.
[0100] In other aspects of this embodiment, a BoNT/D HN region comprises a
polypeptide having, e.g., at
least 70% amino acid identity with amino acids 446-864 of SEQ ID NO: 4, at
least 75% amino acid
identity with amino acids 446-864 of SEQ ID NO: 4, at least 80% amino acid
identity with amino acids
446-864 of SEQ ID NO: 4, at least 85% amino acid identity with amino acids 446-
864 of SEQ ID NO: 4, at
least 90% amino acid identity with amino acids 446-864 of SEQ ID NO: 4 or at
least 95% amino acid
identity with amino acids 446-864 of SEQ ID NO: 4. In yet other aspects of
this embodiment, a BoNT/D
HN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 446-
864 of SEQ ID NO: 4, at most 75% amino acid identity with amino acids 446-864
of SEQ ID NO: 4, at
most 80% amino acid identity with amino acids 446-864 of SEQ ID NO: 4, at most
85% amino acid
identity with amino acids 446-864 of SEQ ID NO: 4, at most 90% amino acid
identity with amino acids
446-864 of SEQ ID NO: 4 or at most 95% amino acid identity with amino acids
446-864 of SEQ ID NO: 4.
[0101] In other aspects of this embodiment, a BoNT/D HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 446-864 of SEQ ID NO: 4. In
other aspects of this
embodiment, a BoNT/D HN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 446-864 of SEQ ID NO: 4. In yet other aspects of this
embodiment, a BoNT/D HN
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 446-864 of SEQ
ID NO: 4. In other aspects of this embodiment, a BoNT/D HN region comprises a
polypeptide having,
e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 non-
contiguous amino acid deletions relative to amino acids 446-864 of SEQ ID NO:
4. In still other aspects
of this embodiment, a BoNT/D HN region comprises a polypeptide having, e.g.,
at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-
contiguous amino acid additions
relative to amino acids 446-864 of SEQ ID NO: 4. In other aspects of this
embodiment, a BoNT/D HN
region comprises a polypeptide having, e.g., at least one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid additions relative to
amino acids 446-864 of SEQ
ID NO: 4.
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[0102] In other aspects of this embodiment, a BoNT/D HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid substitutions relative to amino acids 446-864 of SEQ ID NO: 4. In other
aspects of this embodiment,
a BoNT/D HN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid
substitutions relative to amino acids
446-864 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D HN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid deletions relative to amino acids 446-864 of SEQ
ID NO: 4. In other
aspects of this embodiment, a BoNT/D HN region comprises a polypeptide having,
e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid deletions
relative to amino acids 446-864 of SEQ ID NO: 4. In still other aspects of
this embodiment, a BoNT/D HN
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino
acids 446-864 of SEQ ID
NO: 4. In other aspects of this embodiment, a BoNT/D HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid additions relative to amino acids 446-864 of SEQ ID NO: 4.
[0103] In another embodiment, a Clostridial toxin translocation domain
comprises a BoNT/E HN region.
In an aspect of this embodiment, a BoNT/E HN region comprises amino acids 423-
847 of SEQ ID NO: 5.
In another aspect of this embodiment, a BoNT/E HN region comprises a naturally
occurring BoNT/E HN
region variant, such as, e.g., a HN region from a BoNT/E isoform or a HN
region from a BoNT/E subtype.
In another aspect of this embodiment, a BoNT/E HN region comprises amino acids
423-847 of a naturally
occurring BoNT/E HN region variant of SEQ ID NO: 5, such as, e.g., amino acids
423-847 of a BoNT/E
isoform of SEQ ID NO: 5 or amino acids 423-847 of a BoNT/E subtype of SEQ ID
NO: 5. In still another
aspect of this embodiment, a BoNT/E HN region comprises a non-naturally
occurring BoNT/E HN region
variant, such as, e.g., a conservative BoNT/E HN region variant, a non-
conservative BoNT/E HN region
variant, a BoNT/E chimeric HN region, an active BoNT/E HN region fragment, or
any combination thereof.
In still another aspect of this embodiment, a BoNT/E HN region comprises amino
acids 423-847 of a non-
naturally occurring BoNT/E HN region variant of SEQ ID NO: 5, such as, e.g.,
amino acids 423-847 of a
conservative BoNT/E HN region variant of SEQ ID NO: 5, amino acids 423-847 of
a non-conservative
BoNT/E HN region variant of SEQ ID NO: 5, amino acids 423-847 of an active
BoNT/E HN region fragment
of SEQ ID NO: 5, or any combination thereof.
[0104] In other aspects of this embodiment, a BoNT/E HN region comprises a
polypeptide having, e.g., at
least 70% amino acid identity with amino acids 423-847 of SEQ ID NO: 5, at
least 75% amino acid
identity with amino acids 423-847 of SEQ ID NO: 5, at least 80% amino acid
identity with amino acids
423-847 of SEQ ID NO: 5, at least 85% amino acid identity with amino acids 423-
847 of SEQ ID NO: 5, at
least 90% amino acid identity with amino acids 423-847 of SEQ ID NO: 5 or at
least 95% amino acid
identity with amino acids 423-847 of SEQ ID NO: 5. In yet other aspects of
this embodiment, a BoNT/E
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HN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 423-
847 of SEQ ID NO: 5, at most 75% amino acid identity with amino acids 423-847
of SEQ ID NO: 5, at
most 80% amino acid identity with amino acids 423-847 of SEQ ID NO: 5, at most
85% amino acid
identity with amino acids 423-847 of SEQ ID NO: 5, at most 90% amino acid
identity with amino acids
423-847 of SEQ ID NO: 5 or at most 95% amino acid identity with amino acids
423-847 of SEQ ID NO: 5.
[0105] In other aspects of this embodiment, a BoNT/E HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 423-847 of SEQ ID NO: 5. In
other aspects of this
embodiment, a BoNT/E HN region comprises a polypeptide having, e.g., at least
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino
acid substitutions relative to
amino acids 423-847 of SEQ ID NO: 5. In yet other aspects of this embodiment,
a BoNT/E HN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to amino
acids 423-847 of SEQ ID
NO: 5. In other aspects of this embodiment, a BoNT/E HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 423-847 of SEQ ID NO: 5. In still
other aspects of this
embodiment, a BoNT/E HN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid additions relative
to amino acids 423-847 of SEQ ID NO: 5. In other aspects of this embodiment, a
BoNT/E HN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 423-847 of SEQ ID NO:
5.
[0106] In other aspects of this embodiment, a BoNT/E HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid substitutions relative to amino acids 423-847 of SEQ ID NO: 5. In other
aspects of this embodiment,
a BoNT/E HN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid
substitutions relative to amino acids
423-847 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E HN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid deletions relative to amino acids 423-847 of SEQ
ID NO: 5. In other
aspects of this embodiment, a BoNT/E HN region comprises a polypeptide having,
e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid deletions
relative to amino acids 423-847 of SEQ ID NO: 5. In still other aspects of
this embodiment, a BoNT/E HN
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino
acids 423-847 of SEQ ID
NO: 5. In other aspects of this embodiment, a BoNT/E HN region comprises a
polypeptide having, e.g., at
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least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid additions relative to amino acids 423-847 of SEQ ID NO: 5.
[0107] In another embodiment, a Clostridial toxin translocation domain
comprises a BoNT/F HN region.
In an aspect of this embodiment, a BoNT/F HN region comprises amino acids 440-
866 of SEQ ID NO: 6.
In another aspect of this embodiment, a BoNT/F HN region comprises a naturally
occurring BoNT/F HN
region variant, such as, e.g., a HN region from a BoNT/F isoform or a HN
region from a BoNT/F subtype.
In another aspect of this embodiment, a BoNT/F HN region comprises amino acids
440-866 of a naturally
occurring BoNT/F HN region variant of SEQ ID NO: 6, such as, e.g., amino acids
440-866 of a BoNT/F
isoform of SEQ ID NO: 6 or amino acids 440-866 of a BoNT/F subtype of SEQ ID
NO: 6. In still another
aspect of this embodiment, a BoNT/F HN region comprises a non-naturally
occurring BoNT/F HN region
variant, such as, e.g., a conservative BoNT/F HN region variant, a non-
conservative BoNT/F HN region
variant, a BoNT/F chimeric HN region, an active BoNT/F HN region fragment, or
any combination thereof.
In still another aspect of this embodiment, a BoNT/F HN region comprises amino
acids 440-866 of a non-
naturally occurring BoNT/F HN region variant of SEQ ID NO: 6, such as, e.g.,
amino acids 440-866 of a
conservative BoNT/F HN region variant of SEQ ID NO: 6, amino acids 440-866 of
a non-conservative
BoNT/F HN region variant of SEQ ID NO: 6, amino acids 440-866 of an active
BoNT/F HN region fragment
of SEQ ID NO: 6, or any combination thereof.
[0108] In other aspects of this embodiment, a BoNT/F HN region comprises a
polypeptide having, e.g., at
least 70% amino acid identity with amino acids 440-866 of SEQ ID NO: 6, at
least 75% amino acid
identity with amino acids 440-866 of SEQ ID NO: 6, at least 80% amino acid
identity with amino acids
440-866 of SEQ ID NO: 6, at least 85% amino acid identity with amino acids 440-
866 of SEQ ID NO: 6, at
least 90% amino acid identity with amino acids 440-866 of SEQ ID NO: 6 or at
least 95% amino acid
identity with amino acids 440-866 of SEQ ID NO: 6. In yet other aspects of
this embodiment, a BoNT/F
HN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 440-
866 of SEQ ID NO: 6, at most 75% amino acid identity with amino acids 440-866
of SEQ ID NO: 6, at
most 80% amino acid identity with amino acids 440-866 of SEQ ID NO: 6, at most
85% amino acid
identity with amino acids 440-866 of SEQ ID NO: 6, at most 90% amino acid
identity with amino acids
440-866 of SEQ ID NO: 6 or at most 95% amino acid identity with amino acids
440-866 of SEQ ID NO: 6.
[0109] In other aspects of this embodiment, a BoNT/F HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 440-866 of SEQ ID NO: 6. In
other aspects of this
embodiment, a BoNT/F HN region comprises a polypeptide having, e.g., at least
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino
acid substitutions relative to
amino acids 440-866 of SEQ ID NO: 6. In yet other aspects of this embodiment,
a BoNT/F HN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to amino
acids 440-866 of SEQ ID
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NO: 6. In other aspects of this embodiment, a BoNT/F HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 440-866 of SEQ ID NO: 6. In still
other aspects of this
embodiment, a BoNT/F HN region comprises a polypeptide having, e.g., at most
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino
acid additions relative to
amino acids 440-866 of SEQ ID NO: 6. In other aspects of this embodiment, a
BoNT/F HN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 440-866 of SEQ ID NO:
6.
[0110] In other aspects of this embodiment, a BoNT/F HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid substitutions relative to amino acids 440-866 of SEQ ID NO: 6. In other
aspects of this embodiment,
a BoNT/F HN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid
substitutions relative to amino acids
440-866 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F HN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid deletions relative to amino acids 440-866 of SEQ
ID NO: 6. In other
aspects of this embodiment, a BoNT/F HN region comprises a polypeptide having,
e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid deletions
relative to amino acids 440-866 of SEQ ID NO: 6. In still other aspects of
this embodiment, a BoNT/F HN
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino
acids 440-866 of SEQ ID
NO: 6. In other aspects of this embodiment, a BoNT/F HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid additions relative to amino acids 440-866 of SEQ ID NO: 6.
[0111] In another embodiment, a Clostridial toxin translocation domain
comprises a BoNT/G HN region.
In an aspect of this embodiment, a BoNT/G HN region comprises amino acids 447-
865 of SEQ ID NO: 7.
In another aspect of this embodiment, a BoNT/G HN region comprises a naturally
occurring BoNT/G HN
region variant, such as, e.g., a HN region from a BoNT/G isoform or a HN
region from a BoNT/G subtype.
In another aspect of this embodiment, a BoNT/G HN region comprises amino acids
447-865 of a naturally
occurring BoNT/G HN region variant of SEQ ID NO: 7, such as, e.g., amino acids
447-865 of a BoNT/G
isoform of SEQ ID NO: 7 or amino acids 447-865 of a BoNT/G subtype of SEQ ID
NO: 7. In still another
aspect of this embodiment, a BoNT/G HN region comprises a non-naturally
occurring BoNT/G HN region
variant, such as, e.g., a conservative BoNT/G HN region variant, a non-
conservative BoNT/G HN region
variant, a BoNT/G chimeric HN region, an active BoNT/G HN region fragment, or
any combination thereof.
In still another aspect of this embodiment, a BoNT/G HN region comprises amino
acids 447-865 of a non-
naturally occurring BoNT/G HN region variant of SEQ ID NO: 7, such as, e.g.,
amino acids 447-865 of a
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conservative BoNT/G HN region variant of SEQ ID NO: 7, amino acids 447-865 of
a non-conservative
BoNT/G HN region variant of SEQ ID NO: 7, amino acids 447-865 of an active
BoNT/G HN region
fragment of SEQ ID NO: 7, or any combination thereof.
[0112] In other aspects of this embodiment, a BoNT/G HN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 447-865 of SEQ ID NO: 7, at
least 75% amino acid
identity with amino acids 447-865 of SEQ ID NO: 7, at least 80% amino acid
identity with amino acids
447-865 of SEQ ID NO: 7, at least 85% amino acid identity with amino acids 447-
865 of SEQ ID NO: 7, at
least 90% amino acid identity with amino acids 447-865 of SEQ ID NO: 7 or at
least 95% amino acid
identity with amino acids 447-865 of SEQ ID NO: 7. In yet other aspects of
this embodiment, a BoNT/G
HN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 447-
865 of SEQ ID NO: 7, at most 75% amino acid identity with amino acids 447-865
of SEQ ID NO: 7, at
most 80% amino acid identity with amino acids 447-865 of SEQ ID NO: 7, at most
85% amino acid
identity with amino acids 447-865 of SEQ ID NO: 7, at most 90% amino acid
identity with amino acids
447-865 of SEQ ID NO: 7 or at most 95% amino acid identity with amino acids
447-865 of SEQ ID NO: 7.
[0113] In other aspects of this embodiment, a BoNT/G HN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 447-865 of SEQ ID NO: 7. In
other aspects of this
embodiment, a BoNT/G HN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous
amino acid substitutions
relative to amino acids 447-865 of SEQ ID NO: 7. In yet other aspects of this
embodiment, a BoNT/G HN
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to
amino acids 447-865 of SEQ
ID NO: 7. In other aspects of this embodiment, a BoNT/G HN region comprises a
polypeptide having,
e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 non-
contiguous amino acid deletions relative to amino acids 447-865 of SEQ ID NO:
7. In still other aspects
of this embodiment, a BoNT/G HN region comprises a polypeptide having, e.g.,
at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-
contiguous amino acid additions
relative to amino acids 447-865 of SEQ ID NO: 7. In other aspects of this
embodiment, a BoNT/G HN
region comprises a polypeptide having, e.g., at least one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid additions relative to
amino acids 447-865 of SEQ
ID NO: 7.
[0114] In other aspects of this embodiment, a BoNT/G HN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous
amino acid substitutions relative to amino acids 447-865 of SEQ ID NO: 7. In
other aspects of this
embodiment, a BoNT/G HN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid substitutions relative to
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amino acids 447-865 of SEQ ID NO: 7. In yet other aspects of this embodiment,
a BoNT/G HN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40, 50, 100 or 200 contiguous amino acid deletions relative to amino acids
447-865 of SEQ ID NO: 7.
In other aspects of this embodiment, a BoNT/G HN region comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid
deletions relative to amino acids 447-865 of SEQ ID NO: 7. In still other
aspects of this embodiment, a
BoNT/G HN region comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions
relative to amino acids 447-
865 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G HN region
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to amino acids 447-865 of SEQ ID NO:
7.
[0115] In another embodiment, a Clostridial toxin translocation domain
comprises a TeNT HN region. In
an aspect of this embodiment, a TeNT HN region comprises amino acids 458-881
of SEQ ID NO: 8. In
another aspect of this embodiment, a TeNT HN region comprises a naturally
occurring TeNT HN region
variant, such as, e.g., a HN region from a TeNT isoform or a HN region from a
TeNT subtype. In another
aspect of this embodiment, a TeNT HN region comprises amino acids 458-881 of a
naturally occurring
TeNT HN region variant of SEQ ID NO: 8, such as, e.g., amino acids 458-881 of
a TeNT isoform of SEQ
ID NO: 8 or amino acids 458-881 of a TeNT subtype of SEQ ID NO: 8. In still
another aspect of this
embodiment, a TeNT HN region comprises a non-naturally occurring TeNT HN
region variant, such as,
e.g., a conservative TeNT HN region variant, a non-conservative TeNT HN region
variant, a TeNT chimeric
HN region, an active TeNT HN region fragment, or any combination thereof. In
still another aspect of this
embodiment, a TeNT HN region comprises amino acids 458-881 of a non-naturally
occurring TeNT HN
region variant of SEQ ID NO: 8, such as, e.g., amino acids 458-881 of a
conservative TeNT HN region
variant of SEQ ID NO: 8, amino acids 458-881 of a non-conservative TeNT HN
region variant of SEQ ID
NO: 8, amino acids 458-881 of an active TeNT HN region fragment of SEQ ID NO:
8, or any combination
thereof.
[0116] In other aspects of this embodiment, a TeNT HN region comprises a
polypeptide having, e.g., at
least 70% amino acid identity with amino acids 458-881 of SEQ ID NO: 8, at
least 75% amino acid
identity with amino acids 458-881 of SEQ ID NO: 8, at least 80% amino acid
identity with amino acids
458-881 of SEQ ID NO: 8, at least 85% amino acid identity with amino acids 458-
881 of SEQ ID NO: 8, at
least 90% amino acid identity with amino acids 458-881 of SEQ ID NO: 8 or at
least 95% amino acid
identity with amino acids 458-881 of SEQ ID NO: 8. In yet other aspects of
this embodiment, a TeNT HN
region comprises a polypeptide having, e.g., at most 70% amino acid identity
with amino acids 458-881 of
SEQ ID NO: 8, at most 75% amino acid identity with amino acids 458-881 of SEQ
ID NO: 8, at most 80%
amino acid identity with amino acids 458-881 of SEQ ID NO: 8, at most 85%
amino acid identity with
amino acids 458-881 of SEQ ID NO: 8, at most 90% amino acid identity with
amino acids 458-881 of SEQ
ID NO: 8 or at most 95% amino acid identity with amino acids 458-881 of SEQ ID
NO: 8.
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[0117] In other aspects of this embodiment, a TeNT HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 458-881 of SEQ ID NO: 8. In
other aspects of this
embodiment, a TeNT HN region comprises a polypeptide having, e.g., at least
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino
acid substitutions relative to
amino acids 458-881 of SEQ ID NO: 8. In yet other aspects of this embodiment,
a TeNT HN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to amino
acids 458-881 of SEQ ID
NO: 8. In other aspects of this embodiment, a TeNT HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 non-contiguous
amino acid deletions relative to amino acids 458-881 of SEQ ID NO: 8. In still
other aspects of this
embodiment, a TeNT HN region comprises a polypeptide having, e.g., at most
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino
acid additions relative to
amino acids 458-881 of SEQ ID NO: 8. In other aspects of this embodiment, a
TeNT HN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 non-contiguous amino acid additions relative to amino
acids 458-881 of SEQ ID NO:
8.
[0118] In other aspects of this embodiment, a TeNT HN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid substitutions relative to amino acids 458-881 of SEQ ID NO: 8. In other
aspects of this embodiment,
a TeNT HN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid
substitutions relative to amino acids
458-881 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT HN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid deletions relative to amino acids 458-881 of SEQ
ID NO: 8. In other
aspects of this embodiment, a TeNT HN region comprises a polypeptide having,
e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid deletions
relative to amino acids 458-881 of SEQ ID NO: 8. In still other aspects of
this embodiment, a TeNT HN
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino
acids 458-881 of SEQ ID
NO: 8. In other aspects of this embodiment, a TeNT HN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid additions relative to amino acids 458-881 of SEQ ID NO: 8.
[0119] Aspects of the present invention provide, in part, a translocation
facilitating domain. As used
herein, the term "translocation facilitating domain" means any polypeptide
that can further facilitate the
translocation step of the intoxication process that mediates Clostridial toxin
light chain translocation.
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Thus, a translocation facilitating domain assists the Clostridial toxin
translocation domain in the
movement of a Clostridial toxin light chain across a membrane and encompasses
the movement of a
Clostridial toxin light chain through the membrane of an intracellular vesicle
into the cytoplasm of a cell. A
non-limiting example of a translocation facilitating domain is a Clostridial
toxin translocation facilitating
domain, such as, e.g., a Clostridial toxin HCN region such as, e.g., a BoNT/A
HCN region, a BoNT/B HCN
region, a BoNT/C1 HCN region, a BoNT/D HCN region, a BoNT/E HCN region, a
BoNT/F HCN region, a
BoNT/G HCN region, and a TeNT HCN region. Another non-limiting example of a
translocation facilitating
domain is a viral fusogenic peptide domain found in an enveloped virus, such
as, e.g., an influenzavirus,
an alphavirus, a vesiculovirus, a respirovirus, a morbillivirus, an
avulavirus, a henipavirus, a
metapneumovirus and a foamy virus.
[0120] Thus, in an embodiment, a translocation facilitating domain assists the
Clostridial toxin
translocation domain in the movement of a Clostridial toxin light chain across
a membrane. In aspects of
this embodiment, a translocation facilitating domain assists the Clostridial
toxin translocation domain in
the movement of a Clostridial toxin light chain across a membrane by
increasing the amount of Clostridial
toxin light chain in the cytoplasm by, e.g., at least 10 %, at least 20 %, at
least 30 %, at least 40 %, at
least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or at
least 100 %. In other aspects
of this embodiment, a translocation facilitating domain assists the
Clostridial toxin translocation domain in
the movement of a Clostridial toxin light chain across a membrane by
increasing the amount of Clostridial
toxin light chain in the cytoplasm by, e.g., at least two-fold, at least three-
fold, at least four-fold, at least
five-fold, at least ten-fold or at least twenty-fold. In yet other aspects of
this embodiment, a translocation
facilitating domain assists the Clostridial toxin translocation domain in the
movement of a Clostridial toxin
light chain across a membrane by increasing the amount of Clostridial toxin
light chain in the cytoplasm
by, e.g., at most 10 %, at most 20 %, at most 30 %, at most 40 %, at most 50
%, at most 60 %, at most
70 %, at most 80 %, at most 90 % or at most 100 %. In other aspects of this
embodiment, a translocation
facilitating domain assists the Clostridial toxin translocation domain in the
movement of a Clostridial toxin
light chain across a membrane by increasing the amount of Clostridial toxin
light chain in the cytoplasm
by, e.g., at most two-fold, at most three-fold, at most four-fold, at most
five-fold, at most ten-fold or at most
twenty-fold.
[0121] A Clostridial toxin translocation facilitating domain includes, without
limitation, naturally occurring
Clostridial toxin HCN region variants, such as, e.g., Clostridial toxin HCN
region isoforms and Clostridial
toxin HCN region subtypes; non-naturally occurring Clostridial toxin HCN
region variants, such as, e.g.,
conservative Clostridial toxin HCN region variants, non-conservative
Clostridial toxin HCN region variants,
Clostridial toxin HCN region chimerics, active Clostridial toxin HCN region
fragments thereof, or any
combination thereof.
[0122] As used herein, the term "Clostridial toxin HCN region variant,"
whether naturally-occurring or non-
naturally-occurring, means a Clostridial toxin HCN region that has at least
one amino acid change from the
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corresponding region of the disclosed reference sequences (see Table 1) and
can be described in
percent identity to the corresponding region of that reference sequence.
Unless expressly indicated, all
Clostridial toxin HCN region variants disclosed in the present specification
are capable of further facilitating
the translocation step of the intoxication process that mediates Clostridial
toxin light chain translocation.
As non-limiting examples, a BoNT/A HCN region variant comprising amino acids
874-1110 of SEQ ID NO:
1 will have at least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or
addition, as compared to the amino acid region 874-1110 of SEQ ID NO: 1; a
BoNT/B HCN region variant
comprising amino acids 861-1097of SEQ ID NO: 2 will have at least one amino
acid difference, such as,
e.g., an amino acid substitution, deletion or addition, as compared to the
amino acid region 861-1097of
SEQ ID NO: 2; a BoNT/C1 HCN region variant comprising amino acids 869-1111 of
SEQ ID NO: 3 will
have at least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as
compared to the amino acid region 869-1111 of SEQ ID NO: 3; a BoNT/D HCN
region variant comprising
amino acids 865-1098 of SEQ ID NO: 4 will have at least one amino acid
difference, such as, e.g., an
amino acid substitution, deletion or addition, as compared to the amino acid
region 865-1098 of SEQ ID
NO: 4; a BoNT/E HCN region variant comprising amino acids 848-1085 of SEQ ID
NO: 5 will have at least
one amino acid difference, such as, e.g., an amino acid substitution, deletion
or addition, as compared to
the amino acid region 848-1085 of SEQ ID NO: 5; a BoNT/F HCN region variant
comprising amino acids
867-1105 of SEQ ID NO: 6 will have at least one amino acid difference, such
as, e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid region 867-
1105 of SEQ ID NO: 6; a
BoNT/G HCN region variant comprising amino acids 866-1105 of SEQ ID NO: 7 will
have at least one
amino acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the
amino acid region 866-1105 of SEQ ID NO: 7; and a TeNT HCN region variant
comprising amino acids
882-1127 of SEQ ID NO: 8 will have at least one amino acid difference, such
as, e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid region 882-
1127 of SEQ ID NO: 8.
[0123] It is recognized by those of skill in the art that within each serotype
of Clostridial toxin there can
be naturally occurring Clostridial toxin HCN region variants that differ
somewhat in their amino acid
sequence, and also in the nucleic acids encoding these proteins. For example,
there are presently four
BoNT/A subtypes, BoNT/Al, BoNT/A2, BoNT/A3 and BoNT/A4, with specific HCN
region subtypes
showing approximately 87% amino acid identity when compared to another BoNT/A
HCN region subtype.
As used herein, the term "naturally occurring Clostridial toxin HCN region
variant" means any Clostridial
toxin HCN region produced by a naturally-occurring process, including, without
limitation, Clostridial toxin
HCN region isoforms produced from alternatively-spliced transcripts,
Clostridial toxin HCN region isoforms
produced by spontaneous mutation and Clostridial toxin HCN region subtypes. A
naturally occurring
Clostridial toxin HCN region variant can function in substantially the same
manner as the reference
Clostridial toxin HCN region on which the naturally occurring Clostridial
toxin HCN region variant is based,
and can be substituted for the reference Clostridial toxin HCN region in any
aspect of the present
invention. A naturally occurring Clostridial toxin HCN region variant may
substitute one or more amino
acids, two or more amino acids, three or more amino acids, four or more amino
acids, five or more amino
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acids, ten or more amino acids, 20 or more amino acids, 30 or more amino
acids, 40 or more amino acids
or 50 or more amino acids from the reference Clostridial toxin HCN region on
which the naturally occurring
Clostridial toxin HCN region variant is based. A naturally occurring
Clostridial toxin HCN region variant can
also substitute at least 10 contiguous amino acids, at least 15 contiguous
amino acids, at least 20
contiguous amino acids, or at least 25 contiguous amino acids from the
reference Clostridial toxin HCN
region on which the naturally occurring Clostridial toxin HCN region variant
is based, that possess at least
50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85%
amino acid identity or
95% amino acid identity to the reference Clostridial toxin HCN region on which
the naturally occurring
Clostridial toxin HCN region variant is based.
[0124] A non-limiting examples of a naturally occurring Clostridial toxin HCN
region variant is a Clostridial
toxin HCN region isoform such as, e.g., a BoNT/A HCN region isoform, a BoNT/B
HCN region isoform, a
BoNT/C1 HCN region isoform, a BoNT/D HCN region isoform, a BoNT/E HCN region
isoform, a BoNT/F HCN
region isoform, a BoNT/G HCN region isoform, and a TeNT HCN region isoform. A
Clostridial toxin HCN
region isoform can function in substantially the same manner as the reference
Clostridial toxin HCN region
on which the Clostridial toxin HCN region isoform is based, and can be
substituted for the reference
Clostridial toxin HCN region in any aspect of the present invention.
[0125] Another non-limiting examples of a naturally occurring Clostridial
toxin HCN region variant is a
Clostridial toxin HCN region subtype such as, e.g., a HCN region from subtype
BoNT/Al, BoNT/A2,
BoNT/A3 and BoNT/A4; a HCN region from subtype BoNT/B1, BoNT/B2, BoNT/B
bivalent and BoNT/B
nonproteolytic; a HCN region from subtype BoNT/C1-1 and BoNT/C1-2; a HCN
region from subtype
BoNT/El, BoNT/E2 and BoNT/E3; and a HCN region from subtype BoNT/F1, BoNT/F2,
BoNT/F3 and
BoNT/F4. A Clostridial toxin HCN region subtype can function in substantially
the same manner as the
reference Clostridial toxin HCN region on which the Clostridial toxin HCN
region subtype is based, and can
be substituted for the reference Clostridial toxin HCN region in any aspect of
the present invention.
[0126] As used herein, the term "non-naturally occurring Clostridial toxin HCN
region variant" means any
Clostridial toxin HCN region produced with the aid of human manipulation,
including, without limitation,
Clostridial toxin HCN regions produced by genetic engineering using random
mutagenesis or rational
design and Clostridial toxin HCN regions produced by chemical synthesis. Non-
limiting examples of non-
naturally occurring Clostridial toxin HCN region variants include, e.g.,
conservative Clostridial toxin HCN
region variants, non-conservative Clostridial toxin HCN region variants,
Clostridial toxin HCN region
chimeric variants and active Clostridial toxin HCN region fragments.
[0127] As used herein, the term "conservative Clostridial toxin HCN region
variant" means a Clostridial
toxin HCN region that has at least one amino acid substituted by another amino
acid or an amino acid
analog that has at least one property similar to that of the original amino
acid from the reference
Clostridial toxin HCN region sequence (Table 1). Examples of properties
include, without limitation, similar
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size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity,
hydrogen-bonding capacity, a physicochemical property, of the like, or any
combination thereof. A
conservative Clostridial toxin HCN region variant can function in
substantially the same manner as the
reference Clostridial toxin HCN region on which the conservative Clostridial
toxin HCN region variant is
based, and can be substituted for the reference Clostridial toxin HCN region
in any aspect of the present
invention. A conservative Clostridial toxin HCN region variant may substitute
one or more amino acids,
two or more amino acids, three or more amino acids, four or more amino acids,
five or more amino acids,
ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or
more amino acids, 50 or
more amino acids or 100 or more amino acids from the reference Clostridial
toxin HCN region on which the
conservative Clostridial toxin HCN region variant is based. A conservative
Clostridial toxin HCN region
variant can also substitute at least 10 contiguous amino acids, at least 15
contiguous amino acids, at
least 20 contiguous amino acids, or at least 25 contiguous amino acids from
the reference Clostridial
toxin HCN region on which the conservative Clostridial toxin HCN region
variant is based, that possess at
least 50% amino acid identity, 65% amino acid identity, 75% amino acid
identity, 85% amino acid identity
or 95% amino acid identity to the reference Clostridial toxin HCN region on
which the conservative
Clostridial toxin HCN region variant is based. Non-limiting examples of a
conservative Clostridial toxin HCN
region variant include, e.g., conservative BoNT/A HCN region variants,
conservative BoNT/B HCN region
variants, conservative BoNT/C1 HCN region variants, conservative BoNT/D HCN
region variants,
conservative BoNT/E HCN region variants, conservative BoNT/F HCN region
variants, conservative
BoNT/G HCN region variants, and conservative TeNT HCN region variants.
[0128] As used herein, the term "non-conservative Clostridial toxin HCN region
variant" means a
Clostridial toxin HCN region in which 1) at least one amino acid is deleted
from the reference Clostridial
toxin HCN region on which the non-conservative Clostridial toxin HCN region
variant is based; 2) at least
one amino acid added to the reference Clostridial toxin HCN region on which
the non-conservative
Clostridial toxin HCN region is based; or 3) at least one amino acid is
substituted by another amino acid or
an amino acid analog that does not share any property similar to that of the
original amino acid from the
reference Clostridial toxin HCN region sequence (Table 1). A non-conservative
Clostridial toxin HCN region
variant can function in substantially the same manner as the reference
Clostridial toxin HCN region on
which the non-conservative Clostridial toxin HCN region variant is based, and
can be substituted for the
reference Clostridial toxin HCN region in any aspect of the present invention.
A non-conservative
Clostridial toxin HCN region variant can delete one or more amino acids, two
or more amino acids, three or
more amino acids, four or more amino acids, five or more amino acids, and ten
or more amino acids from
the reference Clostridial toxin HCN region on which the non-conservative
Clostridial toxin HCN region
variant is based. A non-conservative Clostridial toxin HCN region variant can
add one or more amino
acids, two or more amino acids, three or more amino acids, four or more amino
acids, five or more amino
acids, and ten or more amino acids to the reference Clostridial toxin HCN
region on which the non-
conservative Clostridial toxin HCN region variant is based. A non-conservative
Clostridial toxin HCN region
variant may substitute one or more amino acids, two or more amino acids, three
or more amino acids,
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four or more amino acids, five or more amino acids, ten or more amino acids,
20 or more amino acids, 30
or more amino acids, 40 or more amino acids, 50 or more amino acids or 100 or
more amino acids from
the reference Clostridial toxin HCN region on which the non-conservative
Clostridial toxin HCN region
variant is based. A non-conservative Clostridial toxin HCN region variant can
also substitute at least 10
contiguous amino acids, at least 15 contiguous amino acids, at least 20
contiguous amino acids, or at
least 25 contiguous amino acids from the reference Clostridial toxin HCN
region on which the non-
conservative Clostridial toxin HCN region variant is based, that possess at
least 50% amino acid identity,
65% amino acid identity, 75% amino acid identity, 85% amino acid identity or
95% amino acid identity to
the reference Clostridial toxin HCN region on which the non-conservative
Clostridial toxin HCN region
variant is based. Non-limiting examples of a non-conservative Clostridial
toxin HCN region variant include,
e.g., non-conservative BoNT/A HCN region variants, non-conservative BoNT/B HCN
region variants, non-
conservative BoNT/C1 HCN region variants, non-conservative BoNT/D HCN region
variants, non-
conservative BoNT/E HCN region variants, non-conservative BoNT/F HCN region
variants, non-
conservative BoNT/G HCN region variants, and non-conservative TeNT HCN region
variants.
[0129] As used herein, the term "Clostridial toxin HCN region chimeric" means
a polypeptide comprising
at least a portion of a Clostridial toxin HCN region and at least a portion of
at least one other polypeptide to
form a toxin HCN region with at least one property different from the
reference Clostridial toxin HCN regions
of Table 1, with the proviso that this Clostridial toxin HCN region chimeric
is still capable of further
facilitating the translocation step of the intoxication process where the LC
is released from intracellular
vesicles into the cytoplasm of the target cell and thus participate in
executing the overall cellular
mechanism whereby a Clostridial toxin proteolytically cleaves a substrate.
[0130] As used herein, the term "active Clostridial toxin HCN region fragment"
means any of a variety of
Clostridial toxin fragments comprising the HcN region can be useful in aspects
of the present invention
with the proviso that these active fragments can further facilitate the
translocation step of the intoxication
process where the LC is released from intracellular vesicles into the
cytoplasm of the target cell and thus
participate in executing the overall cellular mechanism whereby a Clostridial
toxin proteolytically cleaves a
substrate. The HCN domains from the heavy chains of Clostridial toxins are
approximately 230-250 amino
acids in length and comprise a translocation domain (Table 1). Additionally,
while a specific amino acid
positions have been identified to delineate the boundaries of the Clostridial
toxin HCN region (Table 1), it is
well known in the art that the functional boundaries are not definitive. For
example, amino-terminus of the
HCN domain for all naturally-occurring Clostridial toxins is the HN domain
(translocation domain). In
examining the structure for BoNT/A, a random coil linker region forms a
boundary between the HN and
HCN domains (see FIG. 7A). The BoNT/A HN domain appears to end with an a-helix
comprising amino
acids N859 to 1873. Following the a-helix there is a random coil (1873 to
1878) that leads into the HCN
domain where a(3-sheet begins at position 1878. The above residues define
boundaries at the beginning
or end of defined secondary structures and do not imply that there are not
significant interactions (i.e.,
hydrophobic, H-bond, etc.) between residues in the random coil region and one
or both of the domains
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that it links together. Thus, minimally an amino acid that defines the amino-
terminal boundary of the
BoNT/A HCN domain comprises can be any amino acid present in the amino acid
region Y869 to L879.
Similar analysis indicates that minimally, the amino acid that defines the
amino-terminal boundary of the
BoNT/B HCN domain can be any amino acid present in the amino acid region Y856
to L866; the amino
acid that defines the amino-terminal boundary of the BoNT/C1 HCN domain can be
any amino acid
present in the amino acid region Y864 to L874; the amino acid that defines the
amino-terminal boundary
of the BoNT/D HCN domain can be any amino acid present in the amino acid
region Y860 to L870; the
amino acid that defines the amino-terminal boundary of the BoNT/E HCN domain
can be any amino acid
present in the amino acid region F843 to L853; the amino acid that defines the
amino-terminal boundary
of the BoNT/F HCN domain can be any amino acid present in the amino acid
region L862 to L872; the
amino acid that defines the amino-terminal boundary of the BoNT/G HCN domain
can be any amino acid
present in the amino acid region Y861 to L871; and the amino acid that defines
the amino-terminal
boundary of the TeNT HCN domain can be any amino acid present in the amino
acid region 1877 to L887.
[0131] Similarly, the carboxyl-terminal portion of the HCN domain (i.e., the
fusion point between the HCN
and Hcc domains) all naturally-occurring Clostridial toxins comprises a range
of amino acids. In defining
the boundary of the BoNT/A HCN domain as the beginning or the end of ordered
secondary structure, the
HCN domain could end at Q1091 of an a-helix and the Hcc domain could begin at
K1109 of a(3-strand
(see FIG. 7B). The intervening amino acid sequence between these two domains
comprises a longer
random coil but, this does not imply that the random coil is not structurally
important. In fact, this random
coil has a great deal of interaction with both the HCN and Hcc domains (i.e.,
hydrophobic and H-bonding).
Thus, minimally an amino acid that defines the carboxyl-terminal boundary of
the BoNT/A HCN domain
comprises can be any amino acid present in the amino acid region D1089 to
Y1111. Similar analysis
indicates that minimally, the amino acid that defines the carboxyl-terminal
boundary of the BoNT/B HCN
domain can be any amino acid present in the amino acid region K1076 to Y1098;
the amino acid that
defines the carboxyl-terminal boundary of the BoNT/C1 HCN domain can be any
amino acid present in the
amino acid region N1090 to Y1112; the amino acid that defines the carboxyl-
terminal boundary of the
BoNT/D HCN domain can be any amino acid present in the amino acid region E1077
to Y1099; the amino
acid that defines the carboxyl-terminal boundary of the BoNT/E HCN domain can
be any amino acid
present in the amino acid region S1064 to Y1086; the amino acid that defines
the carboxyl-terminal
boundary of the BoNT/F HCN domain can be any amino acid present in the amino
acid region S1084 to
Y1106; the amino acid that defines the carboxyl-terminal boundary of the
BoNT/G HCN domain can be any
amino acid present in the amino acid region W1084 to Y1106; and the amino acid
that defines the
carboxyl-terminal boundary of the TeNT HCN domain can be any amino acid
present in the amino acid
region T1106 to Y1128.
[0132] Thus, aspects of this embodiment can include Clostridial toxin HCN
regions comprising a
translocation facilitating domain having a length of, e.g., at least 200 amino
acids, at least 225 amino
acids, at least 250 amino acids and at least 275 amino acids. Other aspects of
this embodiment can
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include Clostridial toxin HCN regions comprising translocation facilitating
domain having a length of, e.g.,
at most 200 amino acids, at most 225 amino acids, at most 250 amino acids and
at most 275 amino
acids.
[0133] Any of a variety of sequence alignment methods can be used to determine
percent identity of
naturally-occurring Clostridial toxin HCN region variants and non-naturally-
occurring Clostridial toxin HCN
region variants, including, without limitation, global methods, local methods
and hybrid methods, such as,
e.g., segment approach methods. Protocols to determine percent identity are
routine procedures within
the scope of one skilled in the art and from the teaching herein.
[0134] Thus, in an embodiment, a modified Clostridial toxin disclosed in the
present specification
comprises a Clostridial toxin translocation facilitating domain. In an aspect
of this embodiment, a
Clostridial toxin translocation facilitating domain comprises a naturally
occurring Clostridial toxin HCN
region variant, such as, e.g., a Clostridial toxin HCN region isoform or a
Clostridial toxin HCN region
subtype. In another aspect of this embodiment, a Clostridial toxin
translocation domain comprises a non-
naturally occurring Clostridial toxin HCN region variant, such as, e.g., a
conservative Clostridial toxin HCN
region variant, a non-conservative Clostridial toxin HCN region variant, a
Clostridial toxin chimeric HCN
region, an active Clostridial toxin HCN region fragment, or any combination
thereof.
[0135] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a BoNT/A
HCN region. In an aspect of this embodiment, a BoNT/A HCN region comprises
amino acids 874-1110 of
SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/A HCN region
comprises a naturally
occurring BoNT/A HCN region variant, such as, e.g., a HCN region from a BoNT/A
isoform or a HCN region
from a BoNT/A subtype. In another aspect of this embodiment, a BoNT/A HCN
region comprises amino
acids 874-1110 of a naturally occurring BoNT/A HCN region variant of SEQ ID
NO: 1, such as, e.g., amino
acids 874-1110 of a BoNT/A isoform of SEQ ID NO: 1 or amino acids 874-1110 of
a BoNT/A subtype of
SEQ ID NO: 1. In still another aspect of this embodiment, a BoNT/A HCN region
comprises a non-
naturally occurring BoNT/A HCN region variant, such as, e.g., a conservative
BoNT/A HCN region variant, a
non-conservative BoNT/A HCN region variant, a BoNT/A chimeric HCN region, an
active BoNT/A HCN
region fragment, or any combination thereof. In still another aspect of this
embodiment, a BoNT/A HCN
region comprises amino acids 874-1110 of a non-naturally occurring BoNT/A HCN
region variant of SEQ
ID NO: 1, such as, e.g., amino acids 874-1110 of a conservative BoNT/A HCN
region variant of SEQ ID
NO: 1, amino acids 874-1110 of a non-conservative BoNT/A HCN region variant of
SEQ ID NO: 1, amino
acids 874-1110 of an active BoNT/A HCN region fragment of SEQ ID NO: 1, or any
combination thereof.
[0136] In other aspects of this embodiment, a BoNT/A HCN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 874-1110 of SEQ ID NO: 1, at
least 75% amino acid
identity with amino acids 874-1110 of SEQ ID NO: 1, at least 80% amino acid
identity with amino acids
874-1110 of SEQ ID NO: 1, at least 85% amino acid identity with amino acids
874-1110 of SEQ ID NO: 1,
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at least 90% amino acid identity with amino acids 874-1110 of SEQ ID NO: 1 or
at least 95% amino acid
identity with amino acids 874-1110 of SEQ ID NO: 1. In yet other aspects of
this embodiment, a BoNT/A
HCN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 874-
1110 of SEQ ID NO: 1, at most 75% amino acid identity with amino acids 874-
1110 of SEQ ID NO: 1, at
most 80% amino acid identity with amino acids 874-1110 of SEQ ID NO: 1, at
most 85% amino acid
identity with amino acids 874-1110 of SEQ ID NO: 1, at most 90% amino acid
identity with amino acids
874-1110 of SEQ ID NO: 1 or at most 95% amino acid identity with amino acids
874-1110 of SEQ ID NO:
1.
[0137] In other aspects of this embodiment, a BoNT/A HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 874-1110 of SEQ ID NO: 1. In
other aspects of this
embodiment, a BoNT/A HCN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid substitutions relative to
amino acids 874-1110 of SEQ ID NO: 1. In yet other aspects of this embodiment,
a BoNT/A HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 non-contiguous amino acid deletions relative to amino acids
874-1110 of SEQ ID NO:
1. In other aspects of this embodiment, a BoNT/A HCN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 non-contiguous amino
acid deletions relative to amino acids 874-1110 of SEQ ID NO: 1. In still
other aspects of this
embodiment, a BoNT/A HCN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid additions relative to
amino acids 874-1110 of SEQ ID NO: 1. In other aspects of this embodiment, a
BoNT/A HCN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50 or 100 non-contiguous amino acid additions relative to amino acids 874-
1110 of SEQ ID NO: 1.
[0138] In other aspects of this embodiment, a BoNT/A HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 contiguous amino acid
substitutions relative to amino acids 874-1110 of SEQ ID NO: 1. In other
aspects of this embodiment, a
BoNT/A HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous amino acid substitutions
relative to amino acids 874-
1110 of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A HCN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid deletions relative to amino acids 874-1110 of SEQ ID
NO: 1. In other aspects
of this embodiment, a BoNT/A HCN region comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous
amino acid deletions relative to
amino acids 874-1110 of SEQ ID NO: 1. In still other aspects of this
embodiment, a BoNT/A HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 contiguous amino acid additions relative to amino acids 874-
1110 of SEQ ID NO: 1. In
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other aspects of this embodiment, a BoNT/A HCN region comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid
additions relative to amino acids 874-1110 of SEQ ID NO: 1.
[0139] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a BoNT/B
HCN region. In an aspect of this embodiment, a BoNT/B HCN region comprises
amino acids 861-1097of
SEQ ID NO: 2. In another aspect of this embodiment, a BoNT/B HCN region
comprises a naturally
occurring BoNT/B HCN region variant, such as, e.g., a HCN region from a BoNT/B
isoform or a HCN region
from a BoNT/B subtype. In another aspect of this embodiment, a BoNT/B HCN
region comprises amino
acids 861-1097of a naturally occurring BoNT/B HCN region variant of SEQ ID NO:
2, such as, e.g., amino
acids 861-1097of a BoNT/B isoform of SEQ ID NO: 2 or amino acids 861-1097of a
BoNT/B subtype of
SEQ ID NO: 2. In still another aspect of this embodiment, a BoNT/B HCN region
comprises a non-
naturally occurring BoNT/B HCN region variant, such as, e.g., a conservative
BoNT/B HCN region variant, a
non-conservative BoNT/B HCN region variant, a BoNT/B chimeric HCN region, an
active BoNT/B HCN
region fragment, or any combination thereof. In still another aspect of this
embodiment, a BoNT/B HCN
region comprises amino acids 861-1097of a non-naturally occurring BoNT/B HCN
region variant of SEQ ID
NO: 2, such as, e.g., amino acids 861-1097of a conservative BoNT/B HCN region
variant of SEQ ID NO: 2,
amino acids 861-1097of a non-conservative BoNT/B HCN region variant of SEQ ID
NO: 2, amino acids
861-1097of an active BoNT/B HCN region fragment of SEQ ID NO: 2, or any
combination thereof.
[0140] In other aspects of this embodiment, a BoNT/B HCN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 861-1097of SEQ ID NO: 2, at
least 75% amino acid
identity with amino acids 861-1097of SEQ ID NO: 2, at least 80% amino acid
identity with amino acids
861-1097of SEQ ID NO: 2, at least 85% amino acid identity with amino acids 861-
1097of SEQ ID NO: 2,
at least 90% amino acid identity with amino acids 861-1097of SEQ ID NO: 2 or
at least 95% amino acid
identity with amino acids 861-1097of SEQ ID NO: 2. In yet other aspects of
this embodiment, a BoNT/B
HCN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 861-
1097of SEQ ID NO: 2, at most 75% amino acid identity with amino acids 861-
1097of SEQ ID NO: 2, at
most 80% amino acid identity with amino acids 861-1097of SEQ ID NO: 2, at most
85% amino acid
identity with amino acids 861-1097of SEQ ID NO: 2, at most 90% amino acid
identity with amino acids
861-1097of SEQ ID NO: 2 or at most 95% amino acid identity with amino acids
861-1097of SEQ ID NO:
2.
[0141] In other aspects of this embodiment, a BoNT/B HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 non-contiguous amino
acid substitutions relative to amino acids 861-1097of SEQ ID NO: 2. In other
aspects of this embodiment,
a BoNT/B HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino acid
substitutions relative to amino acids
861-1097of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B HCN
region comprises a
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polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 non-contiguous amino acid deletions relative to amino acids 861-1097of SEQ
ID NO: 2. In other
aspects of this embodiment, a BoNT/B HCN region comprises a polypeptide
having, e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-
contiguous amino acid deletions
relative to amino acids 861-1097of SEQ ID NO: 2. In still other aspects of
this embodiment, a BoNT/B
HCN region comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50 or 100 non-contiguous amino acid additions relative to
amino acids 861-1097of SEQ
ID NO: 2. In other aspects of this embodiment, a BoNT/B HCN region comprises a
polypeptide having,
e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 non-contiguous
amino acid additions relative to amino acids 861-1097of SEQ ID NO: 2.
[0142] In other aspects of this embodiment, a BoNT/B HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 contiguous amino acid
substitutions relative to amino acids 861-1097of SEQ ID NO: 2. In other
aspects of this embodiment, a
BoNT/B HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous amino acid substitutions
relative to amino acids 861-
1097of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B HCN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid deletions relative to amino acids 861-1097of SEQ ID
NO: 2. In other aspects
of this embodiment, a BoNT/B HCN region comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous
amino acid deletions relative to
amino acids 861-1097of SEQ ID NO: 2. In still other aspects of this
embodiment, a BoNT/B HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 contiguous amino acid additions relative to amino acids 861-
1097of SEQ ID NO: 2. In
other aspects of this embodiment, a BoNT/B HCN region comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid
additions relative to amino acids 861-1097of SEQ ID NO: 2.
[0143] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a BoNT/C1
HCN region. In an aspect of this embodiment, a BoNT/C1 HCN region comprises
amino acids 869-1111 of
SEQ ID NO: 3. In another aspect of this embodiment, a BoNT/C1 HCN region
comprises a naturally
occurring BoNT/C1 HCN region variant, such as, e.g., a HCN region from a
BoNT/C1 isoform or a HCN
region from a BoNT/C1 subtype. In another aspect of this embodiment, a BoNT/C1
HCN region comprises
amino acids 869-1111 of a naturally occurring BoNT/C1 HCN region variant of
SEQ ID NO: 3, such as,
e.g., amino acids 869-1111 of a BoNT/C1 isoform of SEQ ID NO: 3 or amino acids
869-1111 of a
BoNT/C1 subtype of SEQ ID NO: 3. In still another aspect of this embodiment, a
BoNT/C1 HCN region
comprises a non-naturally occurring BoNT/C1 HCN region variant, such as, e.g.,
a conservative BoNT/C1
HCN region variant, a non-conservative BoNT/C1 HCN region variant, a BoNT/C1
chimeric HCN region, an
active BoNT/C1 HCN region fragment, or any combination thereof. In still
another aspect of this
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embodiment, a BoNT/C1 HCN region comprises amino acids 869-1111 of a non-
naturally occurring
BoNT/C1 HCN region variant of SEQ ID NO: 3, such as, e.g., amino acids 869-
1111 of a conservative
BoNT/C1 HCN region variant of SEQ ID NO: 3, amino acids 869-1111 of a non-
conservative BoNT/C1 HCN
region variant of SEQ ID NO: 3, amino acids 869-1111 of an active BoNT/C1 HCN
region fragment of SEQ
ID NO: 3, or any combination thereof.
[0144] In other aspects of this embodiment, a BoNT/C1 HCN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 869-1111 of SEQ ID NO: 3, at
least 75% amino acid
identity with amino acids 869-1111 of SEQ ID NO: 3, at least 80% amino acid
identity with amino acids
869-1111 of SEQ ID NO: 3, at least 85% amino acid identity with amino acids
869-1111 of SEQ ID NO: 3,
at least 90% amino acid identity with amino acids 869-1111 of SEQ ID NO: 3 or
at least 95% amino acid
identity with amino acids 869-1111 of SEQ ID NO: 3. In yet other aspects of
this embodiment, a
BoNT/C1 HCN region comprises a polypeptide having, e.g., at most 70% amino
acid identity with amino
acids 869-1111 of SEQ ID NO: 3, at most 75% amino acid identity with amino
acids 869-1111 of SEQ ID
NO: 3, at most 80% amino acid identity with amino acids 869-1111 of SEQ ID NO:
3, at most 85% amino
acid identity with amino acids 869-1111 of SEQ ID NO: 3, at most 90% amino
acid identity with amino
acids 869-1111 of SEQ ID NO: 3 or at most 95% amino acid identity with amino
acids 869-1111 of SEQ
ID NO: 3.
[0145] In other aspects of this embodiment, a BoNT/C1 HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 869-1111 of SEQ ID NO: 3. In
other aspects of this
embodiment, a BoNT/C1 HCN region comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid substitutions relative to
amino acids 869-1111 of SEQ ID NO: 3. In yet other aspects of this embodiment,
a BoNT/C1 HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 non-contiguous amino acid deletions relative to amino acids
869-1111 of SEQ ID NO:
3. In other aspects of this embodiment, a BoNT/C1 HCN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 non-contiguous amino
acid deletions relative to amino acids 869-1111 of SEQ ID NO: 3. In still
other aspects of this
embodiment, a BoNT/C1 HCN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid additions relative to
amino acids 869-1111 of SEQ ID NO: 3. In other aspects of this embodiment, a
BoNT/C1 HCN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50 or 100 non-contiguous amino acid additions relative to amino acids 869-
1111 of SEQ ID NO: 3.
[0146] In other aspects of this embodiment, a BoNT/C1 HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 contiguous amino acid
substitutions relative to amino acids 869-1111 of SEQ ID NO: 3. In other
aspects of this embodiment, a
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BoNT/C1 HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous amino acid substitutions
relative to amino acids 869-
1111 of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 HCN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid deletions relative to amino acids 869-1111 of SEQ ID
NO: 3. In other aspects
of this embodiment, a BoNT/C1 HCN region comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous
amino acid deletions relative to
amino acids 869-1111 of SEQ ID NO: 3. In still other aspects of this
embodiment, a BoNT/C1 HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 contiguous amino acid additions relative to amino acids 869-
1111 of SEQ ID NO: 3. In
other aspects of this embodiment, a BoNT/C1 HCN region comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid
additions relative to amino acids 869-1111 of SEQ ID NO: 3.
[0147] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a BoNT/D
HCN region. In an aspect of this embodiment, a BoNT/D HCN region comprises
amino acids 865-1098 of
SEQ ID NO: 4. In another aspect of this embodiment, a BoNT/D HCN region
comprises a naturally
occurring BoNT/D HCN region variant, such as, e.g., a HCN region from a BoNT/D
isoform or a HCN region
from a BoNT/D subtype. In another aspect of this embodiment, a BoNT/D HCN
region comprises amino
acids 865-1098 of a naturally occurring BoNT/D HCN region variant of SEQ ID
NO: 4, such as, e.g., amino
acids 865-1098 of a BoNT/D isoform of SEQ ID NO: 4 or amino acids 865-1098 of
a BoNT/D subtype of
SEQ ID NO: 4. In still another aspect of this embodiment, a BoNT/D HCN region
comprises a non-
naturally occurring BoNT/D HCN region variant, such as, e.g., a conservative
BoNT/D HCN region variant, a
non-conservative BoNT/D HCN region variant, a BoNT/D chimeric HCN region, an
active BoNT/D HCN
region fragment, or any combination thereof. In still another aspect of this
embodiment, a BoNT/D HCN
region comprises amino acids 865-1098 of a non-naturally occurring BoNT/D HCN
region variant of SEQ
ID NO: 4, such as, e.g., amino acids 865-1098 of a conservative BoNT/D HCN
region variant of SEQ ID
NO: 4, amino acids 865-1098 of a non-conservative BoNT/D HCN region variant of
SEQ ID NO: 4, amino
acids 865-1098 of an active BoNT/D HCN region fragment of SEQ ID NO: 4, or any
combination thereof.
[0148] In other aspects of this embodiment, a BoNT/D HCN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 865-1098 of SEQ ID NO: 4, at
least 75% amino acid
identity with amino acids 865-1098 of SEQ ID NO: 4, at least 80% amino acid
identity with amino acids
865-1098 of SEQ ID NO: 4, at least 85% amino acid identity with amino acids
865-1098 of SEQ ID NO: 4,
at least 90% amino acid identity with amino acids 865-1098 of SEQ ID NO: 4 or
at least 95% amino acid
identity with amino acids 865-1098 of SEQ ID NO: 4. In yet other aspects of
this embodiment, a BoNT/D
HCN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 865-
1098 of SEQ ID NO: 4, at most 75% amino acid identity with amino acids 865-
1098 of SEQ ID NO: 4, at
most 80% amino acid identity with amino acids 865-1098 of SEQ ID NO: 4, at
most 85% amino acid
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identity with amino acids 865-1098 of SEQ ID NO: 4, at most 90% amino acid
identity with amino acids
865-1098 of SEQ ID NO: 4 or at most 95% amino acid identity with amino acids
865-1098 of SEQ ID NO:
4.
[0149] In other aspects of this embodiment, a BoNT/D HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 865-1098 of SEQ ID NO: 4. In
other aspects of this
embodiment, a BoNT/D HCN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid substitutions relative to
amino acids 865-1098 of SEQ ID NO: 4. In yet other aspects of this embodiment,
a BoNT/D HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 non-contiguous amino acid deletions relative to amino acids
865-1098 of SEQ ID NO:
4. In other aspects of this embodiment, a BoNT/D HCN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 non-contiguous amino
acid deletions relative to amino acids 865-1098 of SEQ ID NO: 4. In still
other aspects of this
embodiment, a BoNT/D HCN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid additions relative to
amino acids 865-1098 of SEQ ID NO: 4. In other aspects of this embodiment, a
BoNT/D HCN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50 or 100 non-contiguous amino acid additions relative to amino acids 865-
1098 of SEQ ID NO: 4.
[0150] In other aspects of this embodiment, a BoNT/D HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 contiguous amino acid
substitutions relative to amino acids 865-1098 of SEQ ID NO: 4. In other
aspects of this embodiment, a
BoNT/D HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous amino acid substitutions
relative to amino acids 865-
1098 of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D HCN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid deletions relative to amino acids 865-1098 of SEQ ID
NO: 4. In other aspects
of this embodiment, a BoNT/D HCN region comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous
amino acid deletions relative to
amino acids 865-1098 of SEQ ID NO: 4. In still other aspects of this
embodiment, a BoNT/D HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 contiguous amino acid additions relative to amino acids 865-
1098 of SEQ ID NO: 4. In
other aspects of this embodiment, a BoNT/D HCN region comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid
additions relative to amino acids 865-1098 of SEQ ID NO: 4.
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[0151] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a BoNT/E
HCN region. In an aspect of this embodiment, a BoNT/E HCN region comprises
amino acids 848-1085 of
SEQ ID NO: 5. In another aspect of this embodiment, a BoNT/E HCN region
comprises a naturally
occurring BoNT/E HCN region variant, such as, e.g., a HCN region from a BoNT/E
isoform or a HCN region
from a BoNT/E subtype. In another aspect of this embodiment, a BoNT/E HCN
region comprises amino
acids 848-1085 of a naturally occurring BoNT/E HCN region variant of SEQ ID
NO: 5, such as, e.g., amino
acids 848-1085 of a BoNT/E isoform of SEQ ID NO: 5 or amino acids 848-1085 of
a BoNT/E subtype of
SEQ ID NO: 5. In still another aspect of this embodiment, a BoNT/E HCN region
comprises a non-
naturally occurring BoNT/E HCN region variant, such as, e.g., a conservative
BoNT/E HCN region variant, a
non-conservative BoNT/E HCN region variant, a BoNT/E chimeric HCN region, an
active BoNT/E HCN
region fragment, or any combination thereof. In still another aspect of this
embodiment, a BoNT/E HCN
region comprises amino acids 848-1085 of a non-naturally occurring BoNT/E HCN
region variant of SEQ
ID NO: 5, such as, e.g., amino acids 848-1085 of a conservative BoNT/E HCN
region variant of SEQ ID
NO: 5, amino acids 848-1085 of a non-conservative BoNT/E HCN region variant of
SEQ ID NO: 5, amino
acids 848-1085 of an active BoNT/E HCN region fragment of SEQ ID NO: 5, or any
combination thereof.
[0152] In other aspects of this embodiment, a BoNT/E HCN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 848-1085 of SEQ ID NO: 5, at
least 75% amino acid
identity with amino acids 848-1085 of SEQ ID NO: 5, at least 80% amino acid
identity with amino acids
848-1085 of SEQ ID NO: 5, at least 85% amino acid identity with amino acids
848-1085 of SEQ ID NO: 5,
at least 90% amino acid identity with amino acids 848-1085 of SEQ ID NO: 5 or
at least 95% amino acid
identity with amino acids 848-1085 of SEQ ID NO: 5. In yet other aspects of
this embodiment, a BoNT/E
HCN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 848-
1085 of SEQ ID NO: 5, at most 75% amino acid identity with amino acids 848-
1085 of SEQ ID NO: 5, at
most 80% amino acid identity with amino acids 848-1085 of SEQ ID NO: 5, at
most 85% amino acid
identity with amino acids 848-1085 of SEQ ID NO: 5, at most 90% amino acid
identity with amino acids
848-1085 of SEQ ID NO: 5 or at most 95% amino acid identity with amino acids
848-1085 of SEQ ID NO:
5.
[0153] In other aspects of this embodiment, a BoNT/E HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 848-1085 of SEQ ID NO: 5. In
other aspects of this
embodiment, a BoNT/E HCN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid substitutions relative to
amino acids 848-1085 of SEQ ID NO: 5. In yet other aspects of this embodiment,
a BoNT/E HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 non-contiguous amino acid deletions relative to amino acids
848-1085 of SEQ ID NO:
5. In other aspects of this embodiment, a BoNT/E HCN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 non-contiguous amino
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acid deletions relative to amino acids 848-1085 of SEQ ID NO: 5. In still
other aspects of this
embodiment, a BoNT/E HCN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid additions relative to
amino acids 848-1085 of SEQ ID NO: 5. In other aspects of this embodiment, a
BoNT/E HCN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50 or 100 non-contiguous amino acid additions relative to amino acids 848-
1085 of SEQ ID NO: 5.
[0154] In other aspects of this embodiment, a BoNT/E HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 contiguous amino acid
substitutions relative to amino acids 848-1085 of SEQ ID NO: 5. In other
aspects of this embodiment, a
BoNT/E HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous amino acid substitutions
relative to amino acids 848-
1085 of SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E HCN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid deletions relative to amino acids 848-1085 of SEQ ID
NO: 5. In other aspects
of this embodiment, a BoNT/E HCN region comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous
amino acid deletions relative to
amino acids 848-1085 of SEQ ID NO: 5. In still other aspects of this
embodiment, a BoNT/E HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 contiguous amino acid additions relative to amino acids 848-
1085 of SEQ ID NO: 5. In
other aspects of this embodiment, a BoNT/E HCN region comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid
additions relative to amino acids 848-1085 of SEQ ID NO: 5.
[0155] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a BoNT/F
HCN region. In an aspect of this embodiment, a BoNT/F HCN region comprises
amino acids 867-1105 of
SEQ ID NO: 6. In another aspect of this embodiment, a BoNT/F HCN region
comprises a naturally
occurring BoNT/F HCN region variant, such as, e.g., a HCN region from a BoNT/F
isoform or a HCN region
from a BoNT/F subtype. In another aspect of this embodiment, a BoNT/F HCN
region comprises amino
acids 867-1105 of a naturally occurring BoNT/F HCN region variant of SEQ ID
NO: 6, such as, e.g., amino
acids 867-1105 of a BoNT/F isoform of SEQ ID NO: 6 or amino acids 867-1105 of
a BoNT/F subtype of
SEQ ID NO: 6. In still another aspect of this embodiment, a BoNT/F HCN region
comprises a non-
naturally occurring BoNT/F HCN region variant, such as, e.g., a conservative
BoNT/F HCN region variant, a
non-conservative BoNT/F HCN region variant, a BoNT/F chimeric HCN region, an
active BoNT/F HCN region
fragment, or any combination thereof. In still another aspect of this
embodiment, a BoNT/F HCN region
comprises amino acids 867-1105 of a non-naturally occurring BoNT/F HCN region
variant of SEQ ID NO:
6, such as, e.g., amino acids 867-1105 of a conservative BoNT/F HCN region
variant of SEQ ID NO: 6,
amino acids 867-1105 of a non-conservative BoNT/F HCN region variant of SEQ ID
NO: 6, amino acids
867-1105 of an active BoNT/F HCN region fragment of SEQ ID NO: 6, or any
combination thereof.
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[0156] In other aspects of this embodiment, a BoNT/F HCN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 867-1105 of SEQ ID NO: 6, at
least 75% amino acid
identity with amino acids 867-1105 of SEQ ID NO: 6, at least 80% amino acid
identity with amino acids
867-1105 of SEQ ID NO: 6, at least 85% amino acid identity with amino acids
867-1105 of SEQ ID NO: 6,
at least 90% amino acid identity with amino acids 867-1105 of SEQ ID NO: 6 or
at least 95% amino acid
identity with amino acids 867-1105 of SEQ ID NO: 6. In yet other aspects of
this embodiment, a BoNT/F
HCN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 867-
1105 of SEQ ID NO: 6, at most 75% amino acid identity with amino acids 867-
1105 of SEQ ID NO: 6, at
most 80% amino acid identity with amino acids 867-1105 of SEQ ID NO: 6, at
most 85% amino acid
identity with amino acids 867-1105 of SEQ ID NO: 6, at most 90% amino acid
identity with amino acids
867-1105 of SEQ ID NO: 6 or at most 95% amino acid identity with amino acids
867-1105 of SEQ ID NO:
6.
[0157] In other aspects of this embodiment, a BoNT/F HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 867-1105 of SEQ ID NO: 6. In
other aspects of this
embodiment, a BoNT/F HCN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid substitutions relative to
amino acids 867-1105 of SEQ ID NO: 6. In yet other aspects of this embodiment,
a BoNT/F HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 non-contiguous amino acid deletions relative to amino acids
867-1105 of SEQ ID NO:
6. In other aspects of this embodiment, a BoNT/F HCN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 non-contiguous amino
acid deletions relative to amino acids 867-1105 of SEQ ID NO: 6. In still
other aspects of this
embodiment, a BoNT/F HCN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid additions relative to
amino acids 867-1105 of SEQ ID NO: 6. In other aspects of this embodiment, a
BoNT/F HCN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50 or 100 non-contiguous amino acid additions relative to amino acids 867-
1105 of SEQ ID NO: 6.
[0158] In other aspects of this embodiment, a BoNT/F HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 contiguous amino acid
substitutions relative to amino acids 867-1105 of SEQ ID NO: 6. In other
aspects of this embodiment, a
BoNT/F HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous amino acid substitutions
relative to amino acids 867-
1105 of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F HCN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid deletions relative to amino acids 867-1105 of SEQ ID
NO: 6. In other aspects
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of this embodiment, a BoNT/F HCN region comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous
amino acid deletions relative to
amino acids 867-1105 of SEQ ID NO: 6. In still other aspects of this
embodiment, a BoNT/F HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 contiguous amino acid additions relative to amino acids 867-
1105 of SEQ ID NO: 6. In
other aspects of this embodiment, a BoNT/F HCN region comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid
additions relative to amino acids 867-1105 of SEQ ID NO: 6.
[0159] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a BoNT/G
HCN region. In an aspect of this embodiment, a BoNT/G HCN region comprises
amino acids 866-1105 of
SEQ ID NO: 7. In another aspect of this embodiment, a BoNT/G HCN region
comprises a naturally
occurring BoNT/G HCN region variant, such as, e.g., a HCN region from a BoNT/G
isoform or a HCN region
from a BoNT/G subtype. In another aspect of this embodiment, a BoNT/G HCN
region comprises amino
acids 866-1105 of a naturally occurring BoNT/G HCN region variant of SEQ ID
NO: 7, such as, e.g., amino
acids 866-1105 of a BoNT/G isoform of SEQ ID NO: 7 or amino acids 866-1105 of
a BoNT/G subtype of
SEQ ID NO: 7. In still another aspect of this embodiment, a BoNT/G HCN region
comprises a non-
naturally occurring BoNT/G HCN region variant, such as, e.g., a conservative
BoNT/G HCN region variant,
a non-conservative BoNT/G HCN region variant, a BoNT/G chimeric HCN region, an
active BoNT/G HCN
region fragment, or any combination thereof. In still another aspect of this
embodiment, a BoNT/G HCN
region comprises amino acids 866-1105 of a non-naturally occurring BoNT/G HCN
region variant of SEQ
ID NO: 7, such as, e.g., amino acids 866-1105 of a conservative BoNT/G HCN
region variant of SEQ ID
NO: 7, amino acids 866-1105 of a non-conservative BoNT/G HCN region variant of
SEQ ID NO: 7, amino
acids 866-1105 of an active BoNT/G HCN region fragment of SEQ ID NO: 7, or any
combination thereof.
[0160] In other aspects of this embodiment, a BoNT/G HCN region comprises a
polypeptide having, e.g.,
at least 70% amino acid identity with amino acids 866-1105 of SEQ ID NO: 7, at
least 75% amino acid
identity with amino acids 866-1105 of SEQ ID NO: 7, at least 80% amino acid
identity with amino acids
866-1105 of SEQ ID NO: 7, at least 85% amino acid identity with amino acids
866-1105 of SEQ ID NO: 7,
at least 90% amino acid identity with amino acids 866-1105 of SEQ ID NO: 7 or
at least 95% amino acid
identity with amino acids 866-1105 of SEQ ID NO: 7. In yet other aspects of
this embodiment, a BoNT/G
HCN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 866-
1105 of SEQ ID NO: 7, at most 75% amino acid identity with amino acids 866-
1105 of SEQ ID NO: 7, at
most 80% amino acid identity with amino acids 866-1105 of SEQ ID NO: 7, at
most 85% amino acid
identity with amino acids 866-1105 of SEQ ID NO: 7, at most 90% amino acid
identity with amino acids
866-1105 of SEQ ID NO: 7 or at most 95% amino acid identity with amino acids
866-1105 of SEQ ID NO:
7.
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[0161] In other aspects of this embodiment, a BoNT/G HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 866-1105 of SEQ ID NO: 7. In
other aspects of this
embodiment, a BoNT/G HCN region comprises a polypeptide having, e.g., at least
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid substitutions relative to
amino acids 866-1105 of SEQ ID NO: 7. In yet other aspects of this embodiment,
a BoNT/G HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 non-contiguous amino acid deletions relative to amino acids
866-1105 of SEQ ID NO:
7. In other aspects of this embodiment, a BoNT/G HCN region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 non-contiguous amino
acid deletions relative to amino acids 866-1105 of SEQ ID NO: 7. In still
other aspects of this
embodiment, a BoNT/G HCN region comprises a polypeptide having, e.g., at most
one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino
acid additions relative to
amino acids 866-1105 of SEQ ID NO: 7. In other aspects of this embodiment, a
BoNT/G HCN region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50 or 100 non-contiguous amino acid additions relative to amino acids 866-
1105 of SEQ ID NO: 7.
[0162] In other aspects of this embodiment, a BoNT/G HCN region comprises a
polypeptide having, e.g.,
at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50 or 100 contiguous amino acid
substitutions relative to amino acids 866-1105 of SEQ ID NO: 7. In other
aspects of this embodiment, a
BoNT/G HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous amino acid substitutions
relative to amino acids 866-
1105 of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G HCN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid deletions relative to amino acids 866-1105 of SEQ ID
NO: 7. In other aspects
of this embodiment, a BoNT/G HCN region comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous
amino acid deletions relative to
amino acids 866-1105 of SEQ ID NO: 7. In still other aspects of this
embodiment, a BoNT/G HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 contiguous amino acid additions relative to amino acids 866-
1105 of SEQ ID NO: 7. In
other aspects of this embodiment, a BoNT/G HCN region comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid
additions relative to amino acids 866-1105 of SEQ ID NO: 7.
[0163] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a TeNT HCN
region. In an aspect of this embodiment, a TeNT HCN region comprises amino
acids 882-1127 of SEQ ID
NO: 8. In another aspect of this embodiment, a TeNT HCN region comprises a
naturally occurring TeNT
HCN region variant, such as, e.g., a HCN region from a TeNT isoform or a HCN
region from a TeNT subtype.
In another aspect of this embodiment, a TeNT HCN region comprises amino acids
882-1127 of a naturally
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occurring TeNT HCN region variant of SEQ ID NO: 8, such as, e.g., amino acids
882-1127 of a TeNT
isoform of SEQ ID NO: 8 or amino acids 882-1127 of a TeNT subtype of SEQ ID
NO: 8. In still another
aspect of this embodiment, a TeNT HCN region comprises a non-naturally
occurring TeNT HCN region
variant, such as, e.g., a conservative TeNT HCN region variant, a non-
conservative TeNT HCN region
variant, a TeNT chimeric HCN region, an active TeNT HCN region fragment, or
any combination thereof. In
still another aspect of this embodiment, a TeNT HCN region comprises amino
acids 882-1127 of a non-
naturally occurring TeNT HCN region variant of SEQ ID NO: 8, such as, e.g.,
amino acids 882-1127 of a
conservative TeNT HCN region variant of SEQ ID NO: 8, amino acids 882-1127 of
a non-conservative
TeNT HCN region variant of SEQ ID NO: 8, amino acids 882-1127 of an active
TeNT HCN region fragment
of SEQ ID NO: 8, or any combination thereof.
[0164] In other aspects of this embodiment, a TeNT HCN region comprises a
polypeptide having, e.g., at
least 70% amino acid identity with amino acids 882-1127 of SEQ ID NO: 8, at
least 75% amino acid
identity with amino acids 882-1127 of SEQ ID NO: 8, at least 80% amino acid
identity with amino acids
882-1127 of SEQ ID NO: 8, at least 85% amino acid identity with amino acids
882-1127 of SEQ ID NO: 8,
at least 90% amino acid identity with amino acids 882-1127 of SEQ ID NO: 8 or
at least 95% amino acid
identity with amino acids 882-1127 of SEQ ID NO: 8. In yet other aspects of
this embodiment, a TeNT
HCN region comprises a polypeptide having, e.g., at most 70% amino acid
identity with amino acids 882-
1127 of SEQ ID NO: 8, at most 75% amino acid identity with amino acids 882-
1127 of SEQ ID NO: 8, at
most 80% amino acid identity with amino acids 882-1127 of SEQ ID NO: 8, at
most 85% amino acid
identity with amino acids 882-1127 of SEQ ID NO: 8, at most 90% amino acid
identity with amino acids
882-1127 of SEQ ID NO: 8 or at most 95% amino acid identity with amino acids
882-1127 of SEQ ID NO:
8.
[0165] In other aspects of this embodiment, a TeNT HCN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100, or 200 non-contiguous
amino acid substitutions relative to amino acids 882-1127 of SEQ ID NO: 8. In
other aspects of this
embodiment, a TeNT HCN region comprises a polypeptide having, e.g., at least
one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino acid
substitutions relative to
amino acids 882-1127 of SEQ ID NO: 8. In yet other aspects of this embodiment,
a TeNT HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 non-contiguous amino acid deletions relative to amino acids
882-1127 of SEQ ID NO:
8. In other aspects of this embodiment, a TeNT HcN region comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or
100 non-contiguous amino acid
deletions relative to amino acids 882-1127 of SEQ ID NO: 8. In still other
aspects of this embodiment, a
TeNT HCN region comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 non-contiguous amino acid additions
relative to amino acids 882-
1127 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNT HCN region
comprises a polypeptide
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having, e.g., at least one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50 or 100 non-
contiguous amino acid additions relative to amino acids 882-1127 of SEQ ID NO:
8.
[0166] In other aspects of this embodiment, a TeNT HCN region comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50
or 100 contiguous amino acid
substitutions relative to amino acids 882-1127 of SEQ ID NO: 8. In other
aspects of this embodiment, a
TeNT HCN region comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous amino acid substitutions
relative to amino acids 882-
1127 of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT HCN
region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50 or
100 contiguous amino acid deletions relative to amino acids 882-1127 of SEQ ID
NO: 8. In other aspects
of this embodiment, a TeNT HCN region comprises a polypeptide having, e.g., at
least one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100 contiguous
amino acid deletions relative to
amino acids 882-1127 of SEQ ID NO: 8. In still other aspects of this
embodiment, a TeNT HCN region
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20,
30, 40 , 50 or 100 contiguous amino acid additions relative to amino acids 882-
1127 of SEQ ID NO: 8. In
other aspects of this embodiment, a TeNT HCN region comprises a polypeptide
having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50 or 100
contiguous amino acid additions
relative to amino acids 882-1127 of SEQ ID NO: 8.
[0167] The fusion of the membrane of enveloped viruses to a cellular membrane
is an essential step in
the release of the viral capsule into the cytoplasm of the host cell. This
fusion event is mediated by a
fusogenic peptide segment present in viral glycoproteins located on the viral
membrane and involves
either a pH-dependent or pH-independent process, see, e.g., Frederick M.
Hughson, Structural
Characterization of Viral Fusion Proteins, 5(3) Curr. Biol. 265-274 (1995);
Trudy G. Morrison, Structure
and Function of a Paramyxovirus Fusion Protein, 1614(1) Biochim. Biophys.
Acta. 73-84; David J. Schibli
and Winfried Weissenhorn, Class 1 and Class ll Viral Fusion Protein Structures
Reveal Similar Principles
in Membrane Fusion, 21(6) Mol. Membr. Biol. 361-371 (2004). The fusogenic
peptide domain comprises
a hydrophobic, glycine-rich peptide of approximately 20-30 amino acids that
assist in the insertion of the
viral capsule into a cellular membrane. Thus, an enveloped virus fusogenic
peptide domain can be useful
as a translocation facilitating domain.
[0168] An enveloped virus fusogenic peptide domain includes, without
limitation, naturally occurring
enveloped virus fusogenic peptide domain variants, such as, e.g., enveloped
virus fusogenic peptide
domain isoforms and enveloped virus fusogenic peptide domain subtypes; non-
naturally occurring
enveloped virus fusogenic peptide domain variants, such as, e.g., conservative
enveloped virus fusogenic
peptide domain variants, non-conservative enveloped virus fusogenic peptide
domain variants, enveloped
virus fusogenic peptide domain chimerics, active enveloped virus fusogenic
peptide domain fragments
thereof, or any combination thereof.
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[0169] As used herein, the term "enveloped virus fusogenic peptide domain
variant," whether naturally-
occurring or non-naturally-occurring, means an enveloped virus fusogenic
peptide domain that has at
least one amino acid change from the corresponding region of the disclosed
reference sequences and
can be described in percent identity to the corresponding region of that
reference sequence. Unless
expressly indicated, all Clostridial toxin HcN region variants disclosed in
the present specification are
capable of further facilitating the translocation step of the intoxication
process that mediates Clostridial
toxin light chain translocation. As non-limiting examples, an Influenza virus
A fusogenic peptide domain
variant comprising SEQ ID NO: 194 will have at least one amino acid
difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to SEQ ID NO: 194; a
Semliki Forest virus fusogenic
peptide domain variant comprising SEQ ID NO: 199 will have at least one amino
acid difference, such as,
e.g., an amino acid substitution, deletion or addition, as compared to SEQ ID
NO: 199; an Eastern equine
encephalitis virus fusogenic peptide domain variant comprising SEQ ID NO: 201
will have at least one
amino acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to SEQ
ID NO: 201; a Venezuelan equine encephalitis virus fusogenic peptide domain
variant comprising SEQ ID
NO: 209 will have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or
addition, as compared to SEQ ID NO: 209; a Vesicular stomatitis virus
fusogenic peptide domain variant
comprising SEQ ID NO: 226 will have at least one amino acid difference, such
as, e.g., an amino acid
substitution, deletion or addition, as compared to SEQ ID NO: 226; a Sendai
virus fusogenic peptide
domain variant comprising SEQ ID NO: 237 will have at least one amino acid
difference, such as, e.g., an
amino acid substitution, deletion or addition, as compared to SEQ ID NO: 237;
a Canine distemper virus
fusogenic peptide domain variant comprising SEQ ID NO: 244 will have at least
one amino acid
difference, such as, e.g., an amino acid substitution, deletion or addition,
as compared to SEQ ID NO:
244; a Newcastle disease virus fusogenic peptide domain variant comprising SEQ
ID NO: 254 will have at
least one amino acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as
compared to SEQ ID NO: 254; and a Hendra virus fusogenic peptide domain
variant comprising SEQ ID
NO: 267 will have at least one amino acid difference, such as, e.g., an amino
acid substitution, deletion or
addition, as compared to SEQ ID NO: 267.
[0170] It is recognized by those of skill in the art that for each enveloped
virus there can be naturally
occurring fusogenic peptide domain variants that differ somewhat in their
amino acid sequence, and also
in the nucleic acids encoding these proteins. For example, at least five
naturally-occurring variants of the
fusogenic peptide domain present in the Influenza A virus haemagglutinin are
known (SEQ ID NO: 194 to
SEQ ID NO: 198); at least six naturally-occurring variants of the fusogenic
peptide domain present in the
Eastern equine encephalitis virus El protein are known (SEQ ID NO: 201 to SEQ
ID NO: 206); at least
eight naturally-occurring variants of the fusogenic peptide domain present in
the Venezuelan equine
encephalitis virus El protein are known (SEQ ID NO: 209 to SEQ ID NO: 216); at
least seven naturally-
occurring variants of the fusogenic peptide domain present in the Vesicular
stomatitis virus (VSV)
glycoprotein G are known (SEQ ID NO: 226 to SEQ ID NO: 232); and at least
eleven naturally-occurring
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variants of the fusogenic peptide domain present in the Newcastle disease
virus F protein are known
(SEQ ID NO: 254 to SEQ ID NO: 264). As used herein, the term "enveloped virus
fusogenic peptide
domain variant" means any enveloped virus fusogenic peptide domain produced by
a naturally-occurring
process, including, without limitation, enveloped virus fusogenic peptide
domain isoforms produced from
alternatively-spliced transcripts, enveloped virus fusogenic peptide domain
isoforms produced by
spontaneous mutation and enveloped virus fusogenic peptide domain subtypes. A
naturally occurring
enveloped virus fusogenic peptide domain variant can function in substantially
the same manner as the
reference enveloped virus fusogenic peptide domain on which the naturally
occurring enveloped virus
fusogenic peptide domain variant is based, and can be substituted for the
reference enveloped virus
fusogenic peptide domain in any aspect of the present invention. A naturally
occurring enveloped virus
fusogenic peptide domain variant may substitute one or more amino acids, two
or more amino acids,
three or more amino acids, four or more amino acids, five or more amino acids
or ten or more amino
acids from the reference enveloped virus fusogenic peptide domain on which the
naturally occurring
enveloped virus fusogenic peptide domain is based. A naturally occurring
enveloped virus fusogenic
peptide domain variant can also substitute at least 2 contiguous amino acids,
at least 3 contiguous amino
acids, at least 4 contiguous amino acids or at least 5 contiguous amino acids
from the reference
enveloped virus fusogenic peptide domain on which the naturally occurring
enveloped virus fusogenic
peptide domain variant is based, that possess at least 50% amino acid
identity, 65% amino acid identity,
75% amino acid identity, 85% amino acid identity or 95% amino acid identity to
the reference enveloped
virus fusogenic peptide domain on which the naturally occurring enveloped
virus fusogenic peptide
domain variant is based.
[0171] A non-limiting examples of a naturally occurring enveloped virus
fusogenic peptide domain
variant is an enveloped virus fusogenic peptide domain isoform such as, e.g.,
an influenzavirus fusogenic
peptide domain isoform, an alphavirus fusogenic peptide domain isoform, a
vesiculovirus fusogenic
peptide domain isoform, a respirovirus fusogenic peptide domain isoform, a
morbillivirus fusogenic
peptide domain isoform, an avulavirus fusogenic peptide domain isoform, a
henipavirus fusogenic peptide
domain isoform, a metapneumovirus fusogenic peptide domain isoform and a foamy
virus fusogenic
peptide domain isoform. An enveloped virus fusogenic peptide domain isoform
can function in
substantially the same manner as the reference enveloped virus fusogenic
peptide domain on which the
enveloped virus fusogenic peptide domain isoform is based, and can be
substituted for the reference
enveloped virus fusogenic peptide domain in any aspect of the present
invention.
[0172] A non-limiting examples of a naturally occurring enveloped virus
fusogenic peptide domain
variant is an enveloped virus fusogenic peptide domain subtype such as, e.g.,
an influenzavirus fusogenic
peptide domain subtype, an alphavirus fusogenic peptide domain subtype, a
vesiculovirus fusogenic
peptide domain subtype, a respirovirus fusogenic peptide domain subtype, a
morbillivirus fusogenic
peptide domain subtype, an avulavirus fusogenic peptide domain subtype, a
henipavirus fusogenic
peptide domain subtype, a metapneumovirus fusogenic peptide domain subtype and
a foamy virus
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fusogenic peptide domain subtype. An enveloped virus fusogenic peptide domain
subtype can function in
substantially the same manner as the reference enveloped virus fusogenic
peptide domain on which the
enveloped virus fusogenic peptide domain subtype is based, and can be
substituted for the reference
enveloped virus fusogenic peptide domain in any aspect of the present
invention.
[0173] As used herein, the term "non-naturally occurring enveloped virus
fusogenic peptide domain
variant" means any enveloped virus fusogenic peptide domain produced with the
aid of human
manipulation, including, without limitation, enveloped virus fusogenic peptide
domains produced by
genetic engineering using random mutagenesis or rational design and enveloped
virus fusogenic peptide
domains produced by chemical synthesis. Non-limiting examples of non-naturally
occurring enveloped
virus fusogenic peptide domain variants include, e.g., conservative enveloped
virus fusogenic peptide
domain variants, non-conservative enveloped virus fusogenic peptide domain
variants, enveloped virus
fusogenic peptide domain chimeric variants and active enveloped virus
fusogenic peptide domain
fragments.
[0174] As used herein, the term "conservative enveloped virus fusogenic
peptide domain variant" means
an enveloped virus fusogenic peptide domain that has at least one amino acid
substituted by another
amino acid or an amino acid analog that has at least one property similar to
that of the original amino acid
from the reference enveloped virus fusogenic peptide domain sequence. Examples
of properties include,
without limitation, similar size, topography, charge, hydrophobicity,
hydrophilicity, lipophilicity, covalent-
bonding capacity, hydrogen-bonding capacity, a physicochemical property, of
the like, or any combination
thereof. A conservative enveloped virus fusogenic peptide domain variant can
function in substantially
the same manner as the reference enveloped virus fusogenic peptide domain on
which the conservative
enveloped virus fusogenic peptide domain variant is based, and can be
substituted for the reference
enveloped virus fusogenic peptide domain in any aspect of the present
invention. A conservative
enveloped virus fusogenic peptide domain variant may substitute one or more
amino acids, two or more
amino acids, three or more amino acids, four or more amino acids, five or more
amino acids or ten or
more amino acids from the reference enveloped virus fusogenic peptide domain
on which the
conservative enveloped virus fusogenic peptide domain variant is based. A
conservative enveloped virus
fusogenic peptide domain variant can also substitute at least 2 contiguous
amino acids, at least 3
contiguous amino acids, at least 4 contiguous amino acids or at least 5
contiguous amino acids from the
reference enveloped virus fusogenic peptide domain on which the conservative
enveloped virus
fusogenic peptide domain variant is based, that possess at least 50% amino
acid identity, 65% amino
acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino
acid identity to the
reference enveloped virus fusogenic peptide domain on which the conservative
enveloped virus
fusogenic peptide domain variant is based. Non-limiting examples of a
conservative enveloped virus
fusogenic peptide domain variant include, e.g., conservative influenzavirus
fusogenic peptide domain
variants, conservative alphavirus fusogenic peptide domain variants,
conservative vesiculovirus fusogenic
peptide domain variants, conservative respirovirus fusogenic peptide domain
variants, conservative
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morbillivirus fusogenic peptide domain variants, conservative avulavirus
fusogenic peptide domain
variants, conservative henipavirus fusogenic peptide domain variants,
conservative metapneumovirus
fusogenic peptide domain variants and conservative foamy virus fusogenic
peptide domain variants.
[0175] As used herein, the term "non-conservative enveloped virus fusogenic
peptide domain variant"
means an enveloped virus fusogenic peptide domain in which 1) at least one
amino acid is deleted from
the reference enveloped virus fusogenic peptide domain on which the non-
conservative enveloped virus
fusogenic peptide domain variant is based; 2) at least one amino acid added to
the reference enveloped
virus fusogenic peptide domain on which the non-conservative enveloped virus
fusogenic peptide domain
is based; or 3) at least one amino acid is substituted by another amino acid
or an amino acid analog that
does not share any property similar to that of the original amino acid from
the reference enveloped virus
fusogenic peptide domain sequence. A non-conservative enveloped virus
fusogenic peptide domain
variant can function in substantially the same manner as the reference
enveloped virus fusogenic peptide
domain on which the non-conservative enveloped virus fusogenic peptide domain
variant is based, and
can be substituted for the reference enveloped virus fusogenic peptide domain
in any aspect of the
present invention. A non-conservative enveloped virus fusogenic peptide domain
variant can delete one
or more amino acids, two or more amino acids, three or more amino acids, four
or more amino acids or
five or more amino acids from the reference enveloped virus fusogenic peptide
domain on which the non-
conservative enveloped virus fusogenic peptide domain variant is based. A non-
conservative enveloped
virus fusogenic peptide domain variant can add one or more amino acids, two or
more amino acids, three
or more amino acids, four or more amino acids or five or more amino acids to
the reference enveloped
virus fusogenic peptide domain on which the non-conservative enveloped virus
fusogenic peptide domain
variant is based. A non-conservative enveloped virus fusogenic peptide domain
variant may substitute
one or more amino acids, two or more amino acids, three or more amino acids,
four or more amino acids,
five or more amino acids or ten or more amino acids from the reference
enveloped virus fusogenic
peptide domain on which the non-conservative enveloped virus fusogenic peptide
domain variant is
based. A non-conservative enveloped virus fusogenic peptide domain variant can
also substitute at least
2 contiguous amino acids, at least 3 contiguous amino acids, at least 4
contiguous amino acids or at least
contiguous amino acids from the reference enveloped virus fusogenic peptide
domain on which the
non-conservative enveloped virus fusogenic peptide domain variant is based,
that possess at least 50%
amino acid identity, 65% amino acid identity, 75% amino acid identity, 85%
amino acid identity or 95%
amino acid identity to the reference enveloped virus fusogenic peptide domain
on which the non-
conservative enveloped virus fusogenic peptide domain variant is based. Non-
limiting examples of a non-
conservative enveloped virus fusogenic peptide domain variant include, e.g.,
non-conservative
influenzavirus fusogenic peptide domain variants, non-conservative alphavirus
fusogenic peptide domain
variants, non-conservative vesiculovirus fusogenic peptide domain variants,
non-conservative respirovirus
fusogenic peptide domain variants, non-conservative morbillivirus fusogenic
peptide domain variants,
non-conservative avulavirus fusogenic peptide domain variants, non-
conservative henipavirus fusogenic
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peptide domain variants, non-conservative metapneumovirus fusogenic peptide
domain variants and non-
conservative foamy virus fusogenic peptide domain variants.
[0176] As used herein, the term "enveloped virus fusogenic peptide domain
chimeric" means a
polypeptide comprising at least a portion of an enveloped virus fusogenic
peptide domain and at least a
portion of at least one other polypeptide to form an enveloped virus fusogenic
peptide domain with at
least one property different from the reference enveloped virus fusogenic
peptide domain, with the
proviso that this enveloped virus fusogenic peptide domain chimeric is still
capable of further facilitating
the translocation step of the intoxication process where the LC is released
from intracellular vesicles into
the cytoplasm of the target cell and thus participate in executing the overall
cellular mechanism whereby
a Clostridial toxin proteolytically cleaves a substrate.
[0177] As used herein, the term "active enveloped virus fusogenic peptide
domain fragment" means any
of a variety of enveloped virus fusogenic peptide domain fragments that can
further facilitate the
translocation step of the intoxication process where the LC is released from
intracellular vesicles into the
cytoplasm of the target cell and thus participate in executing the overall
cellular mechanism whereby a
Clostridial toxin proteolytically cleaves a substrate. Enveloped virus
fusogenic peptide domains are
approximately 15-30 amino acids in length. Thus, aspects of this embodiment
can include a translocation
facilitating domain comprising an active enveloped virus fusogenic peptide
domain fragment having a
length of, e.g., at least 10 amino acids, at least 15 amino acids, at least 20
amino acids and at least 25
amino acids. Other aspects of this embodiment can include a translocation
facilitating domain comprising
an active enveloped virus fusogenic peptide domain fragment having a length
of, e.g., at most 10 amino
acids, at most 15 amino acids, at most 20 amino acids and at most 25 amino
acids.
[0178] Any of a variety of sequence alignment methods can be used to determine
percent identity of
naturally-occurring enveloped virus fusogenic peptide domain variants and non-
naturally-occurring
enveloped virus fusogenic peptide domain variants, including, without
limitation, global methods, local
methods and hybrid methods, such as, e.g., segment approach methods. Protocols
to determine percent
identity are routine procedures within the scope of one skilled in the art and
from the teaching herein.
[0179] Thus, in an embodiment, a modified Clostridial toxin disclosed in the
present specification
comprises a Clostridial toxin translocation facilitating domain comprising an
enveloped virus fusogenic
peptide domain. In an aspect of this embodiment, a Clostridial toxin
translocation facilitating domain
comprises a naturally occurring enveloped virus fusogenic peptide domain
variant, such as, e.g., an
enveloped virus fusogenic peptide domain isoform or an enveloped virus
fusogenic peptide domain
subtype. In another aspect of this embodiment, a Clostridial toxin
translocation domain comprises a non-
naturally occurring enveloped virus fusogenic peptide domain variant, such as,
e.g., a conservative
enveloped virus fusogenic peptide domain variant, a non-conservative enveloped
virus fusogenic peptide
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domain variant, an enveloped virus fusogenic peptide domain chimeric, an
active enveloped virus
fusogenic peptide domain fragment, or any combination thereof.
[0180] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises an
influenzavirus fusogenic peptide domain. In another aspect of this embodiment,
an influenzavirus
fusogenic peptide domain comprises a naturally occurring influenzavirus
fusogenic peptide domain
variant, such as, e.g., an influenzavirus fusogenic peptide domain isoform or
an influenzavirus fusogenic
peptide domain subtype. In another aspect of this embodiment, an
influenzavirus fusogenic peptide
domain comprises a naturally occurring influenzavirus fusogenic peptide domain
variant of SEQ ID NO:
194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198, such
as, e.g., an
influenzavirus fusogenic peptide domain isoform of SEQ ID NO: 194, SEQ ID NO:
195, SEQ ID NO: 196,
SEQ ID NO: 197 or SEQ ID NO: 198 or an influenzavirus fusogenic peptide domain
subtype of SEQ ID
NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198. In
still another aspect
of this embodiment, an influenzavirus fusogenic peptide domain comprises a non-
naturally occurring
influenzavirus fusogenic peptide domain variant, such as, e.g., a conservative
influenzavirus fusogenic
peptide domain variant, a non-conservative influenzavirus fusogenic peptide
domain variant, an
influenzavirus fusogenic peptide domain chimeric, an active influenzavirus
fusogenic peptide domain
fragment, or any combination thereof. In still another aspect of this
embodiment, an influenzavirus
fusogenic peptide domain comprises amino acids a non-naturally occurring
influenzavirus fusogenic
peptide domain variant of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ
ID NO: 197 or SEQ
ID NO: 198, such as, e.g., a conservative influenzavirus fusogenic peptide
domain variant of SEQ ID NO:
194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198, a non-
conservative
influenzavirus fusogenic peptide domain variant of SEQ ID NO: 194, SEQ ID NO:
195, SEQ ID NO: 196,
SEQ ID NO: 197 or SEQ ID NO: 198, an active influenzavirus fusogenic peptide
domain fragment of SEQ
ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198,
or any combination
thereof.
[0181] In other aspects of this embodiment, an influenzavirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO:
194, SEQ ID NO: 195, SEQ
ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198, at least 75% amino acid identity
with SEQ ID NO: 194,
SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198, at least 80%
amino acid identity
with SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID
NO: 198, at least
85% amino acid identity with SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196,
SEQ ID NO: 197 or
SEQ ID NO: 198, at least 90% amino acid identity with SEQ ID NO: 194, SEQ ID
NO: 195, SEQ ID NO:
196, SEQ ID NO: 197 or SEQ ID NO: 198 or at least 95% amino acid identity with
SEQ ID NO: 194, SEQ
ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198. In yet other
aspects of this
embodiment, an influenzavirus fusogenic peptide domain comprises a polypeptide
having, e.g., at most
70% amino acid identity with SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196,
SEQ ID NO: 197 or
SEQ ID NO: 198, at most 75% amino acid identity with SEQ ID NO: 194, SEQ ID
NO: 195, SEQ ID NO:
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196, SEQ ID NO: 197 or SEQ ID NO: 198, at most 80% amino acid identity with
SEQ ID NO: 194, SEQ ID
NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198, at most 85% amino
acid identity with
SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO:
198, at most 90%
amino acid identity with SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ
ID NO: 197 or SEQ ID
NO: 198 or at most 95% amino acid identity with SEQ ID NO: 194, SEQ ID NO:
195, SEQ ID NO: 196,
SEQ ID NO: 197 or SEQ ID NO: 198.
[0182] In other aspects of this embodiment, an influenzavirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID
NO: 196, SEQ ID NO: 197
or SEQ ID NO: 198. In other aspects of this embodiment, an influenzavirus
fusogenic peptide domain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine or 10 non-
contiguous amino acid substitutions relative to SEQ ID NO: 194, SEQ ID NO:
195, SEQ ID NO: 196, SEQ
ID NO: 197 or SEQ ID NO: 198. In yet other aspects of this embodiment, an
influenzavirus fusogenic
peptide domain comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight,
nine or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 194, SEQ
ID NO: 195, SEQ ID
NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198. In other aspects of this
embodiment, an influenzavirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 non-contiguous amino acid deletions relative to SEQ
ID NO: 194, SEQ ID NO:
195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198. In still other aspects
of this embodiment, an
influenzavirus fusogenic peptide domain comprises a polypeptide having, e.g.,
at most one, two, three,
four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions
relative to SEQ ID NO: 194,
SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198. In other
aspects of this
embodiment, an influenzavirus fusogenic peptide domain comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous
amino acid additions relative to
SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO:
198.
[0183] In other aspects of this embodiment, an influenzavirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous amino
acid substitutions relative to SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196,
SEQ ID NO: 197 or
SEQ ID NO: 198. In other aspects of this embodiment, an influenzavirus
fusogenic peptide domain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine or 10
contiguous amino acid substitutions relative to SEQ ID NO: 194, SEQ ID NO:
195, SEQ ID NO: 196, SEQ
ID NO: 197 or SEQ ID NO: 198. In yet other aspects of this embodiment, an
influenzavirus fusogenic
peptide domain comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight,
nine or 10 contiguous amino acid deletions relative to SEQ ID NO: 194, SEQ ID
NO: 195, SEQ ID NO:
196, SEQ ID NO: 197 or SEQ ID NO: 198. In other aspects of this embodiment, an
influenzavirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 contiguous amino acid deletions relative to SEQ ID
NO: 194, SEQ ID NO: 195,
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SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198. In still other aspects of
this embodiment, an
influenzavirus fusogenic peptide domain comprises a polypeptide having, e.g.,
at most one, two, three,
four, five, six, seven, eight, nine or 10 contiguous amino acid additions
relative to SEQ ID NO: 194, SEQ
ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198. In other aspects
of this embodiment,
an influenzavirus fusogenic peptide domain comprises a polypeptide having,
e.g., at least one, two, three,
four, five, six, seven, eight, nine or 10 contiguous amino acid additions
relative to SEQ ID NO: 194, SEQ
ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198.
[0184] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises an
alphavirus fusogenic peptide domain. In another aspect of this embodiment, an
alphavirus fusogenic
peptide domain comprises a naturally occurring alphavirus fusogenic peptide
domain variant, such as,
e.g., an alphavirus fusogenic peptide domain isoform or an alphavirus
fusogenic peptide domain subtype.
In another aspect of this embodiment, an alphavirus fusogenic peptide domain
comprises a naturally
occurring alphavirus fusogenic peptide domain variant of SEQ ID NO: 199, SEQ
ID NO: 200, SEQ ID NO:
201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID
NO: 206, SEQ ID
NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ
ID NO: 212, SEQ
ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217,
SEQ ID NO: 218,
SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO:
223, SEQ ID NO:
224 or SEQ ID NO: 225, such as, e.g., an alphavirus fusogenic peptide domain
isoform of SEQ ID NO:
199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID
NO: 204, SEQ ID
NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ
ID NO: 210, SEQ
ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215,
SEQ ID NO: 216,
SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO:
221, SEQ ID NO:
222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225 or an alphavirus
fusogenic peptide domain
subtype of SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ
ID NO: 203, SEQ
ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208,
SEQ ID NO: 209,
SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:
214, SEQ ID NO:
215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID
NO: 220, SEQ ID
NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225. In
still another aspect
of this embodiment, an alphavirus fusogenic peptide domain comprises a non-
naturally occurring
alphavirus fusogenic peptide domain variant, such as, e.g., a conservative
alphavirus fusogenic peptide
domain variant, a non-conservative alphavirus fusogenic peptide domain
variant, an alphavirus fusogenic
peptide domain chimeric, an active alphavirus fusogenic peptide domain
fragment, or any combination
thereof. In still another aspect of this embodiment, an alphavirus fusogenic
peptide domain comprises
amino acids a non-naturally occurring alphavirus fusogenic peptide domain
variant of SEQ ID NO: 199,
SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO:
204, SEQ ID NO:
205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID
NO: 210, SEQ ID
NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ
ID NO: 216, SEQ
ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221,
SEQ ID NO: 222,
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SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225, such as, e.g., a
conservative alphavirus fusogenic
peptide domain variant of SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ
ID NO: 202, SEQ ID
NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ
ID NO: 208, SEQ
ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213,
SEQ ID NO: 214,
SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO:
219, SEQ ID NO:
220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID
NO: 225, a non-
conservative alphavirus fusogenic peptide domain variant of SEQ ID NO: 199,
SEQ ID NO: 200, SEQ ID
NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ
ID NO: 206, SEQ
ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,
SEQ ID NO: 212,
SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO:
217, SEQ ID NO:
218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID
NO: 223, SEQ ID
NO: 224 or SEQ ID NO: 225, an active alphavirus fusogenic peptide domain
fragment of SEQ ID NO:
199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID
NO: 204, SEQ ID
NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ
ID NO: 210, SEQ
ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215,
SEQ ID NO: 216,
SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO:
221, SEQ ID NO:
222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225, or any combination
thereof.
[0185] In other aspects of this embodiment, an alphavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO:
199, SEQ ID NO: 200, SEQ
ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205,
SEQ ID NO: 206,
SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:
211, SEQ ID NO:
212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID
NO: 217, SEQ ID
NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ
ID NO: 223, SEQ
ID NO: 224 or SEQ ID NO: 225, at least 75% amino acid identity with SEQ ID NO:
199, SEQ ID NO: 200,
SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO:
205, SEQ ID NO:
206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID
NO: 211, SEQ ID
NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ
ID NO: 217, SEQ
ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222,
SEQ ID NO: 223,
SEQ ID NO: 224 or SEQ ID NO: 225, at least 80% amino acid identity with SEQ ID
NO: 194, SEQ ID NO:
195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198, at least 85% amino acid
identity with SEQ ID
NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ
ID NO: 204, SEQ
ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209,
SEQ ID NO: 210,
SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO:
215, SEQ ID NO:
216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID
NO: 221, SEQ ID
NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225, at least 90% amino
acid identity with
SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO:
203, SEQ ID NO:
204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID
NO: 209, SEQ ID
NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ
ID NO: 215, SEQ
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ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220,
SEQ ID NO: 221,
SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225 or at least
95% amino acid
identity with SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,
SEQ ID NO: 203,
SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO:
208, SEQ ID NO:
209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID
NO: 214, SEQ ID
NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ
ID NO: 220, SEQ
ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225.
In yet other
aspects of this embodiment, an alphavirus fusogenic peptide domain comprises a
polypeptide having,
e.g., at most 70% amino acid identity with SEQ ID NO: 199, SEQ ID NO: 200, SEQ
ID NO: 201, SEQ ID
NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ
ID NO: 207, SEQ
ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212,
SEQ ID NO: 213,
SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:
218, SEQ ID NO:
219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID
NO: 224 or SEQ ID
NO: 225, at most 75% amino acid identity with SEQ ID NO: 199, SEQ ID NO: 200,
SEQ ID NO: 201, SEQ
ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206,
SEQ ID NO: 207,
SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO:
212, SEQ ID NO:
213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID
NO: 218, SEQ ID
NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ
ID NO: 224 or
SEQ ID NO: 225, at most 80% amino acid identity with SEQ ID NO: 199, SEQ ID
NO: 200, SEQ ID NO:
201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID
NO: 206, SEQ ID
NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ
ID NO: 212, SEQ
ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217,
SEQ ID NO: 218,
SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO:
223, SEQ ID NO:
224 or SEQ ID NO: 225, at most 85% amino acid identity with SEQ ID NO: 199,
SEQ ID NO: 200, SEQ ID
NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ
ID NO: 206, SEQ
ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,
SEQ ID NO: 212,
SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO:
217, SEQ ID NO:
218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID
NO: 223, SEQ ID
NO: 224 or SEQ ID NO: 225, at most 90% amino acid identity with SEQ ID NO:
199, SEQ ID NO: 200,
SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO:
205, SEQ ID NO:
206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID
NO: 211, SEQ ID
NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ
ID NO: 217, SEQ
ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222,
SEQ ID NO: 223,
SEQ ID NO: 224 or SEQ ID NO: 225 or at most 95% amino acid identity with SEQ
ID NO: 199, SEQ ID
NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ
ID NO: 205, SEQ
ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210,
SEQ ID NO: 211,
SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO:
216, SEQ ID NO:
217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID
NO: 222, SEQ ID
NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225.
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[0186] In other aspects of this embodiment, an alphavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID
NO: 201, SEQ ID NO:
202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID
NO: 207, SEQ ID
NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ
ID NO: 213, SEQ
ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218,
SEQ ID NO: 219,
SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224
or SEQ ID NO:
225. In other aspects of this embodiment, an alphavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID
NO: 201, SEQ ID NO:
202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID
NO: 207, SEQ ID
NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ
ID NO: 213, SEQ
ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218,
SEQ ID NO: 219,
SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224
or SEQ ID NO:
225. In yet other aspects of this embodiment, an alphavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid deletions relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO:
201, SEQ ID NO: 202,
SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO:
207, SEQ ID NO:
208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID
NO: 213, SEQ ID
NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ
ID NO: 219, SEQ
ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or
SEQ ID NO: 225.
In other aspects of this embodiment, an alphavirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine or
10 non-contiguous amino acid
deletions relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID
NO: 202, SEQ ID NO:
203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID
NO: 208, SEQ ID
NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ
ID NO: 214, SEQ
ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219,
SEQ ID NO: 220,
SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO:
225. In still other
aspects of this embodiment, an alphavirus fusogenic peptide domain comprises a
polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-
contiguous amino acid additions
relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,
SEQ ID NO: 203, SEQ
ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208,
SEQ ID NO: 209,
SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:
214, SEQ ID NO:
215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID
NO: 220, SEQ ID
NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225. In
other aspects of
this embodiment, an alphavirus fusogenic peptide domain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous
amino acid additions relative to
SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO:
203, SEQ ID NO:
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204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID
NO: 209, SEQ ID
NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ
ID NO: 215, SEQ
ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220,
SEQ ID NO: 221,
SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225.
[0187] In other aspects of this embodiment, an alphavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous amino
acid substitutions relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201,
SEQ ID NO: 202, SEQ
ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207,
SEQ ID NO: 208,
SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO:
213, SEQ ID NO:
214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID
NO: 219, SEQ ID
NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ
ID NO: 225. In
other aspects of this embodiment, an alphavirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
substitutions relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ
ID NO: 202, SEQ ID
NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ
ID NO: 208, SEQ
ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213,
SEQ ID NO: 214,
SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO:
219, SEQ ID NO:
220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID
NO: 225. In yet
other aspects of this embodiment, an alphavirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
deletions relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID
NO: 202, SEQ ID NO:
203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID
NO: 208, SEQ ID
NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ
ID NO: 214, SEQ
ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219,
SEQ ID NO: 220,
SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO:
225. In other
aspects of this embodiment, an alphavirus fusogenic peptide domain comprises a
polypeptide having,
e.g., at least one, two, three, four, five, six, seven, eight, nine or 10
contiguous amino acid deletions
relative to SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,
SEQ ID NO: 203, SEQ
ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208,
SEQ ID NO: 209,
SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:
214, SEQ ID NO:
215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID
NO: 220, SEQ ID
NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225. In
still other aspects
of this embodiment, an alphavirus fusogenic peptide domain comprises a
polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine or 10 contiguous
amino acid additions relative to
SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO:
203, SEQ ID NO:
204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID
NO: 209, SEQ ID
NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ
ID NO: 215, SEQ
ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220,
SEQ ID NO: 221,
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SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225. In other
aspects of this
embodiment, an alphavirus fusogenic peptide domain comprises a polypeptide
having, e.g., at least one,
two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid
additions relative to SEQ ID NO:
199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID
NO: 204, SEQ ID
NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ
ID NO: 210, SEQ
ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215,
SEQ ID NO: 216,
SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO:
221, SEQ ID NO:
222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225.
[0188] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a
vesiculovirus fusogenic peptide domain. In another aspect of this embodiment,
a vesiculovirus fusogenic
peptide domain comprises a naturally occurring vesiculovirus fusogenic peptide
domain variant, such as,
e.g., a vesiculovirus fusogenic peptide domain isoform or a vesiculovirus
fusogenic peptide domain
subtype. In another aspect of this embodiment, a vesiculovirus fusogenic
peptide domain comprises a
naturally occurring vesiculovirus fusogenic peptide domain variant of SEQ ID
NO: 226, SEQ ID NO: 227,
SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO:
232, SEQ ID NO:
233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, such as, e.g., a
vesiculovirus fusogenic
peptide domain isoform of SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ
ID NO: 229, SEQ
ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234,
SEQ ID NO: 235 or
SEQ ID NO: 236 or a vesiculovirus fusogenic peptide domain subtype of SEQ ID
NO: 226, SEQ ID NO:
227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID
NO: 232, SEQ ID
NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236. In still another
aspect of this
embodiment, a vesiculovirus fusogenic peptide domain comprises a non-naturally
occurring vesiculovirus
fusogenic peptide domain variant, such as, e.g., a conservative vesiculovirus
fusogenic peptide domain
variant, a non-conservative vesiculovirus fusogenic peptide domain variant, a
vesiculovirus fusogenic
peptide domain chimeric, an active vesiculovirus fusogenic peptide domain
fragment, or any combination
thereof. In still another aspect of this embodiment, a vesiculovirus fusogenic
peptide domain comprises
amino acids a non-naturally occurring vesiculovirus fusogenic peptide domain
variant of SEQ ID NO: 226,
SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO:
231, SEQ ID NO:
232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, such
as, e.g., a
conservative vesiculovirus fusogenic peptide domain variant of SEQ ID NO: 226,
SEQ ID NO: 227, SEQ
ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232,
SEQ ID NO: 233,
SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, a non-conservative
vesiculovirus fusogenic
peptide domain variant of SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ
ID NO: 229, SEQ ID
NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ
ID NO: 235 or
SEQ ID NO: 236, an active vesiculovirus fusogenic peptide domain fragment of
SEQ ID NO: 226, SEQ ID
NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ
ID NO: 232, SEQ
ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, or any
combination thereof.
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[0189] In other aspects of this embodiment, a vesiculovirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO:
226, SEQ ID NO: 227, SEQ
ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232,
SEQ ID NO: 233,
SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, at least 75% amino acid
identity with SEQ ID NO:
226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID
NO: 231, SEQ ID
NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, at
least 80% amino
acid identity with SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO:
229, SEQ ID NO:
230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID
NO: 235 or SEQ ID
NO: 236, at least 85% amino acid identity with SEQ ID NO: 226, SEQ ID NO: 227,
SEQ ID NO: 228, SEQ
ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233,
SEQ ID NO: 234,
SEQ ID NO: 235 or SEQ ID NO: 236, at least 90% amino acid identity with SEQ ID
NO: 226, SEQ ID NO:
227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID
NO: 232, SEQ ID
NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236 or at least 95%
amino acid identity with
SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO:
230, SEQ ID NO:
231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID
NO: 236. In yet
other aspects of this embodiment, a vesiculovirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at most 70% amino acid identity with SEQ ID NO: 226, SEQ ID NO:
227, SEQ ID NO: 228,
SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO:
233, SEQ ID NO:
234, SEQ ID NO: 235 or SEQ ID NO: 236, at most 75% amino acid identity with
SEQ ID NO: 226, SEQ ID
NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ
ID NO: 232, SEQ
ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, at most 80%
amino acid identity with
SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO:
230, SEQ ID NO:
231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID
NO: 236, at most
85% amino acid identity with SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228,
SEQ ID NO: 229, SEQ
ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234,
SEQ ID NO: 235 or
SEQ ID NO: 236, at most 90% amino acid identity with SEQ ID NO: 226, SEQ ID
NO: 227, SEQ ID NO:
228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID
NO: 233, SEQ ID
NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236 or at most 95% amino acid identity
with SEQ ID NO: 226,
SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO:
231, SEQ ID NO:
232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236.
[0190] In other aspects of this embodiment, a vesiculovirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID
NO: 228, SEQ ID NO:
229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID
NO: 234, SEQ ID
NO: 235 or SEQ ID NO: 236. In other aspects of this embodiment, a
vesiculovirus fusogenic peptide
domain comprises a polypeptide having, e.g., at least one, two, three, four,
five, six, seven, eight, nine or
non-contiguous amino acid substitutions relative to SEQ ID NO: 226, SEQ ID NO:
227, SEQ ID NO:
228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID
NO: 233, SEQ ID
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NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236. In yet other aspects of this
embodiment, a vesiculovirus
fusogenic peptide domain comprises a polypeptide having, e.g., at most one,
two, three, four, five, six,
seven, eight, nine or 10 non-contiguous amino acid deletions relative to SEQ
ID NO: 226, SEQ ID NO:
227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID
NO: 232, SEQ ID
NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236. In other aspects of
this embodiment, a
vesiculovirus fusogenic peptide domain comprises a polypeptide having, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions
relative to SEQ ID NO: 226,
SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO:
231, SEQ ID NO:
232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236. In
still other aspects of
this embodiment, a vesiculovirus fusogenic peptide domain comprises a
polypeptide having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous
amino acid additions relative to
SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO:
230, SEQ ID NO:
231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID
NO: 236. In other
aspects of this embodiment, a vesiculovirus fusogenic peptide domain comprises
a polypeptide having,
e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-
contiguous amino acid additions
relative to SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229,
SEQ ID NO: 230, SEQ
ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or
SEQ ID NO: 236.
[0191] In other aspects of this embodiment, a vesiculovirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous amino
acid substitutions relative to SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228,
SEQ ID NO: 229, SEQ
ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234,
SEQ ID NO: 235 or
SEQ ID NO: 236. In other aspects of this embodiment, a vesiculovirus fusogenic
peptide domain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine or 10
contiguous amino acid substitutions relative to SEQ ID NO: 226, SEQ ID NO:
227, SEQ ID NO: 228, SEQ
ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233,
SEQ ID NO: 234,
SEQ ID NO: 235 or SEQ ID NO: 236. In yet other aspects of this embodiment, a
vesiculovirus fusogenic
peptide domain comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight,
nine or 10 contiguous amino acid deletions relative to SEQ ID NO: 226, SEQ ID
NO: 227, SEQ ID NO:
228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID
NO: 233, SEQ ID
NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236. In other aspects of this
embodiment, a vesiculovirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 contiguous amino acid deletions relative to SEQ ID
NO: 226, SEQ ID NO: 227,
SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO:
232, SEQ ID NO:
233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236. In still other aspects
of this embodiment, a
vesiculovirus fusogenic peptide domain comprises a polypeptide having, e.g.,
at most one, two, three,
four, five, six, seven, eight, nine or 10 contiguous amino acid additions
relative to SEQ ID NO: 226, SEQ
ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231,
SEQ ID NO: 232,
SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236. In other
aspects of this
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embodiment, a vesiculovirus fusogenic peptide domain comprises a polypeptide
having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino
acid additions relative to SEQ ID
NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ
ID NO: 231, SEQ
ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236.
[0192] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a
respirovirus fusogenic peptide domain. In another aspect of this embodiment, a
respirovirus fusogenic
peptide domain comprises a naturally occurring respirovirus fusogenic peptide
domain variant, such as,
e.g., a respirovirus fusogenic peptide domain isoform or a respirovirus
fusogenic peptide domain subtype.
In another aspect of this embodiment, a respirovirus fusogenic peptide domain
comprises a naturally
occurring respirovirus fusogenic peptide domain variant of SEQ ID NO: 237, SEQ
ID NO: 238, SEQ ID
NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243,
such as, e.g., a
respirovirus fusogenic peptide domain isoform of SEQ ID NO: 237, SEQ ID NO:
238, SEQ ID NO: 239,
SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243 or a
respirovirus fusogenic
peptide domain subtype of SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ
ID NO: 240, SEQ
ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243. In still another aspect of this
embodiment, a
respirovirus fusogenic peptide domain comprises a non-naturally occurring
respirovirus fusogenic peptide
domain variant, such as, e.g., a conservative respirovirus fusogenic peptide
domain variant, a non-
conservative respirovirus fusogenic peptide domain variant, a respirovirus
fusogenic peptide domain
chimeric, an active respirovirus fusogenic peptide domain fragment, or any
combination thereof. In still
another aspect of this embodiment, a respirovirus fusogenic peptide domain
comprises amino acids a
non-naturally occurring respirovirus fusogenic peptide domain variant of SEQ
ID NO: 237, SEQ ID NO:
238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID
NO: 243, such as,
e.g., a conservative respirovirus fusogenic peptide domain variant of SEQ ID
NO: 237, SEQ ID NO: 238,
SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO:
243, a non-
conservative respirovirus fusogenic peptide domain variant of SEQ ID NO: 237,
SEQ ID NO: 238, SEQ ID
NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243, an
active respirovirus
fusogenic peptide domain fragment of SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID
NO: 239, SEQ ID NO:
240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243, or any combination
thereof.
[0193] In other aspects of this embodiment, a respirovirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO:
237, SEQ ID NO: 238, SEQ
ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243,
at least 75% amino
acid identity with SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO:
240, SEQ ID NO:
241, SEQ ID NO: 242 or SEQ ID NO: 243, at least 80% amino acid identity with
SEQ ID NO: 237, SEQ ID
NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ
ID NO: 243, at
least 85% amino acid identity with SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO:
239, SEQ ID NO: 240,
SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243, at least 90% amino acid
identity with SEQ ID NO:
237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID
NO: 242 or SEQ ID
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NO: 243 or at least 95% amino acid identity with SEQ ID NO: 237, SEQ ID NO:
238, SEQ ID NO: 239,
SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243. In yet other
aspects of this
embodiment, a respirovirus fusogenic peptide domain comprises a polypeptide
having, e.g., at most 70%
amino acid identity with SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ
ID NO: 240, SEQ ID
NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243, at most 75% amino acid identity
with SEQ ID NO: 237,
SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242
or SEQ ID NO:
243, at most 80% amino acid identity with SEQ ID NO: 237, SEQ ID NO: 238, SEQ
ID NO: 239, SEQ ID
NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243, at most 85% amino
acid identity with
SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO:
241, SEQ ID NO:
242 or SEQ ID NO: 243, at most 90% amino acid identity with SEQ ID NO: 237,
SEQ ID NO: 238, SEQ ID
NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243 or
at most 95% amino
acid identity with SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO:
240, SEQ ID NO:
241, SEQ ID NO: 242 or SEQ ID NO: 243.
[0194] In other aspects of this embodiment, a respirovirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID
NO: 239, SEQ ID NO:
240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243. In other aspects of
this embodiment, a
respirovirus fusogenic peptide domain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions
relative to SEQ ID NO: 237,
SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242
or SEQ ID NO:
243. In yet other aspects of this embodiment, a respirovirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid deletions relative to SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO:
239, SEQ ID NO: 240,
SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243. In other aspects of this
embodiment, a
respirovirus fusogenic peptide domain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or 10 non-contiguous amino acid deletions
relative to SEQ ID NO: 237, SEQ
ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or
SEQ ID NO: 243.
In still other aspects of this embodiment, a respirovirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid additions relative to SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO:
239, SEQ ID NO: 240,
SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243. In other aspects of this
embodiment, a
respirovirus fusogenic peptide domain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or 10 non-contiguous amino acid additions
relative to SEQ ID NO: 237, SEQ
ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or
SEQ ID NO: 243.
[0195] In other aspects of this embodiment, a respirovirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous amino
acid substitutions relative to SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239,
SEQ ID NO: 240, SEQ
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ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243. In other aspects of this
embodiment, a respirovirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 contiguous amino acid substitutions relative to SEQ
ID NO: 237, SEQ ID NO:
238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID
NO: 243. In yet
other aspects of this embodiment, a respirovirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
deletions relative to SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID
NO: 240, SEQ ID NO:
241, SEQ ID NO: 242 or SEQ ID NO: 243. In other aspects of this embodiment, a
respirovirus fusogenic
peptide domain comprises a polypeptide having, e.g., at least one, two, three,
four, five, six, seven, eight,
nine or 10 contiguous amino acid deletions relative to SEQ ID NO: 237, SEQ ID
NO: 238, SEQ ID NO:
239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243. In
still other aspects of
this embodiment, a respirovirus fusogenic peptide domain comprises a
polypeptide having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino
acid additions relative to SEQ ID
NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ
ID NO: 242 or
SEQ ID NO: 243. In other aspects of this embodiment, a respirovirus fusogenic
peptide domain
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine or 10
contiguous amino acid additions relative to SEQ ID NO: 237, SEQ ID NO: 238,
SEQ ID NO: 239, SEQ ID
NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 243.
[0196] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a
morbillivirus fusogenic peptide domain. In another aspect of this embodiment,
a morbillivirus fusogenic
peptide domain comprises a naturally occurring morbillivirus fusogenic peptide
domain variant, such as,
e.g., a morbillivirus fusogenic peptide domain isoform or a morbillivirus
fusogenic peptide domain
subtype. In another aspect of this embodiment, a morbillivirus fusogenic
peptide domain comprises a
naturally occurring morbillivirus fusogenic peptide domain variant of SEQ ID
NO: 244, SEQ ID NO: 245,
SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO:
250, SEQ ID NO:
251, SEQ ID NO: 252 or SEQ ID NO: 253, such as, e.g., a morbillivirus
fusogenic peptide domain isoform
of SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO:
248, SEQ ID NO:
249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253 or a
morbillivirus fusogenic
peptide domain subtype of SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ
ID NO: 247, SEQ
ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or
SEQ ID NO: 253.
In still another aspect of this embodiment, a morbillivirus fusogenic peptide
domain comprises a non-
naturally occurring morbillivirus fusogenic peptide domain variant, such as,
e.g., a conservative
morbillivirus fusogenic peptide domain variant, a non-conservative
morbillivirus fusogenic peptide domain
variant, a morbillivirus fusogenic peptide domain chimeric, an active
morbillivirus fusogenic peptide
domain fragment, or any combination thereof. In still another aspect of this
embodiment, a morbillivirus
fusogenic peptide domain comprises amino acids a non-naturally occurring
morbillivirus fusogenic
peptide domain variant of SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ
ID NO: 247, SEQ ID
NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ
ID NO: 253,
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such as, e.g., a conservative morbillivirus fusogenic peptide domain variant
of SEQ ID NO: 244, SEQ ID
NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ
ID NO: 250, SEQ
ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253, a non-conservative morbillivirus
fusogenic peptide
domain variant of SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO:
247, SEQ ID NO:
248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID
NO: 253, an
active morbillivirus fusogenic peptide domain fragment of SEQ ID NO: 244, SEQ
ID NO: 245, SEQ ID NO:
246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID
NO: 251, SEQ ID
NO: 252 or SEQ ID NO: 253, or any combination thereof.
[0197] In other aspects of this embodiment, a morbillivirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO:
244, SEQ ID NO: 245, SEQ
ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250,
SEQ ID NO: 251,
SEQ ID NO: 252 or SEQ ID NO: 253, at least 75% amino acid identity with SEQ ID
NO: 244, SEQ ID NO:
245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID
NO: 250, SEQ ID
NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253, at least 80% amino acid identity
with SEQ ID NO: 244,
SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO:
249, SEQ ID NO:
250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253, at least 85% amino acid
identity with SEQ ID
NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ
ID NO: 249, SEQ
ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253, at least 90%
amino acid identity with
SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO:
248, SEQ ID NO:
249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253 or at
least 95% amino acid
identity with SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247,
SEQ ID NO: 248,
SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO:
253. In yet other
aspects of this embodiment, a morbillivirus fusogenic peptide domain comprises
a polypeptide having,
e.g., at most 70% amino acid identity with SEQ ID NO: 244, SEQ ID NO: 245, SEQ
ID NO: 246, SEQ ID
NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ
ID NO: 252 or
SEQ ID NO: 253, at most 75% amino acid identity with SEQ ID NO: 244, SEQ ID
NO: 245, SEQ ID NO:
246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID
NO: 251, SEQ ID
NO: 252 or SEQ ID NO: 253, at most 80% amino acid identity with SEQ ID NO:
244, SEQ ID NO: 245,
SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO:
250, SEQ ID NO:
251, SEQ ID NO: 252 or SEQ ID NO: 253, at most 85% amino acid identity with
SEQ ID NO: 244, SEQ ID
NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ
ID NO: 250, SEQ
ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253, at most 90% amino acid identity
with SEQ ID NO: 244,
SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO:
249, SEQ ID NO:
250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253 or at most 95% amino
acid identity with SEQ
ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248,
SEQ ID NO: 249,
SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253.
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[0198] In other aspects of this embodiment, a morbillivirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID
NO: 246, SEQ ID NO:
247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID
NO: 252 or SEQ ID
NO: 253. In other aspects of this embodiment, a morbillivirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at least one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID
NO: 246, SEQ ID NO:
247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID
NO: 252 or SEQ ID
NO: 253. In yet other aspects of this embodiment, a morbillivirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid deletions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO:
246, SEQ ID NO: 247,
SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252
or SEQ ID NO:
253. In other aspects of this embodiment, a morbillivirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid deletions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO:
246, SEQ ID NO: 247,
SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252
or SEQ ID NO:
253. In still other aspects of this embodiment, a morbillivirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid additions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO:
246, SEQ ID NO: 247,
SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252
or SEQ ID NO:
253. In other aspects of this embodiment, a morbillivirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid additions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO:
246, SEQ ID NO: 247,
SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252
or SEQ ID NO:
253.
[0199] In other aspects of this embodiment, a morbillivirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous amino
acid substitutions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246,
SEQ ID NO: 247, SEQ
ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or
SEQ ID NO: 253.
In other aspects of this embodiment, a morbillivirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
substitutions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ
ID NO: 247, SEQ ID
NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ
ID NO: 253. In
yet other aspects of this embodiment, a morbillivirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
deletions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID
NO: 247, SEQ ID NO:
248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID
NO: 253. In other
aspects of this embodiment, a morbillivirus fusogenic peptide domain comprises
a polypeptide having,
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e.g., at least one, two, three, four, five, six, seven, eight, nine or 10
contiguous amino acid deletions
relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247,
SEQ ID NO: 248, SEQ
ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253.
In still other
aspects of this embodiment, a morbillivirus fusogenic peptide domain comprises
a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine or 10
contiguous amino acid additions
relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247,
SEQ ID NO: 248, SEQ
ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253.
In other aspects of
this embodiment, a morbillivirus fusogenic peptide domain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino
acid additions relative to SEQ ID
NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ
ID NO: 249, SEQ
ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253.
[0200] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises an
avulavirus fusogenic peptide domain. In another aspect of this embodiment, an
avulavirus fusogenic
peptide domain comprises a naturally occurring avulavirus fusogenic peptide
domain variant, such as,
e.g., an avulavirus fusogenic peptide domain isoform or an avulavirus
fusogenic peptide domain subtype.
In another aspect of this embodiment, an avulavirus fusogenic peptide domain
comprises a naturally
occurring avulavirus fusogenic peptide domain variant of SEQ ID NO: 254, SEQ
ID NO: 255, SEQ ID NO:
256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID
NO: 261, SEQ ID
NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266,
such as, e.g., an
avulavirus fusogenic peptide domain isoform of SEQ ID NO: 254, SEQ ID NO: 255,
SEQ ID NO: 256,
SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO:
261, SEQ ID NO:
262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266 or an
avulavirus fusogenic
peptide domain subtype of SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ
ID NO: 257, SEQ
ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262,
SEQ ID NO: 263,
SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266. In still another aspect of
this embodiment, an
avulavirus fusogenic peptide domain comprises a non-naturally occurring
avulavirus fusogenic peptide
domain variant, such as, e.g., a conservative avulavirus fusogenic peptide
domain variant, a non-
conservative avulavirus fusogenic peptide domain variant, an avulavirus
fusogenic peptide domain
chimeric, an active avulavirus fusogenic peptide domain fragment, or any
combination thereof. In still
another aspect of this embodiment, an avulavirus fusogenic peptide domain
comprises amino acids a
non-naturally occurring avulavirus fusogenic peptide domain variant of SEQ ID
NO: 254, SEQ ID NO:
255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID
NO: 260, SEQ ID
NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ
ID NO: 266,
such as, e.g., a conservative avulavirus fusogenic peptide domain variant of
SEQ ID NO: 254, SEQ ID
NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ
ID NO: 260, SEQ
ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or
SEQ ID NO: 266, a
non-conservative avulavirus fusogenic peptide domain variant of SEQ ID NO:
254, SEQ ID NO: 255, SEQ
ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260,
SEQ ID NO: 261,
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SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO:
266, an active
avulavirus fusogenic peptide domain fragment of SEQ ID NO: 254, SEQ ID NO:
255, SEQ ID NO: 256,
SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO:
261, SEQ ID NO:
262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266, or any
combination
thereof.
[0201] In other aspects of this embodiment, an avulavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO:
254, SEQ ID NO: 255, SEQ
ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260,
SEQ ID NO: 261,
SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO:
266, at least 75%
amino acid identity with SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ
ID NO: 257, SEQ ID
NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ
ID NO: 263, SEQ
ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266, at least 80% amino acid identity
with SEQ ID NO: 254,
SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO:
259, SEQ ID NO:
260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID
NO: 265 or SEQ ID
NO: 266, at least 85% amino acid identity with SEQ ID NO: 254, SEQ ID NO: 255,
SEQ ID NO: 256, SEQ
ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261,
SEQ ID NO: 262,
SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266, at least 90%
amino acid identity
with SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID
NO: 258, SEQ ID
NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ
ID NO: 264, SEQ
ID NO: 265 or SEQ ID NO: 266 or at least 95% amino acid identity with SEQ ID
NO: 254, SEQ ID NO:
255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID
NO: 260, SEQ ID
NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ
ID NO: 266. In
yet other aspects of this embodiment, an avulavirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at most 70% amino acid identity with SEQ ID NO: 254, SEQ ID NO:
255, SEQ ID NO: 256,
SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO:
261, SEQ ID NO:
262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266, at most
75% amino acid
identity with SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257,
SEQ ID NO: 258,
SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO:
263, SEQ ID NO:
264, SEQ ID NO: 265 or SEQ ID NO: 266, at most 80% amino acid identity with
SEQ ID NO: 254, SEQ ID
NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ
ID NO: 260, SEQ
ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or
SEQ ID NO: 266,
at most 85% amino acid identity with SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID
NO: 256, SEQ ID NO:
257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID
NO: 262, SEQ ID
NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266, at most 90% amino
acid identity with
SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO:
258, SEQ ID NO:
259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID
NO: 264, SEQ ID
NO: 265 or SEQ ID NO: 266 or at most 95% amino acid identity with SEQ ID NO:
254, SEQ ID NO: 255,
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SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO:
260, SEQ ID NO:
261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID
NO: 266.
[0202] In other aspects of this embodiment, an avulavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID
NO: 256, SEQ ID NO:
257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID
NO: 262, SEQ ID
NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266. In other aspects of
this embodiment, an
avulavirus fusogenic peptide domain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or 10 non-contiguous amino acid substitutions
relative to SEQ ID NO: 254,
SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO:
259, SEQ ID NO:
260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID
NO: 265 or SEQ ID
NO: 266. In yet other aspects of this embodiment, an avulavirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid deletions relative to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO:
256, SEQ ID NO: 257,
SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO:
262, SEQ ID NO:
263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266. In other aspects of
this embodiment, an
avulavirus fusogenic peptide domain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or 10 non-contiguous amino acid deletions
relative to SEQ ID NO: 254, SEQ
ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259,
SEQ ID NO: 260,
SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265
or SEQ ID NO:
266. In still other aspects of this embodiment, an avulavirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid additions relative to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO:
256, SEQ ID NO: 257,
SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO:
262, SEQ ID NO:
263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266. In other aspects of
this embodiment, an
avulavirus fusogenic peptide domain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or 10 non-contiguous amino acid additions
relative to SEQ ID NO: 254, SEQ
ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259,
SEQ ID NO: 260,
SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265
or SEQ ID NO:
266.
[0203] In other aspects of this embodiment, an avulavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous amino
acid substitutions relative to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256,
SEQ ID NO: 257, SEQ
ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262,
SEQ ID NO: 263,
SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266. In other aspects of this
embodiment, an
avulavirus fusogenic peptide domain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or 10 contiguous amino acid substitutions
relative to SEQ ID NO: 254, SEQ ID
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NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ
ID NO: 260, SEQ
ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or
SEQ ID NO: 266.
In yet other aspects of this embodiment, an avulavirus fusogenic peptide
domain comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
deletions relative to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID
NO: 257, SEQ ID NO:
258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID
NO: 263, SEQ ID
NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266. In other aspects of this
embodiment, an avulavirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 contiguous amino acid deletions relative to SEQ ID
NO: 254, SEQ ID NO: 255,
SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO:
260, SEQ ID NO:
261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID
NO: 266. In still
other aspects of this embodiment, an avulavirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
additions relative to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID
NO: 257, SEQ ID NO:
258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID
NO: 263, SEQ ID
NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266. In other aspects of this
embodiment, an avulavirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 contiguous amino acid additions relative to SEQ ID
NO: 254, SEQ ID NO: 255,
SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO:
260, SEQ ID NO:
261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID
NO: 266.
[0204] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a
henipavirus fusogenic peptide domain. In another aspect of this embodiment, a
henipavirus fusogenic
peptide domain comprises a naturally occurring henipavirus fusogenic peptide
domain variant, such as,
e.g., a henipavirus fusogenic peptide domain isoform or a henipavirus
fusogenic peptide domain subtype.
In another aspect of this embodiment, a henipavirus fusogenic peptide domain
comprises a naturally
occurring henipavirus fusogenic peptide domain variant of SEQ ID NO: 267 or
SEQ ID NO: 268, such as,
e.g., a henipavirus fusogenic peptide domain isoform of SEQ ID NO: 267 or SEQ
ID NO: 268 or a
henipavirus fusogenic peptide domain subtype of SEQ ID NO: 267 or SEQ ID NO:
268. In still another
aspect of this embodiment, a henipavirus fusogenic peptide domain comprises a
non-naturally occurring
henipavirus fusogenic peptide domain variant, such as, e.g., a conservative
henipavirus fusogenic
peptide domain variant, a non-conservative henipavirus fusogenic peptide
domain variant, a henipavirus
fusogenic peptide domain chimeric, an active henipavirus fusogenic peptide
domain fragment, or any
combination thereof. In still another aspect of this embodiment, a henipavirus
fusogenic peptide domain
comprises amino acids a non-naturally occurring henipavirus fusogenic peptide
domain variant of SEQ ID
NO: 267 or SEQ ID NO: 268, such as, e.g., a conservative henipavirus fusogenic
peptide domain variant
of SEQ ID NO: 267 or SEQ ID NO: 268, a non-conservative henipavirus fusogenic
peptide domain variant
of SEQ ID NO: 267 or SEQ ID NO: 268, an active henipavirus fusogenic peptide
domain fragment of SEQ
ID NO: 267 or SEQ ID NO: 268, or any combination thereof.
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[0205] In other aspects of this embodiment, a henipavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO: 267
or SEQ ID NO: 268, at
least 75% amino acid identity with SEQ ID NO: 267 or SEQ ID NO: 268, at least
80% amino acid identity
with SEQ ID NO: 267 or SEQ ID NO: 268, at least 85% amino acid identity with
SEQ ID NO: 267 or SEQ
ID NO: 268, at least 90% amino acid identity with SEQ ID NO: 267 or SEQ ID NO:
268 or at least 95%
amino acid identity with SEQ ID NO: 267 or SEQ ID NO: 268. In yet other
aspects of this embodiment, a
henipavirus fusogenic peptide domain comprises a polypeptide having, e.g., at
most 70% amino acid
identity with SEQ ID NO: 267 or SEQ ID NO: 268, at most 75% amino acid
identity with SEQ ID NO: 267
or SEQ ID NO: 268, at most 80% amino acid identity with SEQ ID NO: 267 or SEQ
ID NO: 268, at most
85% amino acid identity with SEQ ID NO: 267 or SEQ ID NO: 268, at most 90%
amino acid identity with
SEQ ID NO: 267 or SEQ ID NO: 268 or at most 95% amino acid identity with SEQ
ID NO: 267 or SEQ ID
NO: 268.
[0206] In other aspects of this embodiment, a henipavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 267 or SEQ ID NO: 268. In
other aspects of this
embodiment, a henipavirus fusogenic peptide domain comprises a polypeptide
having, e.g., at least one,
two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino
acid substitutions relative to SEQ
ID NO: 267 or SEQ ID NO: 268. In yet other aspects of this embodiment, a
henipavirus fusogenic peptide
domain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine or
non-contiguous amino acid deletions relative to SEQ ID NO: 267 or SEQ ID NO:
268. In other aspects
of this embodiment, a henipavirus fusogenic peptide domain comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or 10 non-
contiguous amino acid deletions relative
to SEQ ID NO: 267 or SEQ ID NO: 268. In still other aspects of this
embodiment, a henipavirus fusogenic
peptide domain comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight,
nine or 10 non-contiguous amino acid additions relative to SEQ ID NO: 267 or
SEQ ID NO: 268. In other
aspects of this embodiment, a henipavirus fusogenic peptide domain comprises a
polypeptide having,
e.g., at least one, two, three, four, five, six, seven, eight, nine or 10 non-
contiguous amino acid additions
relative to SEQ ID NO: 267 or SEQ ID NO: 268.
[0207] In other aspects of this embodiment, a henipavirus fusogenic peptide
domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous amino
acid substitutions relative to SEQ ID NO: 267 or SEQ ID NO: 268. In other
aspects of this embodiment, a
henipavirus fusogenic peptide domain comprises a polypeptide having, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or 10 contiguous amino acid substitutions
relative to SEQ ID NO: 267 or SEQ
ID NO: 268. In yet other aspects of this embodiment, a henipavirus fusogenic
peptide domain comprises
a polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous
amino acid deletions relative to SEQ ID NO: 267 or SEQ ID NO: 268. In other
aspects of this
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embodiment, a henipavirus fusogenic peptide domain comprises a polypeptide
having, e.g., at least one,
two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid
deletions relative to SEQ ID NO:
267 or SEQ ID NO: 268. In still other aspects of this embodiment, a
henipavirus fusogenic peptide
domain comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine or
contiguous amino acid additions relative to SEQ ID NO: 267 or SEQ ID NO: 268.
In other aspects of
this embodiment, a henipavirus fusogenic peptide domain comprises a
polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino
acid additions relative to SEQ ID
NO: 267 or SEQ ID NO: 268.
[0208] In another embodiment, a Clostridial toxin translocation facilitating
domain comprises a
metapneumovirus fusogenic peptide domain. In another aspect of this
embodiment, a metapneumovirus
fusogenic peptide domain comprises a naturally occurring metapneumovirus
fusogenic peptide domain
variant, such as, e.g., a metapneumovirus fusogenic peptide domain isoform or
a metapneumovirus
fusogenic peptide domain subtype. In another aspect of this embodiment, a
metapneumovirus fusogenic
peptide domain comprises a naturally occurring metapneumovirus fusogenic
peptide domain variant of
SEQ ID NO: 269, such as, e.g., a metapneumovirus fusogenic peptide domain
isoform of SEQ ID NO:
269 or a metapneumovirus fusogenic peptide domain subtype of SEQ ID NO: 269.
In still another aspect
of this embodiment, a metapneumovirus fusogenic peptide domain comprises a non-
naturally occurring
metapneumovirus fusogenic peptide domain variant, such as, e.g., a
conservative metapneumovirus
fusogenic peptide domain variant, a non-conservative metapneumovirus fusogenic
peptide domain
variant, a metapneumovirus fusogenic peptide domain chimeric, an active
metapneumovirus fusogenic
peptide domain fragment, or any combination thereof. In still another aspect
of this embodiment, a
metapneumovirus fusogenic peptide domain comprises amino acids a non-naturally
occurring
metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269, such as,
e.g., a conservative
metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269, a non-
conservative
metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269, an active
metapneumovirus
fusogenic peptide domain fragment of SEQ ID NO: 269, or any combination
thereof.
[0209] In other aspects of this embodiment, a metapneumovirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ ID NO:
269, at least 75% amino acid
identity with SEQ ID NO: 269, at least 80% amino acid identity with SEQ ID NO:
269, at least 85% amino
acid identity with SEQ ID NO: 269, at least 90% amino acid identity with SEQ
ID NO: 269 or at least 95%
amino acid identity with SEQ ID NO: 269. In yet other aspects of this
embodiment, a metapneumovirus
fusogenic peptide domain comprises a polypeptide having, e.g., at most 70%
amino acid identity with
SEQ ID NO: 269, at most 75% amino acid identity with SEQ ID NO: 269, at most
80% amino acid identity
with SEQ ID NO: 269, at most 85% amino acid identity with SEQ ID NO: 269, at
most 90% amino acid
identity with SEQ ID NO: 269 or at most 95% amino acid identity with SEQ ID
NO: 269.
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[0210] In other aspects of this embodiment, a metapneumovirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid substitutions relative to SEQ ID NO: 269. In other aspects of this
embodiment, a
metapneumovirus fusogenic peptide domain comprises a polypeptide having, e.g.,
at least one, two,
three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid
substitutions relative to SEQ ID
NO: 269. In yet other aspects of this embodiment, a metapneumovirus fusogenic
peptide domain
comprises a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine or 10 non-
contiguous amino acid deletions relative to SEQ ID NO: 269. In other aspects
of this embodiment, a
metapneumovirus fusogenic peptide domain comprises a polypeptide having, e.g.,
at least one, two,
three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid
deletions relative to SEQ ID NO:
269. In still other aspects of this embodiment, a metapneumovirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 non-contiguous
amino acid additions relative to SEQ ID NO: 269. In other aspects of this
embodiment, a
metapneumovirus fusogenic peptide domain comprises a polypeptide having, e.g.,
at least one, two,
three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid
additions relative to SEQ ID NO:
269.
[0211] In other aspects of this embodiment, a metapneumovirus fusogenic
peptide domain comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine or 10 contiguous amino
acid substitutions relative to SEQ ID NO: 269. In other aspects of this
embodiment, a metapneumovirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 contiguous amino acid substitutions relative to SEQ
ID NO: 269. In yet other
aspects of this embodiment, a metapneumovirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
deletions relative to SEQ ID NO: 269. In other aspects of this embodiment, a
metapneumovirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 contiguous amino acid deletions relative to SEQ ID
NO: 269. In still other
aspects of this embodiment, a metapneumovirus fusogenic peptide domain
comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven, eight, nine or
10 contiguous amino acid
additions relative to SEQ ID NO: 269. In other aspects of this embodiment, a
metapneumovirus
fusogenic peptide domain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or 10 contiguous amino acid additions relative to SEQ ID
NO: 269.
[0212] Aspects of the present invention provide, in part, an altered targeting
domain. As used herein,
the term "altered targeting domain" means any polypeptide that can selectively
bind to a non-Clostridial
toxin receptor present on a non-Clostridial toxin target cell and initiate the
overall internalization
mechanism whereby the modified Clostridial toxin disclosed in the present
specification intoxicates a
target cell. As used herein, the term "selectively" means having a highly
preferred activity or effect. As
used herein, the term "selectively bind" means a molecule is able to bind its
target receptor under
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physiological conditions, or in vitro conditions substantially approximating
physiological conditions, to a
statistically significantly greater degree relative to other, non-target
receptors. Thus, with reference to an
altered targeting domain of the present specification, there is a
discriminatory binding of the altered
targeting domain to a non-Clostridial toxin receptor presence in a non-
Clostridial toxin target cell.
[0213] An altered targeting domain disclosed in the present specification
facilitates the binding activity of
the modified Clostridial toxins disclosed in the present specification to a
non-Clostridial toxin receptor
located at the surface of a Clostridial toxin target cell. As used herein, the
term "binding activity" means
that one molecule is directly or indirectly contacting another molecule via at
least one intermolecular or
intramolecular force, including, without limitation, a covalent bond, an ionic
bond, a metallic bond, a
hydrogen bond, a hydrophobic interaction, a van der Waals interaction, and the
like, or any combination
thereof. "Bound" and "bind" are considered terms for binding.
[0214] As used herein, the term "binding affinity" means how strong a
molecule's binding activity is for a
particular receptor. In general, high binding affinity results from greater
intermolecular force between a
binding domain and its receptor while low binding affinity involves less
intermolecular force between the
ligand and its receptor. High binding affinity involves a longer residence
time for the binding domain at its
receptor binding site than is the case for low binding affinity. As such, a
molecule with a high binding
affinity means a lower concentration of that molecule is required to maximally
occupy the binding sites of
a receptor and trigger a physiological response. Conversely, low binding
affinity means a relatively high
concentration of a molecule is required before the receptor binding sites of a
receptor is maximally
occupied and the maximum physiological response is achieved. Thus, modified
Clostridial toxins with
increased binding activity due to high binding affinity will allow
administration of reduced doses of the
toxin, thereby reducing or preventing unwanted side-effects associated with
toxin dispersal into non-
targeted areas.
[0215] As used herein, the term "binding specificity" means how specific a
molecule's binding activity is
one particular receptor. In general, high binding specificity results in a
more exclusive interaction with
one particular receptor or subgroup of receptors while low binding specificity
results in a more
promiscuous interaction with a larger group of receptors. As such, a molecule
with a high binding
specificity means that molecule will occupy the binding sites of a particular
receptor and trigger a
physiological response. Conversely, low binding specificity means a molecule
will occupy the binding
sites of a many receptors and trigger a multitude of physiological responses.
Thus, modified Clostridial
toxins with increased binding activity due to high binding specificity will
only target non-Clostridial toxin
receptors present on a subgroup of non-Clostridial toxin target cells, thereby
reducing the side effects
associated with the targeting of all non-Clostridial toxin target cells.
[0216] In addition to its altered targeting activity, replacement of a
naturally-occurring targeting domain
with an altered target domain disclosed in the specification has an added
advantage of reducing the
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likelihood of the modified toxin from eliciting an immunogenic response.
Regions found in the Hcc
targeting domain are bound by neutralizing anti-BoNT/A antibodies, see, e.g.,
M. Zouhair Atassi et al.,
Mapping of the Antibody-binding Regions on Botulinum Neurotoxin H-chain Domain
855-1296 with Anti-
toxin Antibodies from Three Host Species, 15 J. PROT. CHEM. 691-700, (1996);
M. Zouhair Atassi &
Behzod Z. Dolimbek, Mapping of the Antibody-binding Profile on Botulinum
Neurotoxin A HN-domain
(residues 449-859) with Anti-toxin Antibodies from Four Host Species.
Antigenic Profile of the Entire H-
chain of Botulinum Neurotoxin A, 23(1) PROTEIN J. 39-52, (2004). Therefore,
elimination of this targeting
domain will reduce the likelihood of an immunogenic response because 1) the
Clostridial toxin Hcc
targeting domain is absent; 2) an altered targeting domain derived from a
human will most likely not elicit
an immunogenic response in a patient because it is a human polypeptide.
[0217] As used herein, the term "non-Clostridial toxin target celP" means a
cell that is not a naturally
occurring cell that a naturally occurring Clostridial toxin is capable of
intoxicating, including, without
limitation, sensory neurons; autonomic neurons, such as, e.g., sympathetic
neurons and parasympathetic
neurons; and non-neuronal cells, such as, e.g., anterior pituitary cells;
adrenal cells, such as. e.g.,
chromaffin cells of the adrenal medulla; pancreatic cells, such as. e.g.,
pancreatic acinar cells, pancreatic
islet (3 cells; ovarian cells; kidney cells, such as. e.g., inner medullary
collecting duct (IMCD) cells;
stomach cells, such as, e.g., enterochromaffin cells; blood cells, such as.
e.g., eurythrocytes, leucocytes,
platelets, neutrophils, eosinophils, mast cells; epithelial cells, such as.
e.g., those of the apical plasma
membrane; fibroblasts; thyroid cells; chondrocytes; muscle cells; hepatocytes;
glandular cells such as,
e.g., pituitary cells, chromaffin cells.
[0218] It is envisioned that any and all altered targeting domains that
exhibits a binding activity for a non-
Clostridial toxin receptor present on a non-Clostridial toxin target cell can
be used to practice aspects of
the present invention, including, without limitation, polypeptides that
selectively bind to a receptor present
on a sensory neuron, an autonomic neuron or a non-neuronal cell. Polypeptides
useful as altered
targeting domains useful to practice aspect of the present invention include,
without limitation, an opioid
peptide, such as, e.g., an enkephalin, a bovine adrenomedullary-22 (BAM22)
peptide, an endomorphin,
an endorphin, a dynorphin, a nociceptin or a hemorphin; a melanocortin
peptide, such as, e.g., an a-
melanocyte stimulating hormones (a-MSH), a(3-melanocyte stimulating hormones
((3-MSH), a y-
melanocyte stimulating hormones (y-MSH), an adrenocorticotropin (ACTH), a
Corticotropin-like
intermediary peptide (CLIP), a(3-lipotropin ((3-LPH) and a y-lipotropin (y-
LPH); a galanin, such as, e.g., a
galanin and a galanin message-associated peptide (GMAP); a granin, such as,
e.g., a chromogranin A
peptide like a(3-granin, a vasostatin, a chromostatin, a pancreastatin, a WE-
14, a catestatin, a parastatin
and a GE-25, a chromogranin B (secretogranin I) peptide like a GAWK peptide,
an adrenomedullary
peptide and a secretolytin and a chromogranin C (secretogranin II) peptide
like secretoneurin, EM66 and
manserin; a tachykinin peptide, such as, e.g., Substance P, neuropeptide K
(NPK), neuropeptide gamma
(NP gamma), neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L),
neurokinin B (NKB), a
hemokinin and a endokinin; a cholecystokinin, such as, e.q., a cholecystokinin
58, a cholecystokinin 39,
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a cholecystokinin 33, a cholecystokinin 12 and a cholecystokinin 8; a
Neuropeptide Y related peptide,
such as, e.g., a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide
(PP) and a Pancreatic
icosapeptide (PIP); , a kinin peptide, such as, e.g., a bradykinin, a
kallidan, a desArg9 bradykinin and a
desArg10 bradykinin; a protease activated receptor (PAR) peptide, such as,
e.g., a PAR1 peptide, a PAR2
peptide, a PAR3 peptide and a PAR4 peptide; a corticotropin-releasing hormone;
a thyrotropin-releasing
hormone; a somatostatin; a leukemia inhibitor factor (LIF); and an interleukin-
1 ( IL1).
[0219] An altered targeting domain includes, without limitation, naturally
occurring altered targeting
domain variants, such as, e.g., altered targeting domain isoforms; non-
naturally occurring altered
targeting domain variants, such as, e.g., conservative altered targeting
domain variants, non-conservative
altered targeting domain variants, altered targeting domain chimerics, active
altered targeting domain
fragments thereof, or any combination thereof.
[0220] As used herein, the term "variant," when used to describe an altered
targeting domain variant,
whether naturally-occurring or non-naturally-occurring, means an altered
targeting domain that has at
least one amino acid change from the corresponding region of the disclosed
reference sequences and
can be described in percent identity to the corresponding region of that
reference sequence. Unless
expressly indicated, all altered targeting domain variants disclosed in the
present specification are
capable of selectively binding to a non-Clostridial toxin receptor present on
a non-Clostridial toxin target
cell and initiate the overall internalization mechanism whereby a modified
Clostridial toxin disclosed in the
present specification intoxicates a non-Clostridial toxin target cell. As non-
limiting examples, an
endorphin-(3 variant derived from SEQ ID NO: 17 will have at least one amino
acid difference, such as,
e.g., an amino acid substitution, deletion or addition, as compared to SEQ ID
NO: 17; a Dymorphin A
variant derived from SEQ ID NO: 21 will have at least one amino acid
difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to SEQ ID NO: 21; a
nociceptin variant derived from
SEQ ID NO: 52 will have at least one amino acid difference, such as, e.g., an
amino acid substitution,
deletion or addition, as compared to SEQ ID NO: 52; a galanin variant derived
from SEQ ID NO: 72 will
have at least one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as
compared to SEQ ID NO: 72; an adrenomedullary peptide variant derived from SEQ
ID NO: 83 will have
at least one amino acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as
compared to SEQ ID NO: 83; a Substance P variant derived from SEQ ID NO: 88
will have at least one
amino acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to SEQ
ID NO: 88; an endokinin variant derived from SEQ ID NO: 97 will have at least
one amino acid difference,
such as, e.g., an amino acid substitution, deletion or addition, as compared
to SEQ ID NO: 97; a CCK
variant derived from SEQ ID NO: 100 will have at least one amino acid
difference, such as, e.g., an amino
acid substitution, deletion or addition, as compared to SEQ ID NO: 100; a NPY
variant derived from SEQ
ID NO: 116 will have at least one amino acid difference, such as, e.g., an
amino acid substitution, deletion
or addition, as compared to SEQ ID NO: 116; and a PP variant derived from SEQ
ID NO: 118 will have at
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least one amino acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as
compared to SEQ ID NO: 118.
[0221] It is recognized by those of skill in the art that there can be
naturally occurring altered targeting
domain variants that differ somewhat in their amino acid sequence, and also in
the nucleic acids encoding
these proteins. As used herein, the term "naturally occurring altered
targeting domain variant" means any
altered targeting domain produced by a naturally-occurring process, including,
without limitation, altered
targeting domain isoforms produced from alternatively-spliced transcripts,
altered targeting domain
isoforms produced by spontaneous mutation and altered targeting domain
subtypes. A naturally
occurring altered targeting domain variant can function in substantially the
same manner as the reference
altered targeting domain on which the naturally occurring altered targeting
domain variant is based, and
can be substituted for the reference altered targeting domain in any aspect of
the present invention. A
naturally occurring altered targeting domain variant may substitute one or
more amino acids, two or more
amino acids, three or more amino acids, four or more amino acids, five or more
amino acids, ten or more
amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino
acids, 50 or more amino
acids or 100 or more amino acids from the reference altered targeting domain
on which the naturally
occurring altered targeting domain variant is based. A naturally occurring
altered targeting domain variant
can also substitute, e.g., at least 2 contiguous amino acids, at least 3
contiguous amino acids, at least 4
contiguous amino acids, at least 5 contiguous amino acids, at least 10
contiguous amino acids, at least
15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25
contiguous amino acids
from the reference altered targeting domain on which the naturally occurring
altered targeting domain
variant is based, that possess at least 50% amino acid identity, 65% amino
acid identity, 75% amino acid
identity, 85% amino acid identity or 95% amino acid identity to the reference
altered targeting domain on
which the naturally occurring altered targeting domain variant is based.
[0222] A non-limiting example of a naturally occurring altered targeting
domain variant is an altered
targeting domain isoform such as, e.g., an opioid peptide, a melanocortin
peptide, a galanin, a granin, a
tachykinin peptide, a cholecystokinin, a Neuropeptide Y related peptide, a
kinin peptide, a protease
activated receptor (PAR) peptide, a corticotropin-releasing hormone, a
thyrotropin-releasing hormone and
somatostatin. An altered targeting domain isoform can function in
substantially the same manner as the
reference altered targeting domain on which the altered targeting domain
isoform is based, and can be
substituted for the reference altered targeting domain in any aspect of the
present invention.
[0223] As used herein, the term "non-naturally occurring altered targeting
domain variant" means any
altered targeting domain produced with the aid of human manipulation,
including, without limitation,
altered targeting domains produced by genetic engineering using random
mutagenesis or rational design
and altered targeting domains produced by chemical synthesis. Non-limiting
examples of non-naturally
occurring altered targeting domain variants include, e.g., conservative
altered targeting domain variants,
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non-conservative altered targeting domain variants, altered targeting domain
chimeric variants and active
altered targeting domain fragments.
[0224] As used herein, the term "conservative altered targeting domain
variant" means an altered
targeting domain that has at least one amino acid substituted by another amino
acid or an amino acid
analog that has at least one property similar to that of the original amino
acid from the reference altered
targeting domain sequence. Examples of properties include, without limitation,
similar size, topography,
charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding
capacity, hydrogen-bonding capacity,
a physicochemical property, of the like, or any combination thereof. A
conservative altered targeting
domain variant can function in substantially the same manner as the reference
altered targeting domain
on which the conservative altered targeting domain variant is based, and can
be substituted for the
reference altered targeting domain in any aspect of the present invention. A
conservative altered
targeting domain variant may substitute one or more amino acids, two or more
amino acids, three or more
amino acids, four or more amino acids, five or more amino acids, ten or more
amino acids, 20 or more
amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino
acids, 100 or more
amino acids, 200 or more amino acids, 300 or more amino acids, 400 or more
amino acids, or 500 or
more amino acids from the reference altered targeting domain on which the
conservative altered targeting
domain variant is based. A conservative altered targeting domain variant can
also substitute, e.g., at
least 2 contiguous amino acids, at least 3 contiguous amino acids, at least 4
contiguous amino acids, at
least 5 contiguous amino acids, at least 10 contiguous amino acids, at least
15 contiguous amino acids,
at least 20 contiguous amino acids, or at least 25 contiguous amino acids from
the reference altered
targeting domain on which the conservative altered targeting domain variant is
based, that possess at
least 50% amino acid identity, 65% amino acid identity, 75% amino acid
identity, 85% amino acid identity
or 95% amino acid identity to the reference altered targeting domain on which
the conservative altered
targeting domain variant is based. Non-limiting examples of a conservative
altered targeting domain
variant include, e.g., conservative opioid peptide variants, conservative
melanocortin peptide variants,
conservative galanin variants, conservative granin variants, conservative
tachykinin peptide variants,
conservative cholecystokinin variants, conservative Neuropeptide Y related
peptide variants, conservative
kinin peptide variants, conservative PAR peptide variants, conservative
corticotropin-releasing hormone
variants, conservative thyrotropin-releasing hormone variants and conservative
somatostatin variants.
[0225] As used herein, the term "non-conservative altered targeting domain
variant" means an altered
targeting domain in which 1) at least one amino acid is deleted from the
reference altered targeting
domain on which the non-conservative altered targeting domain variant is
based; 2) at least one amino
acid added to the reference altered targeting domain on which the non-
conservative altered targeting
domain is based; or 3) at least one amino acid is substituted by another amino
acid or an amino acid
analog that does not share any property similar to that of the original amino
acid from the reference
altered targeting domain sequence. A non-conservative altered targeting domain
variant can function in
substantially the same manner as the reference altered targeting domain on
which the non-conservative
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altered targeting domain variant is based, and can be substituted for the
reference altered targeting
domain in any aspect of the present invention. A non-conservative altered
targeting domain variant can
delete one or more amino acids, two or more amino acids, three or more amino
acids, four or more amino
acids, five or more amino acids, and ten or more amino acids from the
reference altered targeting domain
on which the non-conservative altered targeting domain variant is based. A non-
conservative altered
targeting domain variant can add one or more amino acids, two or more amino
acids, three or more
amino acids, four or more amino acids, five or more amino acids, and ten or
more amino acids to the
reference altered targeting domain on which the non-conservative altered
targeting domain variant is
based. A non-conservative altered targeting domain variant may substitute one
or more amino acids, two
or more amino acids, three or more amino acids, four or more amino acids, five
or more amino acids, ten
or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or
more amino acids, 50 or
more amino acids, 100 or more amino acids, 200 or more amino acids, 300 or
more amino acids, 400 or
more amino acids, or 500 or more amino acids from the reference altered
targeting domain on which the
non-conservative altered targeting domain variant is based. A non-conservative
altered targeting domain
variant can also substitute, e.g., at least 2 contiguous amino acids, at least
3 contiguous amino acids, at
least 4 contiguous amino acids, at least 5 contiguous amino acids, at least 10
contiguous amino acids, at
least 15 contiguous amino acids, at least 20 contiguous amino acids, or at
least 25 contiguous amino
acids from the reference altered targeting domain on which the non-
conservative altered targeting domain
variant is based, that possess at least 50% amino acid identity, 65% amino
acid identity, 75% amino acid
identity, 85% amino acid identity or 95% amino acid identity to the reference
altered targeting domain on
which the non-conservative altered targeting domain variant is based. Non-
limiting examples of a non-
conservative altered targeting domain variant include, e.g., non-conservative
opioid peptide variants, non-
conservative melanocortin peptide variants, non-conservative galanin variants,
non-conservative granin
variants, non-conservative tachykinin peptide variants, non-conservative
cholecystokinin variants, non-
conservative Neuropeptide Y related peptide variants, non-conservative kinin
peptide variants, non-
conservative PAR peptide variants, non-conservative corticotropin-releasing
hormone variants, non-
conservative thyrotropin-releasing hormone variants and non-conservative
somatostatin variants.
[0226] As used herein, the term "altered targeting domain chimeric" means a
polypeptide comprising at
least a portion of an altered targeting domain and at least a portion of at
least one other polypeptide to
form an altered targeting domain with at least one property different from the
reference altered targeting
domain, with the proviso that this altered targeting domain chimeric is still
capable of selectively binding to
a non-Clostridial toxin receptor present on a non-Clostridial toxin target
cell and initiate the overall
internalization mechanism whereby a modified Clostridial toxin intoxicates a
target cell.
[0227] As used herein, the term "active altered targeting domain fragment"
means any of a variety of
altered targeting domain fragments can be useful in aspects of the present
invention with the proviso that
these active fragments are still capable of selectively binding to a non-
Clostridial toxin receptor present on
a non-Clostridial toxin target cell and initiate the overall internalization
mechanism whereby a Clostridial
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toxin intoxicates a target cell. Thus, aspects of this embodiment can include
altered targeting domains
comprising a length of, e.g., at least 5 amino acids, at least 10 amino acids,
at least 20 amino acids, at
least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at
least 100 amino acids, at least
150 amino acids, at least 200 amino acids, at least 250 amino acids, at least
300 amino acids, at least
350 amino acids, at least 400 amino acids and at least 450 amino acids. Other
aspects of this
embodiment can include altered targeting domains comprising a length of, e.g.,
at most 5 amino acids, at
most 10 amino acids, at most 20 amino acids, at most 30 amino acids, at most
40 amino acids, at most
50 amino acids, at most 100 amino acids, at most 150 amino acids, at most 200
amino acids, at most 250
amino acids, at most 300 amino acids, at most 350 amino acids, at most 400
amino acids and at most
450 amino acids.
[0228] Any of a variety of sequence alignment methods can be used to determine
percent identity of
naturally-occurring altered targeting domain variants and non-naturally-
occurring altered targeting domain
variants, including, without limitation, global methods, local methods and
hybrid methods, such as, e.g.,
segment approach methods. Protocols to determine percent identity are routine
procedures within the
scope of one skilled in the art and from the teaching herein.
[0229] Thus, in an embodiment, a modified Clostridial toxin disclosed in the
present specification
comprises an altered targeting domain. In an aspect of this embodiment, an
altered targeting domain
comprises a naturally occurring altered targeting domain variant, such as,
e.g., an altered targeting
domain isoform or an altered targeting domain subtype. In another aspect of
this embodiment, a
Clostridial toxin altered targeting domain comprises a non-naturally occurring
altered targeting domain
variant, such as, e.g., a conservative altered targeting domain variant, a non-
conservative altered
targeting domain variant, an altered targeting domain chimeric, an active
altered targeting domain
fragment, or any combination thereof.
[0230] An example of an altered targeting domain disclosed in the present
specification is, e.g., a opioid
peptide, such as, e.g., an enkephalin, an endomorphin, an endorphin, a
dynorphin, a nociceptin or a
hemorphin. Thus, in an embodiment, an altered targeting domain is derived from
an opioid peptide.
[0231] In another embodiment, an opioid peptide comprising an altered
targeting domain is an
enkephalin. In aspects of this embodiment, an enkephalin comprising an altered
targeting domain is
derived from a Leu-enkephalin, a Met-enkephalin, a Met-enkephalin MRGL or a
Met-enkephalin MRF. In
other aspects of this embodiment, an enkephalin comprising an altered
targeting domain is SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.
[0232] In other aspects of this embodiment, an enkephalin comprising an
altered targeting domain has,
e.g., at least 70% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO: 11 or SEQ ID NO:
12, at least 75% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11 or SEQ ID NO:
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12, at least 80% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11 or SEQ ID NO:
12, at least 85% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11 or SEQ ID NO:
12, at least 90% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11 or SEQ ID NO:
12 or at least 95% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO: 11 or SEQ ID
NO: 12. In yet other aspects of this embodiment, an enkephalin comprising an
altered targeting domain
has, e.g., at most 70% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11 or SEQ ID
NO: 12, at most 75% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO: 11 or SEQ ID
NO: 12, at most 80% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO: 11 or SEQ ID
NO: 12, at most 85% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO: 11 or SEQ ID
NO: 12, at most 90% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO: 11 or SEQ ID
NO: 12 or at most 95% amino acid identity with SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11 or SEQ
ID NO: 12.
[0233] In other aspects of this embodiment, an enkephalin comprising an
altered targeting domain has,
e.g., at least one, two or three non-contiguous amino acid substitutions
relative to SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In other aspects of this embodiment,
an enkephalin
comprising an altered targeting domain has, e.g., at most one, two or three
non-contiguous amino acid
substitutions relative to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID
NO: 12. In yet other
aspects of this embodiment, an enkephalin comprising an altered targeting
domain has, e.g., at least one,
two or three non-contiguous amino acid deletions relative to SEQ ID NO: 9, SEQ
ID NO: 10, SEQ ID NO:
11 or SEQ ID NO: 12. In yet other aspects of this embodiment, an enkephalin
comprising an altered
targeting domain has, e.g., at most one, two or three non-contiguous amino
acid deletions relative to SEQ
ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In still other
aspects of this embodiment,
an enkephalin comprising an altered targeting domain has, e.g., at least one,
two or three non-contiguous
amino acid additions relative to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or
SEQ ID NO: 12. In yet
other aspects of this embodiment, an enkephalin comprising an altered
targeting domain has, e.g., at
most one, two or three non-contiguous amino acid additions relative to SEQ ID
NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11 or SEQ ID NO: 12.
[0234] In other aspects of this embodiment, an enkephalin comprising an
altered targeting domain has,
e.g., at least one, two or three contiguous amino acid substitutions relative
to SEQ ID NO: 9, SEQ ID NO:
10, SEQ ID NO: 11 or SEQ ID NO: 12. In other aspects of this embodiment, an
enkephalin comprising an
altered targeting domain has, e.g., at most one, two or three contiguous amino
acid substitutions relative
to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In yet other
aspects of this
embodiment, an enkephalin comprising an altered targeting domain has, e.g., at
least one, two or three
contiguous amino acid deletions relative to SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO: 11 or SEQ ID
NO: 12. In yet other aspects of this embodiment, an enkephalin comprising an
altered targeting domain
has, e.g., at most one, two or three contiguous amino acid deletions relative
to SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In still other aspects of this
embodiment, an enkephalin
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comprising an altered targeting domain has, e.g., at least one, two or three
contiguous amino acid
additions relative to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:
12. In yet other
aspects of this embodiment, an enkephalin comprising an altered targeting
domain has, e.g., at most one,
two or three contiguous amino acid additions relative to SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11
or SEQ ID NO: 12.
[0235] In another embodiment, an opioid peptide comprising an altered
targeting domain is a bovine
adrenomedullary-22 (BAM22) peptide. In aspects of this embodiment, a BAM22
peptide comprising an
altered targeting domain is derived from a BAM22 peptide (1-12), a BAM22
peptide (6-22), a BAM22
peptide (8-22) or a BAM22 peptide (1-22). In other aspects of this embodiment,
a BAM22 peptide
comprising an altered targeting domain comprises amino acids 1-12, amino acids
6-22, amino acids 8-22
or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177.
[0236] In other aspects of this embodiment, a peptide comprising an altered
targeting domain has, e.g.,
at least 70% amino acid identity with amino acids 1-12, amino acids 6-22,
amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-
22 of SEQ ID NO: 176;
or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 177, at least
75% amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-
22 or amino acids 1-22
of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ
ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 175;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 176; or amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 177, at least 80%
amino acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22
or amino acids 1-22 of
SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 173; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-
22 of SEQ ID NO: 174;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 175; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 176; or amino acids
1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:
177, at least 85% amino
acid identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ ID
NO: 172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-
22 of SEQ ID NO: 173;
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amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 174; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 175; amino acids 1-
12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 176;
or amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 177, at
least 90% amino acid
identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 173;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 174; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 175; amino acids 1-
12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 176;
or amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 177 or at
least 95% amino acid
identity with amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 173;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 174; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 175; amino acids 1-
12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 176;
or amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 177.
[0237] In yet other aspects of this embodiment, a peptide comprising an
altered targeting domain has,
e.g., at most 70% amino acid identity with amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
177, at most 75% amino acid identity with amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
177, at most 80% amino acid identity with amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
177, at most 85% amino acid identity with amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
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22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
177, at most 90% amino acid identity with amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
177 or at most 95% amino acid identity with amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
177.
[0238] In other aspects of this embodiment, a peptide comprising an altered
targeting domain has, e.g.,
at least one, two, three, four or five non-contiguous amino acid substitutions
relative to amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 172;
amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 173; amino
acids 1-12, amino acids 6-
22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 174; amino acids 1-12,
amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino
acids 6-22, amino acids 8-
22 or amino acids 1-22 of SEQ ID NO: 176; or amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 177. In other aspects of this embodiment, a
peptide comprising an
altered targeting domain has, e.g., at most one, two, three, four or five non-
contiguous amino acid
substitutions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22
or amino acids 1-22 of
SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 173; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-
22 of SEQ ID NO: 174;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 175; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 176; or amino acids
1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:
177. In yet other aspects
of this embodiment, a peptide comprising an altered targeting domain has,
e.g., at least one, two, three,
four or five non-contiguous amino acid deletions relative to amino acids 1-12,
amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino
acids 6-22, amino acids 8-
22 or amino acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22
or amino acids 1-22 of
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SEQ ID NO: 177. In yet other aspects of this embodiment, a peptide comprising
an altered targeting
domain has, e.g., at most one, two, three, four or five non-contiguous amino
acid deletions relative to
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 172; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 173; amino acids 1-
12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 174;
amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 175; amino
acids 1-12, amino acids 6-
22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 176; or amino acids 1-
12, amino acids 6-22,
amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 177. In still other aspects
of this embodiment, a
peptide comprising an altered targeting domain has, e.g., at least one, two,
three, four or five non-
contiguous amino acid additions relative to amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
177. In yet other aspects of this embodiment, a peptide comprising an altered
targeting domain has, e.g.,
at most one, two, three, four or five non-contiguous amino acid additions
relative to amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 172;
amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 173; amino
acids 1-12, amino acids 6-
22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 174; amino acids 1-12,
amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino
acids 6-22, amino acids 8-
22 or amino acids 1-22 of SEQ ID NO: 176; or amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 177.
[0239] In other aspects of this embodiment, a peptide comprising an altered
targeting domain has, e.g.,
at least one, two, three, four or five contiguous amino acid substitutions
relative to amino acids 1-12,
amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 172;
amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 173; amino
acids 1-12, amino acids 6-
22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 174; amino acids 1-12,
amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino
acids 6-22, amino acids 8-
22 or amino acids 1-22 of SEQ ID NO: 176; or amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 177. In other aspects of this embodiment, a
peptide comprising an
altered targeting domain has, e.g., at most one, two, three, four or five
contiguous amino acid
substitutions relative to amino acids 1-12, amino acids 6-22, amino acids 8-22
or amino acids 1-22 of
SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 173; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-
22 of SEQ ID NO: 174;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 175; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 176; or amino acids
1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:
177. In yet other aspects
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of this embodiment, a peptide comprising an altered targeting domain has,
e.g., at least one, two, three,
four or five contiguous amino acid deletions relative to amino acids 1-12,
amino acids 6-22, amino acids
8-22 or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of SEQ
ID NO: 177. In yet other aspects of this embodiment, a peptide comprising an
altered targeting domain
has, e.g., at most one, two, three, four or five contiguous amino acid
deletions relative to amino acids 1-
12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 172;
amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 173; amino
acids 1-12, amino acids 6-
22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 174; amino acids 1-12,
amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino
acids 6-22, amino acids 8-
22 or amino acids 1-22 of SEQ ID NO: 176; or amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 177. In still other aspects of this embodiment,
a peptide comprising an
altered targeting domain has, e.g., at least one, two, three, four or five
contiguous amino acid additions
relative to amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 172;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 173; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 174; amino acids 1-
12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 175;
amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 176; or amino
acids 1-12, amino acids
6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 177. In yet other
aspects of this embodiment,
a peptide comprising an altered targeting domain has, e.g., at most one, two,
three, four or five
contiguous amino acid additions relative to amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID NO:
177.
[0240] In another embodiment, an opioid peptide comprising an altered
targeting domain is an
endomorphin. In aspects of this embodiment, an endomorphin comprising an
altered targeting domain is
derived from an endomorphin-1 or an endomorphin-2. In other aspects of this
embodiment, an
endomorphin comprising an altered targeting domain is SEQ ID NO: 13 or SEQ ID
NO: 14.
[0241] In other aspects of this embodiment, an endomorphin comprising an
altered targeting domain
has, e.g., at least 70% amino acid identity with SEQ ID NO: 13 or SEQ ID NO:
14, at least 75% amino
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acid identity with SEQ ID NO: 13 or SEQ ID NO: 14, at least 80% amino acid
identity with SEQ ID NO: 13
or SEQ ID NO: 14, at least 85% amino acid identity with SEQ ID NO: 13 or SEQ
ID NO: 14, at least 90%
amino acid identity with SEQ ID NO: 13 or SEQ ID NO: 14 or at least 95% amino
acid identity with SEQ
ID NO: 13 or SEQ ID NO: 14. In yet other aspects of this embodiment, an
endomorphin comprising an
altered targeting domain has, e.g., at most 70% amino acid identity with SEQ
ID NO: 13 or SEQ ID NO:
14, at most 75% amino acid identity with SEQ ID NO: 13 or SEQ ID NO: 14, at
most 80% amino acid
identity with SEQ ID NO: 13 or SEQ ID NO: 14, at most 85% amino acid identity
with SEQ ID NO: 13 or
SEQ ID NO: 14, at most 90% amino acid identity with SEQ ID NO: 13 or SEQ ID
NO: 14 or at most 95%
amino acid identity with SEQ ID NO: 13 or SEQ ID NO: 14.
[0242] In other aspects of this embodiment, an endomorphin comprising an
altered targeting domain
has, e.g., at least one, two or three non-contiguous amino acid substitutions
relative to SEQ ID NO: 13 or
SEQ ID NO: 14. In other aspects of this embodiment, an endomorphin comprising
an altered targeting
domain has, e.g., at most one, two or three non-contiguous amino acid
substitutions relative to SEQ ID
NO: 13 or SEQ ID NO: 14. In yet other aspects of this embodiment, an
endomorphin comprising an
altered targeting domain has, e.g., at least one, two or three non-contiguous
amino acid deletions relative
to SEQ ID NO: 13 or SEQ ID NO: 14. In yet other aspects of this embodiment, an
endomorphin
comprising an altered targeting domain has, e.g., at most one, two or three
non-contiguous amino acid
deletions relative to SEQ ID NO: 13 or SEQ ID NO: 14. In still other aspects
of this embodiment, an
endomorphin comprising an altered targeting domain has, e.g., at least one,
two or three non-contiguous
amino acid additions relative to SEQ ID NO: 13 or SEQ ID NO: 14. In yet other
aspects of this
embodiment, an endomorphin comprising an altered targeting domain has, e.g.,
at most one, two or three
non-contiguous amino acid additions relative to SEQ ID NO: 13 or SEQ ID NO:
14.
[0243] In other aspects of this embodiment, an endomorphin comprising an
altered targeting domain
has, e.g., at least one, two or three contiguous amino acid substitutions
relative to SEQ ID NO: 13 or SEQ
ID NO: 14. In other aspects of this embodiment, an endomorphin comprising an
altered targeting domain
has, e.g., at most one, two or three contiguous amino acid substitutions
relative to SEQ ID NO: 13 or
SEQ ID NO: 14. In yet other aspects of this embodiment, an endomorphin
comprising an altered
targeting domain has, e.g., at least one, two or three contiguous amino acid
deletions relative to SEQ ID
NO: 13 or SEQ ID NO: 14. In yet other aspects of this embodiment, an
endomorphin comprising an
altered targeting domain has, e.g., at most one, two or three contiguous amino
acid deletions relative to
SEQ ID NO: 13 or SEQ ID NO: 14. In still other aspects of this embodiment, an
endomorphin comprising
an altered targeting domain has, e.g., at least one, two or three contiguous
amino acid additions relative
to SEQ ID NO: 13 or SEQ ID NO: 14. In yet other aspects of this embodiment, an
endomorphin
comprising an altered targeting domain has, e.g., at most one, two or three
contiguous amino acid
additions relative to SEQ ID NO: 13 or SEQ ID NO: 14.
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[0244] In another embodiment, an opioid peptide comprising an altered
targeting domain is an
endorphin. In aspects of this embodiment, an endorphin comprising an altered
targeting domain is
derived from an endorphin-a, a neoendorphin-a, an endorphin-(3, a neoendorphin-
(3 or an endorphin-y. In
other aspects of this embodiment, an enkephalin comprising an altered
targeting domain is SEQ ID NO:
15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:
20.
[0245] In other aspects of this embodiment, an endorphin comprising an altered
targeting domain has,
e.g., at least 70% amino acid identity with SEQ ID NO: 15, SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19 or SEQ ID NO: 20, at least 75% amino acid identity with SEQ
ID NO: 15, SEQ ID NO:
16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, at least 80%
amino acid identity
with SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19
or SEQ ID NO:
20, at least 85% amino acid identity with SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO: 17, SEQ ID NO:
18, SEQ ID NO: 19 or SEQ ID NO: 20, at least 90% amino acid identity with SEQ
ID NO: 15, SEQ ID NO:
16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 or at least
95% amino acid
identity with SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ
ID NO: 19 or SEQ ID
NO: 20. In yet other aspects of this embodiment, an endorphin comprising an
altered targeting domain
has, e.g., at most 70% amino acid identity with SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID
NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, at most 75% amino acid identity with
SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, at most
80% amino acid
identity with SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ
ID NO: 19 or SEQ ID
NO: 20, at most 85% amino acid identity with SEQ ID NO: 15, SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID
NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, at most 90% amino acid identity with
SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 or at
most 95% amino acid
identity with SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ
ID NO: 19 or SEQ ID
NO: 20.
[0246] In other aspects of this embodiment, an endorphin comprising an altered
targeting domain has,
e.g., at least one, two, three, four or five non-contiguous amino acid
substitutions relative to SEQ ID NO:
15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:
20. In other
aspects of this embodiment, an endorphin comprising an altered targeting
domain has, e.g., at most one,
two, three, four or five non-contiguous amino acid substitutions relative to
SEQ ID NO: 15, SEQ ID NO:
16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. In yet other
aspects of this
embodiment, an endorphin comprising an altered targeting domain has, e.g., at
least one, two, three, four
or five non-contiguous amino acid deletions relative to SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. In yet other aspects of this
embodiment, an
endorphin comprising an altered targeting domain has, e.g., at most one, two,
three, four or five non-
contiguous amino acid deletions relative to SEQ ID NO: 15, SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19 or SEQ ID NO: 20. In still other aspects of this embodiment,
an endorphin comprising
an altered targeting domain has, e.g., at least one, two, three, four or five
non-contiguous amino acid
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additions relative to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19 or
SEQ ID NO: 20. In yet other aspects of this embodiment, an endorphin
comprising an altered targeting
domain has, e.g., at most one, two, three, four or five non-contiguous amino
acid additions relative to
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or
SEQ ID NO: 20.
[0247] In other aspects of this embodiment, an endorphin comprising an altered
targeting domain has,
e.g., at least one, two, three, four or five contiguous amino acid
substitutions relative to SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
In other aspects of
this embodiment, an endorphin comprising an altered targeting domain has,
e.g., at most one, two, three,
four or five contiguous amino acid substitutions relative to SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO:
17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. In yet other aspects of
this embodiment, an
endorphin comprising an altered targeting domain has, e.g., at least one, two,
three, four or five
contiguous amino acid deletions relative to SEQ ID NO: 15, SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19 or SEQ ID NO: 20. In yet other aspects of this embodiment,
an endorphin comprising
an altered targeting domain has, e.g., at most one, two, three, four or five
contiguous amino acid
deletions relative to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19 or
SEQ ID NO: 20. In still other aspects of this embodiment, an endorphin
comprising an altered targeting
domain has, e.g., at least one, two, three, four or five contiguous amino acid
additions relative to SEQ ID
NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID
NO: 20. In yet
other aspects of this embodiment, an endorphin comprising an altered targeting
domain has, e.g., at most
one, two, three, four or five contiguous amino acid additions relative to SEQ
ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
[0248] In another embodiment, an opioid peptide comprising an altered
targeting domain is a dynorphin.
In aspects of this embodiment, a dynorphin comprising an altered targeting
domain is derived from a
dynorphin A, a dynorphin B (leumorphin) or a rimorphin. In other aspects of
this embodiment, a
dynorphin comprising an altered targeting domain is SEQ ID NO: 21, SEQ ID NO:
22, SEQ ID NO: 23,
SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ
ID NO: 29, SEQ
ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID
NO: 35, SEQ ID
NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:
41, SEQ ID NO:
42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47,
SEQ ID NO: 48,
SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51.
[0249] In other aspects of this embodiment, a dynorphin comprising an altered
targeting domain has,
e.g., at least 70% amino acid identity with SEQ ID NO: 21, SEQ ID NO: 30 or
SEQ ID NO: 46, at least
75% amino acid identity with SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46, at
least 80% amino acid
identity with SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46, at least 85%
amino acid identity with
SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46, at least 90% amino acid
identity with SEQ ID NO: 21,
SEQ ID NO: 30 or SEQ ID NO: 46 or at least 95% amino acid identity with SEQ ID
NO: 21, SEQ ID NO:
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30 or SEQ ID NO: 46. In yet other aspects of this embodiment, a dynorphin
comprising an altered
targeting domain has, e.g., at most 70% amino acid identity with SEQ ID NO:
21, SEQ ID NO: 30 or SEQ
ID NO: 46, at most 75% amino acid identity with SEQ ID NO: 21, SEQ ID NO: 30
or SEQ ID NO: 46, at
most 80% amino acid identity with SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO:
46, at most 85%
amino acid identity with SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46, at
most 90% amino acid
identity with SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46 or at most 95%
amino acid identity with
SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46.
[0250] In other aspects of this embodiment, a dynorphin comprising an altered
targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid
substitutions relative to SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46. In
other aspects of this
embodiment, a dynorphin comprising an altered targeting domain has, e.g., at
most one, two, three, four,
five, six, seven, eight, nine or ten non-contiguous amino acid substitutions
relative to SEQ ID NO: 21,
SEQ ID NO: 30 or SEQ ID NO: 46. In yet other aspects of this embodiment, a
dynorphin comprising an
altered targeting domain has, e.g., at least one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid deletions relative to SEQ ID NO: 21, SEQ ID NO: 30 or
SEQ ID NO: 46. In yet
other aspects of this embodiment, a dynorphin comprising an altered targeting
domain has, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten non-contiguous
amino acid deletions relative to
SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46. In still other aspects of this
embodiment, a dynorphin
comprising an altered targeting domain has, e.g., at least one, two, three,
four, five, six, seven, eight, nine
or ten non-contiguous amino acid additions relative to SEQ ID NO: 21, SEQ ID
NO: 30 or SEQ ID NO: 46.
In yet other aspects of this embodiment, a dynorphin comprising an altered
targeting domain has, e.g., at
most one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid additions relative
to SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46.
[0251] In other aspects of this embodiment, a dynorphin comprising an altered
targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions
relative to SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46. In other aspects of
this embodiment, a
dynorphin comprising an altered targeting domain has, e.g., at most one, two,
three, four, five, six, seven,
eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO:
21, SEQ ID NO: 30 or SEQ
ID NO: 46. In yet other aspects of this embodiment, a dynorphin comprising an
altered targeting domain
has, e.g., at least one, two, three, four, five, six, seven, eight, nine or
ten contiguous amino acid deletions
relative to SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46. In yet other
aspects of this embodiment, a
dynorphin comprising an altered targeting domain has, e.g., at most one, two,
three, four, five, six, seven,
eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 21,
SEQ ID NO: 30 or SEQ ID
NO: 46. In still other aspects of this embodiment, a dynorphin comprising an
altered targeting domain
has, e.g., at least one, two, three, four, five, six, seven, eight, nine or
ten contiguous amino acid additions
relative to SEQ ID NO: 21, SEQ ID NO: 30 or SEQ ID NO: 46. In yet other
aspects of this embodiment, a
dynorphin comprising an altered targeting domain has, e.g., at most one, two,
three, four, five, six, seven,
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eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 21,
SEQ ID NO: 30 or SEQ ID
NO: 46.
[0252] In another embodiment, an opioid peptide comprising an altered
targeting domain is a nociceptin.
In aspects of this embodiment, a nociceptin comprising an altered targeting
domain is derived from a
nociceptin RK, a nociceptin, a neuropeptide 1, a neuropeptide 2 or a
neuropeptide 3. In other aspects of
this embodiment, a nociceptin comprising an altered targeting domain is SEQ ID
NO: 52, SEQ ID NO: 53,
SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ
ID NO: 59, SEQ
ID NO: 60 or SEQ ID NO: 61.
[0253] In other aspects of this embodiment, a nociceptin comprising an altered
targeting domain has,
e.g., at least 70% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59, SEQ
ID NO: 60 or SEQ ID
NO: 61, at least 75% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ ID
NO: 61, at least 80% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ ID
NO: 61, at least 85% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ ID
NO: 61, at least 90% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ ID
NO: 61 or at least 95% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ
ID NO: 61. In yet other aspects of this embodiment, a nociceptin comprising an
altered targeting domain
has, e.g., at most 70% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ
ID NO: 61, at most 75% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ
ID NO: 61, at most 80% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ
ID NO: 61, at most 85% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ
ID NO: 61, at most 90% amino acid identity with SEQ ID NO: 52, SEQ ID NO: 59,
SEQ ID NO: 60 or SEQ
ID NO: 61 or at most 95% amino acid identity with SEQ ID NO: 52, SEQ ID NO:
59, SEQ ID NO: 60 or
SEQ ID NO: 61.
[0254] In other aspects of this embodiment, a nociceptin comprising an altered
targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid
substitutions relative to SEQ ID NO: 52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ
ID NO: 61. In other
aspects of this embodiment, a nociceptin comprising an altered targeting
domain has, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten non-contiguous amino
acid substitutions relative to
SEQ ID NO: 52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61. In yet other
aspects of this
embodiment, a nociceptin comprising an altered targeting domain has, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or ten non-contiguous amino acid deletions
relative to SEQ ID NO: 52, SEQ ID
NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61. In yet other aspects of this
embodiment, a nociceptin
comprising an altered targeting domain has, e.g., at most one, two, three,
four, five, six, seven, eight, nine
or ten non-contiguous amino acid deletions relative to SEQ ID NO: 52, SEQ ID
NO: 59, SEQ ID NO: 60 or
SEQ ID NO: 61. In still other aspects of this embodiment, a nociceptin
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten non-contiguous amino
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acid additions relative to SEQ ID NO: 52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ
ID NO: 61. In yet other
aspects of this embodiment, a nociceptin comprising an altered targeting
domain has, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten non-contiguous amino
acid additions relative to SEQ ID
NO: 52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61.
[0255] In other aspects of this embodiment, a nociceptin comprising an altered
targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions
relative to SEQ ID NO: 52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61. In
other aspects of this
embodiment, a nociceptin comprising an altered targeting domain has, e.g., at
most one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid substitutions
relative to SEQ ID NO: 52, SEQ ID
NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61. In yet other aspects of this
embodiment, a nociceptin
comprising an altered targeting domain has, e.g., at least one, two, three,
four, five, six, seven, eight, nine
or ten contiguous amino acid deletions relative to SEQ ID NO: 52, SEQ ID NO:
59, SEQ ID NO: 60 or
SEQ ID NO: 61. In yet other aspects of this embodiment, a nociceptin
comprising an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten contiguous amino acid
deletions relative to SEQ ID NO: 52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID
NO: 61. In still other
aspects of this embodiment, a nociceptin comprising an altered targeting
domain has, e.g., at least one,
two, three, four, five, six, seven, eight, nine or ten contiguous amino acid
additions relative to SEQ ID NO:
52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61. In yet other aspects of
this embodiment, a
nociceptin comprising an altered targeting domain has, e.g., at most one, two,
three, four, five, six, seven,
eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 52,
SEQ ID NO: 59, SEQ ID
NO: 60 or SEQ ID NO: 61.
[0256] Another example of an altered targeting domain disclosed in the present
specification is, e.g., a
melanocortin peptide, such as, e.g., a melanocyte stimulating hormone, an
adrenocorticotropin, a
Corticotropin-like intermediary peptide) or a lipotropin. Thus, in an
embodiment, an altered targeting
domain is derived from a melanocortin peptide.
[0257] In another embodiment, a melanocortin peptide comprising an altered
targeting domain is a
melanocyte stimulating hormone. In aspects of this embodiment, a melanocyte
stimulating hormone
comprising an altered targeting domain is derived from an a-melanocyte
stimulating hormones (a-MSH), a
(3-melanocyte stimulating hormones ((3-MSH), a y-melanocyte stimulating
hormones (y-MSH). In other
aspects of this embodiment, a melanocyte stimulating hormone comprising an
altered targeting domain is
SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64.
[0258] In other aspects of this embodiment, a melanocyte stimulating hormone
comprising an altered
targeting domain has, e.g., at least 70% amino acid identity with SEQ ID NO:
62, SEQ ID NO: 63 or SEQ
ID NO: 64, at least 75% amino acid identity with SEQ ID NO: 62, SEQ ID NO: 63
or SEQ ID NO: 64, at
least 80% amino acid identity with SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO:
64, at least 85%
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amino acid identity with SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64, at
least 90% amino acid
identity with SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64 or at least 95%
amino acid identity with
SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64. In yet other aspects of this
embodiment, a
melanocyte stimulating hormone comprising an altered targeting domain has,
e.g., at most 70% amino
acid identity with SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64, at most 75%
amino acid identity
with SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64, at most 80% amino acid
identity with SEQ ID
NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64, at most 85% amino acid identity with
SEQ ID NO: 62, SEQ ID
NO: 63 or SEQ ID NO: 64, at most 90% amino acid identity with SEQ ID NO: 62,
SEQ ID NO: 63 or SEQ
ID NO: 64 or at most 95% amino acid identity with SEQ ID NO: 62, SEQ ID NO: 63
or SEQ ID NO: 64.
[0259] In other aspects of this embodiment, a melanocyte stimulating hormone
comprising an altered
targeting domain has, e.g., at least one, two, three, four or five non-
contiguous amino acid substitutions
relative to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64. In other aspects of
this embodiment, a
melanocyte stimulating hormone comprising an altered targeting domain has,
e.g., at most one, two,
three, four or five non-contiguous amino acid substitutions relative to SEQ ID
NO: 62, SEQ ID NO: 63 or
SEQ ID NO: 64. In yet other aspects of this embodiment, a melanocyte
stimulating hormone comprising
an altered targeting domain has, e.g., at least one, two, three, four or five
non-contiguous amino acid
deletions relative to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64. In yet
other aspects of this
embodiment, a melanocyte stimulating hormone comprising an altered targeting
domain has, e.g., at
most one, two, three, four or five non-contiguous amino acid deletions
relative to SEQ ID NO: 62, SEQ ID
NO: 63 or SEQ ID NO: 64. In still other aspects of this embodiment, a
melanocyte stimulating hormone
comprising an altered targeting domain has, e.g., at least one, two, three,
four or five non-contiguous
amino acid additions relative to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO:
64. In yet other aspects
of this embodiment, a melanocyte stimulating hormone comprising an altered
targeting domain has, e.g.,
at most one, two, three, four or five non-contiguous amino acid additions
relative to SEQ ID NO: 62, SEQ
ID NO: 63 or SEQ ID NO: 64.
[0260] In other aspects of this embodiment, a melanocyte stimulating hormone
comprising an altered
targeting domain has, e.g., at least one, two, three, four or five contiguous
amino acid substitutions
relative to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64. In other aspects of
this embodiment, a
melanocyte stimulating hormone comprising an altered targeting domain has,
e.g., at most one, two,
three, four or five contiguous amino acid substitutions relative to SEQ ID NO:
62, SEQ ID NO: 63 or SEQ
ID NO: 64. In yet other aspects of this embodiment, a melanocyte stimulating
hormone comprising an
altered targeting domain has, e.g., at least one, two, three, four or five
contiguous amino acid deletions
relative to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64. In yet other
aspects of this embodiment, a
melanocyte stimulating hormone comprising an altered targeting domain has,
e.g., at most one, two,
three, four or five contiguous amino acid deletions relative to SEQ ID NO: 62,
SEQ ID NO: 63 or SEQ ID
NO: 64. In still other aspects of this embodiment, a melanocyte stimulating
hormone comprising an
altered targeting domain has, e.g., at least one, two, three, four or five
contiguous amino acid additions
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relative to SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO: 64. In yet other
aspects of this embodiment, a
melanocyte stimulating hormone comprising an altered targeting domain has,
e.g., at most one, two,
three, four or five contiguous amino acid additions relative to SEQ ID NO: 62,
SEQ ID NO: 63 or SEQ ID
NO: 64.
[0261] In another embodiment, a melanocortin peptide comprising an altered
targeting domain is an
adrenocorticotropin. In aspects of this embodiment, an adrenocorticotropin
comprising an altered
targeting domain is derived from an adrenocorticotropin (ACTH) or a
Corticotropin-like intermediary
peptide (CLIP). In other aspects of this embodiment, an adrenocorticotropin
comprising an altered
targeting domain is SEQ ID NO: 65 or SEQ ID NO: 66.
[0262] In other aspects of this embodiment, an adrenocorticotropin comprising
an altered targeting
domain has, e.g., at least 70% amino acid identity with SEQ ID NO: 65 or SEQ
ID NO: 66, at least 75%
amino acid identity with SEQ ID NO: 65 or SEQ ID NO: 66, at least 80% amino
acid identity with SEQ ID
NO: 65 or SEQ ID NO: 66, at least 85% amino acid identity with SEQ ID NO: 65
or SEQ ID NO: 66, at
least 90% amino acid identity with SEQ ID NO: 65 or SEQ ID NO: 66 or at least
95% amino acid identity
with SEQ ID NO: 65 or SEQ ID NO: 66. In yet other aspects of this embodiment,
an adrenocorticotropin
comprising an altered targeting domain has, e.g., at most 70% amino acid
identity with SEQ ID NO: 65 or
SEQ ID NO: 66, at most 75% amino acid identity with SEQ ID NO: 65 or SEQ ID
NO: 66, at most 80%
amino acid identity with SEQ ID NO: 65 or SEQ ID NO: 66, at most 85% amino
acid identity with SEQ ID
NO: 65 or SEQ ID NO: 66, at most 90% amino acid identity with SEQ ID NO: 65 or
SEQ ID NO: 66 or at
most 95% amino acid identity with SEQ ID NO: 65 or SEQ ID NO: 66.
[0263] In other aspects of this embodiment, an adrenocorticotropin comprising
an altered targeting
domain has, e.g., at least one, two, three, four or five non-contiguous amino
acid substitutions relative to
SEQ ID NO: 65 or SEQ ID NO: 66. In other aspects of this embodiment, an
adrenocorticotropin
comprising an altered targeting domain has, e.g., at most one, two, three,
four or five non-contiguous
amino acid substitutions relative to SEQ ID NO: 65 or SEQ ID NO: 66. In yet
other aspects of this
embodiment, an adrenocorticotropin comprising an altered targeting domain has,
e.g., at least one, two,
three, four or five non-contiguous amino acid deletions relative to SEQ ID NO:
65 or SEQ ID NO: 66. In
yet other aspects of this embodiment, an adrenocorticotropin comprising an
altered targeting domain has,
e.g., at most one, two, three, four or five non-contiguous amino acid
deletions relative to SEQ ID NO: 65
or SEQ ID NO: 66. In still other aspects of this embodiment, an
adrenocorticotropin comprising an altered
targeting domain has, e.g., at least one, two, three, four or five non-
contiguous amino acid additions
relative to SEQ ID NO: 65 or SEQ ID NO: 66. In yet other aspects of this
embodiment, an
adrenocorticotropin comprising an altered targeting domain has, e.g., at most
one, two, three, four or five
non-contiguous amino acid additions relative to SEQ ID NO: 65 or SEQ ID NO:
66.
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[0264] In other aspects of this embodiment, an adrenocorticotropin comprising
an altered targeting
domain has, e.g., at least one, two, three, four or five contiguous amino acid
substitutions relative to SEQ
ID NO: 65 or SEQ ID NO: 66. In other aspects of this embodiment, an
adrenocorticotropin comprising an
altered targeting domain has, e.g., at most one, two, three, four or five
contiguous amino acid
substitutions relative to SEQ ID NO: 65 or SEQ ID NO: 66. In yet other aspects
of this embodiment, an
adrenocorticotropin comprising an altered targeting domain has, e.g., at least
one, two, three, four or five
contiguous amino acid deletions relative to SEQ ID NO: 65 or SEQ ID NO: 66. In
yet other aspects of this
embodiment, an adrenocorticotropin comprising an altered targeting domain has,
e.g., at most one, two,
three, four or five contiguous amino acid deletions relative to SEQ ID NO: 65
or SEQ ID NO: 66. In still
other aspects of this embodiment, an adrenocorticotropin comprising an altered
targeting domain has,
e.g., at least one, two, three, four or five contiguous amino acid additions
relative to SEQ ID NO: 65 or
SEQ ID NO: 66. In yet other aspects of this embodiment, an adrenocorticotropin
comprising an altered
targeting domain has, e.g., at most one, two, three, four or five contiguous
amino acid additions relative to
SEQ ID NO: 65 or SEQ ID NO: 66.
[0265] In another embodiment, a melanocortin peptide comprising an altered
targeting domain is a
lipotropin. In aspects of this embodiment, a lipotropin comprising an altered
targeting domain is derived
from a(3-lipotropin ((3-LPH) or a y-lipotropin (y-LPH). In other aspects of
this embodiment, a lipotropin
comprising an altered targeting domain is SEQ ID NO: 67 or SEQ ID NO: 68.
[0266] In other aspects of this embodiment, a lipotropin comprising an altered
targeting domain has,
e.g., at least 70% amino acid identity with SEQ ID NO: 67 or SEQ ID NO: 68, at
least 75% amino acid
identity with SEQ ID NO: 67 or SEQ ID NO: 68, at least 80% amino acid identity
with SEQ ID NO: 67 or
SEQ ID NO: 68, at least 85% amino acid identity with SEQ ID NO: 67 or SEQ ID
NO: 68, at least 90%
amino acid identity with SEQ ID NO: 67 or SEQ ID NO: 68 or at least 95% amino
acid identity with SEQ
ID NO: 67 or SEQ ID NO: 68. In yet other aspects of this embodiment, a
lipotropin comprising an altered
targeting domain has, e.g., at most 70% amino acid identity with SEQ ID NO: 67
or SEQ ID NO: 68, at
most 75% amino acid identity with SEQ ID NO: 67 or SEQ ID NO: 68, at most 80%
amino acid identity
with SEQ ID NO: 67 or SEQ ID NO: 68, at most 85% amino acid identity with SEQ
ID NO: 67 or SEQ ID
NO: 68, at most 90% amino acid identity with SEQ ID NO: 67 or SEQ ID NO: 68 or
at most 95% amino
acid identity with SEQ ID NO: 67 or SEQ ID NO: 68.
[0267] In other aspects of this embodiment, a lipotropin comprising an altered
targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid
substitutions relative to SEQ ID NO: 67 or SEQ ID NO: 68. In other aspects of
this embodiment, a
lipotropin comprising an altered targeting domain has, e.g., at most one, two,
three, four, five, six, seven,
eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID
NO: 67 or SEQ ID NO: 68.
In yet other aspects of this embodiment, a lipotropin comprising an altered
targeting domain has, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid deletions relative
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to SEQ ID NO: 67 or SEQ ID NO: 68. In yet other aspects of this embodiment, a
lipotropin comprising an
altered targeting domain has, e.g., at most one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid deletions relative to SEQ ID NO: 67 or SEQ ID NO: 68. In
still other aspects of
this embodiment, a lipotropin comprising an altered targeting domain has,
e.g., at least one, two, three,
four, five, six, seven, eight, nine or ten non-contiguous amino acid additions
relative to SEQ ID NO: 67 or
SEQ ID NO: 68. In yet other aspects of this embodiment, a lipotropin
comprising an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten non-contiguous amino
acid additions relative to SEQ ID NO: 67 or SEQ ID NO: 68.
[0268] In other aspects of this embodiment, a lipotropin comprising an altered
targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions
relative to SEQ ID NO: 67 or SEQ ID NO: 68. In other aspects of this
embodiment, a lipotropin
comprising an altered targeting domain has, e.g., at most one, two, three,
four, five, six, seven, eight, nine
or ten contiguous amino acid substitutions relative to SEQ ID NO: 67 or SEQ ID
NO: 68. In yet other
aspects of this embodiment, a lipotropin comprising an altered targeting
domain has, e.g., at least one,
two, three, four, five, six, seven, eight, nine or ten contiguous amino acid
deletions relative to SEQ ID NO:
67 or SEQ ID NO: 68. In yet other aspects of this embodiment, a lipotropin
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid deletions relative to SEQ ID NO: 67 or SEQ ID NO: 68. In still
other aspects of this
embodiment, a lipotropin comprising an altered targeting domain has, e.g., at
least one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid additions relative
to SEQ ID NO: 67 or SEQ ID
NO: 68. In yet other aspects of this embodiment, a lipotropin comprising an
altered targeting domain has,
e.g., at most one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid additions
relative to SEQ ID NO: 67 or SEQ ID NO: 68.
[0269] In another embodiment, a melanocortin peptide comprising an altered
targeting domain is a
neuropeptide derived from a melanocortin peptide. In aspects of this
embodiment, a melanocortin
peptide derived neuropeptide comprising an altered targeting domain is SEQ ID
NO: 69, SEQ ID NO: 70
or SEQ ID NO: 71.
[0270] In other aspects of this embodiment, a melanocortin peptide derived
neuropeptide comprising an
altered targeting domain has, e.g., at least 70% amino acid identity with SEQ
ID NO: 69, SEQ ID NO: 70
or SEQ ID NO: 71, at least 75% amino acid identity with SEQ ID NO: 69, SEQ ID
NO: 70 or SEQ ID NO:
71, at least 80% amino acid identity with SEQ ID NO: 69, SEQ ID NO: 70 or SEQ
ID NO: 71, at least 85%
amino acid identity with SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, at
least 90% amino acid
identity with SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71 or at least 95%
amino acid identity with
SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In yet other aspects of this
embodiment, a
melanocortin peptide derived neuropeptide comprising an altered targeting
domain has, e.g., at most 70%
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amino acid identity with SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, at
most 75% amino acid
identity with SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, at most 80% amino
acid identity with
SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, at most 85% amino acid identity
with SEQ ID NO: 69,
SEQ ID NO: 70 or SEQ ID NO: 71, at most 90% amino acid identity with SEQ ID
NO: 69, SEQ ID NO: 70
or SEQ ID NO: 71 or at most 95% amino acid identity with SEQ ID NO: 69, SEQ ID
NO: 70 or SEQ ID
NO: 71.
[0271] In other aspects of this embodiment, a melanocortin peptide derived
neuropeptide comprising an
altered targeting domain has, e.g., at least one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid substitutions relative to SEQ ID NO: 69, SEQ ID NO: 70
or SEQ ID NO: 71. In
other aspects of this embodiment, a melanocortin peptide derived neuropeptide
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten non-
contiguous amino acid substitutions relative to SEQ ID NO: 69, SEQ ID NO: 70
or SEQ ID NO: 71. In yet
other aspects of this embodiment, a melanocortin peptide derived neuropeptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten non-contiguous
amino acid deletions relative to SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO:
71. In yet other aspects
of this embodiment, a melanocortin peptide derived neuropeptide comprising an
altered targeting domain
has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid
deletions relative to SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In still
other aspects of this
embodiment, a melanocortin peptide derived neuropeptide comprising an altered
targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid additions
relative to SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In yet other
aspects of this embodiment, a
melanocortin peptide derived neuropeptide comprising an altered targeting
domain has, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten non-contiguous amino
acid additions relative to SEQ ID
NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.
[0272] In other aspects of this embodiment, a melanocortin peptide derived
neuropeptide comprising an
altered targeting domain has, e.g., at least one, two, three, four, five, six,
seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 69, SEQ ID NO: 70
or SEQ ID NO: 71. In
other aspects of this embodiment, a melanocortin peptide derived neuropeptide
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid substitutions relative to SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID
NO: 71. In yet other
aspects of this embodiment, a melanocortin peptide derived neuropeptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten contiguous amino acid
deletions relative to SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In yet
other aspects of this
embodiment, a melanocortin peptide derived neuropeptide comprising an altered
targeting domain has,
e.g., at most one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid deletions
relative to SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In still other
aspects of this embodiment, a
melanocortin peptide derived neuropeptide comprising an altered targeting
domain has, e.g., at least one,
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two, three, four, five, six, seven, eight, nine or ten contiguous amino acid
additions relative to SEQ ID NO:
69, SEQ ID NO: 70 or SEQ ID NO: 71. In yet other aspects of this embodiment, a
melanocortin peptide
derived neuropeptide comprising an altered targeting domain has, e.g., at most
one, two, three, four, five,
six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ
ID NO: 69, SEQ ID NO: 70
or SEQ ID NO: 71.
[0273] In another embodiment, a peptide comprising an altered targeting domain
is a galanin. In
aspects of this embodiment, a galanin comprising an altered targeting domain
is derived from a galanin or
a galanin message-associated peptide (GMAP). In other aspects of this
embodiment, a galanin
comprising an altered targeting domain is SEQ ID NO: 72 or SEQ ID NO: 73.
[0274] In other aspects of this embodiment, a galanin comprising an altered
targeting domain has, e.g.,
at least 70% amino acid identity with SEQ ID NO: 72 or SEQ ID NO: 73, at least
75% amino acid identity
with SEQ ID NO: 72 or SEQ ID NO: 73, at least 80% amino acid identity with SEQ
ID NO: 72 or SEQ ID
NO: 73, at least 85% amino acid identity with SEQ ID NO: 72 or SEQ ID NO: 73,
at least 90% amino acid
identity with SEQ ID NO: 72 or SEQ ID NO: 73 or at least 95% amino acid
identity with SEQ ID NO: 72 or
SEQ ID NO: 73. In yet other aspects of this embodiment, a galanin comprising
an altered targeting
domain has, e.g., at most 70% amino acid identity with SEQ ID NO: 72 or SEQ ID
NO: 73, at most 75%
amino acid identity with SEQ ID NO: 72 or SEQ ID NO: 73, at most 80% amino
acid identity with SEQ ID
NO: 72 or SEQ ID NO: 73, at most 85% amino acid identity with SEQ ID NO: 72 or
SEQ ID NO: 73, at
most 90% amino acid identity with SEQ ID NO: 72 or SEQ ID NO: 73 or at most
95% amino acid identity
with SEQ ID NO: 72 or SEQ ID NO: 73.
[0275] In other aspects of this embodiment, a galanin comprising an altered
targeting domain has, e.g.,
at least one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid substitutions
relative to SEQ ID NO: 72 or SEQ ID NO: 73. In other aspects of this
embodiment, a galanin comprising
an altered targeting domain has, e.g., at most one, two, three, four, five,
six, seven, eight, nine or ten non-
contiguous amino acid substitutions relative to SEQ ID NO: 72 or SEQ ID NO:
73. In yet other aspects of
this embodiment, a galanin comprising an altered targeting domain has, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions
relative to SEQ ID NO: 72 or
SEQ ID NO: 73. In yet other aspects of this embodiment, a galanin comprising
an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten non-contiguous amino
acid deletions relative to SEQ ID NO: 72 or SEQ ID NO: 73. In still other
aspects of this embodiment, a
galanin comprising an altered targeting domain has, e.g., at least one, two,
three, four, five, six, seven,
eight, nine or ten non-contiguous amino acid additions relative to SEQ ID NO:
72 or SEQ ID NO: 73. In
yet other aspects of this embodiment, a galanin comprising an altered
targeting domain has, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten non-contiguous
amino acid additions relative to
SEQ ID NO: 72 or SEQ ID NO: 73.
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[0276] In other aspects of this embodiment, a galanin comprising an altered
targeting domain has, e.g.,
at least one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions
relative to SEQ ID NO: 72 or SEQ ID NO: 73. In other aspects of this
embodiment, a galanin comprising
an altered targeting domain has, e.g., at most one, two, three, four, five,
six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 72 or SEQ ID NO:
73. In yet other aspects of
this embodiment, a galanin comprising an altered targeting domain has, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine or ten contiguous amino acid deletions
relative to SEQ ID NO: 72 or SEQ
ID NO: 73. In yet other aspects of this embodiment, a galanin comprising an
altered targeting domain
has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid deletions
relative to SEQ ID NO: 72 or SEQ ID NO: 73. In still other aspects of this
embodiment, a galanin
comprising an altered targeting domain has, e.g., at least one, two, three,
four, five, six, seven, eight, nine
or ten contiguous amino acid additions relative to SEQ ID NO: 72 or SEQ ID NO:
73. In yet other aspects
of this embodiment, a galanin comprising an altered targeting domain has,
e.g., at most one, two, three,
four, five, six, seven, eight, nine or ten contiguous amino acid additions
relative to SEQ ID NO: 72 or SEQ
ID NO: 73.
[0277] Another example of an altered targeting domain disclosed in the present
specification is, e.g., a
granin peptide, such as, e.g., a chromogranin A, a chromogranin B
(secretogranin I) or a chromogranin C
(secretogranin II). Thus, in an embodiment, an altered targeting domain is
derived from a granin peptide.
[0278] In another embodiment, a granin peptide comprising an altered targeting
domain is a
chromogranin A peptide. In aspects of this embodiment, a chromogranin A
peptide comprising an altered
targeting domain is derived from a(3-granin, a vasostatin, a chromostatin, a
pancreastatin, a WE-14, a
catestatin, a parastatin or a GE-25. In other aspects of this embodiment, a
chromogranin A peptide
comprising an altered targeting domain is SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID
NO: 76, SEQ ID NO:
77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81.
[0279] In other aspects of this embodiment, a chromogranin A peptide
comprising an altered targeting
domain has, e.g., at least 70% amino acid identity with SEQ ID NO: 74, SEQ ID
NO: 75, SEQ ID NO: 76,
SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81,
at least 75% amino
acid identity with SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,
SEQ ID NO: 78, SEQ
ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81, at least 80% amino acid identity
with SEQ ID NO: 74, SEQ
ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID
NO: 80 or SEQ ID
NO: 81, at least 85% amino acid identity with SEQ ID NO: 74, SEQ ID NO: 75,
SEQ ID NO: 76, SEQ ID
NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81, at least
90% amino acid
identity with SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ
ID NO: 78, SEQ ID
NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81 or at least 95% amino acid identity
with SEQ ID NO: 74, SEQ
ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID
NO: 80 or SEQ ID
NO: 81. In yet other aspects of this embodiment, a chromogranin A peptide
comprising an altered
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targeting domain has, e.g., at most 70% amino acid identity with SEQ ID NO:
74, SEQ ID NO: 75, SEQ ID
NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID
NO: 81, at most
75% amino acid identity with SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ
ID NO: 77, SEQ ID
NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81, at most 80% amino acid
identity with SEQ ID
NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:
79, SEQ ID NO:
80 or SEQ ID NO: 81, at most 85% amino acid identity with SEQ ID NO: 74, SEQ
ID NO: 75, SEQ ID NO:
76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO:
81, at most 90%
amino acid identity with SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID
NO: 77, SEQ ID NO:
78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81 or at most 95% amino acid
identity with SEQ ID
NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:
79, SEQ ID NO:
80 or SEQ ID NO: 81.
[0280] In other aspects of this embodiment, a chromogranin A peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten non-contiguous amino
acid substitutions relative to SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76,
SEQ ID NO: 77, SEQ ID
NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81. In other aspects of
this embodiment, a
chromogranin A peptide comprising an altered targeting domain has, e.g., at
most one, two, three, four,
five, six, seven, eight, nine or ten non-contiguous amino acid substitutions
relative to SEQ ID NO: 74,
SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ
ID NO: 80 or
SEQ ID NO: 81. In yet other aspects of this embodiment, a chromogranin A
peptide comprising an
altered targeting domain has, e.g., at least one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid deletions relative to SEQ ID NO: 74, SEQ ID NO: 75, SEQ
ID NO: 76, SEQ ID NO:
77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81. In yet other
aspects of this
embodiment, a chromogranin A peptide comprising an altered targeting domain
has, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten non-contiguous amino
acid deletions relative to SEQ ID
NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:
79, SEQ ID NO:
80 or SEQ ID NO: 81. In still other aspects of this embodiment, a chromogranin
A peptide comprising an
altered targeting domain has, e.g., at least one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid additions relative to SEQ ID NO: 74, SEQ ID NO: 75, SEQ
ID NO: 76, SEQ ID NO:
77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81. In yet other
aspects of this
embodiment, a chromogranin A peptide comprising an altered targeting domain
has, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten non-contiguous amino
acid additions relative to SEQ ID
NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:
79, SEQ ID NO:
80 or SEQ ID NO: 81.
[0281] In other aspects of this embodiment, a chromogranin A peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten contiguous amino acid
substitutions relative to SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID
NO: 77, SEQ ID NO:
78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81. In other aspects of this
embodiment, a
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chromogranin A peptide comprising an altered targeting domain has, e.g., at
most one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid substitutions
relative to SEQ ID NO: 74, SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO:
80 or SEQ ID
NO: 81. In yet other aspects of this embodiment, a chromogranin A peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid deletions relative to SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76,
SEQ ID NO: 77, SEQ
ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81. In yet other aspects
of this embodiment,
a chromogranin A peptide comprising an altered targeting domain has, e.g., at
most one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid deletions relative
to SEQ ID NO: 74, SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO:
80 or SEQ ID
NO: 81. In still other aspects of this embodiment, a chromogranin A peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid additions relative to SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76,
SEQ ID NO: 77, SEQ
ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81. In yet other aspects
of this embodiment,
a chromogranin A peptide comprising an altered targeting domain has, e.g., at
most one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid additions relative
to SEQ ID NO: 74, SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO:
80 or SEQ ID
NO: 81.
[0282] In another embodiment, a granin peptide comprising an altered targeting
domain is a
chromogranin B peptide. In aspects of this embodiment, a chromogranin B
peptide comprising an altered
targeting domain is derived from a GAWK peptide, an adrenomedullary peptide or
a secretolytin. In other
aspects of this embodiment, a chromogranin B peptide comprising an altered
targeting domain is SEQ ID
NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
[0283] In other aspects of this embodiment, a chromogranin B peptide
comprising an altered targeting
domain has, e.g., at least 70% amino acid identity with SEQ ID NO: 82, SEQ ID
NO: 83, SEQ ID NO: 84,
SEQ ID NO: 85 or SEQ ID NO: 86, at least 75% amino acid identity with SEQ ID
NO: 82, SEQ ID NO: 83,
SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, at least 80% amino acid
identity with SEQ ID NO: 82,
SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, at least 85%
amino acid identity with
SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86,
at least 90% amino
acid identity with SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85
or SEQ ID NO: 86 or
at least 95% amino acid identity with SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO:
84, SEQ ID NO: 85 or
SEQ ID NO: 86. In yet other aspects of this embodiment, a chromogranin B
peptide comprising an
altered targeting domain has, e.g., at most 70% amino acid identity with SEQ
ID NO: 82, SEQ ID NO: 83,
SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, at most 75% amino acid identity
with SEQ ID NO: 82,
SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, at most 80%
amino acid identity with
SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86,
at most 85% amino
acid identity with SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85
or SEQ ID NO: 86, at
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most 90% amino acid identity with SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,
SEQ ID NO: 85 or
SEQ ID NO: 86 or at most 95% amino acid identity with SEQ ID NO: 82, SEQ ID
NO: 83, SEQ ID NO: 84,
SEQ ID NO: 85 or SEQ ID NO: 86.
[0284] In other aspects of this embodiment, a chromogranin B peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten non-contiguous amino
acid substitutions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,
SEQ ID NO: 85 or SEQ ID
NO: 86. In other aspects of this embodiment, a chromogranin B peptide
comprising an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten non-contiguous amino
acid substitutions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,
SEQ ID NO: 85 or SEQ ID
NO: 86. In yet other aspects of this embodiment, a chromogranin B peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten non-contiguous
amino acid deletions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,
SEQ ID NO: 85 or SEQ
ID NO: 86. In yet other aspects of this embodiment, a chromogranin B peptide
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten non-
contiguous amino acid deletions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ
ID NO: 84, SEQ ID NO:
85 or SEQ ID NO: 86. In still other aspects of this embodiment, a chromogranin
B peptide comprising an
altered targeting domain has, e.g., at least one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid additions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ
ID NO: 84, SEQ ID NO:
85 or SEQ ID NO: 86. In yet other aspects of this embodiment, a chromogranin B
peptide comprising an
altered targeting domain has, e.g., at most one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid additions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ
ID NO: 84, SEQ ID NO:
85 or SEQ ID NO: 86.
[0285] In other aspects of this embodiment, a chromogranin B peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten contiguous amino acid
substitutions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID
NO: 85 or SEQ ID NO:
86. In other aspects of this embodiment, a chromogranin B peptide comprising
an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten contiguous amino acid
substitutions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID
NO: 85 or SEQ ID NO:
86. In yet other aspects of this embodiment, a chromogranin B peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten contiguous amino acid
deletions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:
85 or SEQ ID NO: 86.
In yet other aspects of this embodiment, a chromogranin B peptide comprising
an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten contiguous amino acid
deletions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:
85 or SEQ ID NO: 86.
In still other aspects of this embodiment, a chromogranin B peptide comprising
an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten contiguous amino acid
additions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:
85 or SEQ ID NO: 86.
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In yet other aspects of this embodiment, a chromogranin B peptide comprising
an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten contiguous amino acid
additions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:
85 or SEQ ID NO: 86.
[0286] In another embodiment, a granin peptide comprising an altered targeting
domain is a
chromogranin C peptide. In aspects of this embodiment, a chromogranin C
peptide comprising an altered
targeting domain is derived from a secretoneurin. In other aspects of this
embodiment, a chromogranin C
peptide comprising an altered targeting domain is SEQ ID NO: 87.
[0287] In other aspects of this embodiment, a chromogranin C peptide
comprising an altered targeting
domain has, e.g., at least 70% amino acid identity with SEQ ID NO: 87, at
least 75% amino acid identity
with SEQ ID NO: 87, at least 80% amino acid identity with SEQ ID NO: 87, at
least 85% amino acid
identity with SEQ ID NO: 87, at least 90% amino acid identity with SEQ ID NO:
87 or at least 95% amino
acid identity with SEQ ID NO: 87. In yet other aspects of this embodiment, a
chromogranin C peptide
comprising an altered targeting domain has, e.g., at most 70% amino acid
identity with SEQ ID NO: 87, at
most 75% amino acid identity with SEQ ID NO: 87, at most 80% amino acid
identity with SEQ ID NO: 87,
at most 85% amino acid identity with SEQ ID NO: 87, at most 90% amino acid
identity with SEQ ID NO:
87 or at most 95% amino acid identity with SEQ ID NO: 87.
[0288] In other aspects of this embodiment, a chromogranin C peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten non-contiguous amino
acid substitutions relative to SEQ ID NO: 87. In other aspects of this
embodiment, a chromogranin C
peptide comprising an altered targeting domain has, e.g., at most one, two,
three, four, five, six, seven,
eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID
NO: 87. In yet other
aspects of this embodiment, a chromogranin C peptide comprising an altered
targeting domain has, e.g.,
at least one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid deletions
relative to SEQ ID NO: 87. In yet other aspects of this embodiment, a
chromogranin C peptide
comprising an altered targeting domain has, e.g., at most one, two, three,
four, five, six, seven, eight, nine
or ten non-contiguous amino acid deletions relative to SEQ ID NO: 87. In still
other aspects of this
embodiment, a chromogranin C peptide comprising an altered targeting domain
has, e.g., at least one,
two, three, four, five, six, seven, eight, nine or ten non-contiguous amino
acid additions relative to SEQ ID
NO: 87. In yet other aspects of this embodiment, a chromogranin C peptide
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten non-
contiguous amino acid additions relative to SEQ ID NO: 87.
[0289] In other aspects of this embodiment, a chromogranin C peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten contiguous amino acid
substitutions relative to SEQ ID NO: 87. In other aspects of this embodiment,
a chromogranin C peptide
comprising an altered targeting domain has, e.g., at most one, two, three,
four, five, six, seven, eight, nine
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or ten contiguous amino acid substitutions relative to SEQ ID NO: 87. In yet
other aspects of this
embodiment, a chromogranin C peptide comprising an altered targeting domain
has, e.g., at least one,
two, three, four, five, six, seven, eight, nine or ten contiguous amino acid
deletions relative to SEQ ID NO:
87. In yet other aspects of this embodiment, a chromogranin C peptide
comprising an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten contiguous amino acid
deletions relative to SEQ ID NO: 87. In still other aspects of this
embodiment, a chromogranin C peptide
comprising an altered targeting domain has, e.g., at least one, two, three,
four, five, six, seven, eight, nine
or ten contiguous amino acid additions relative to SEQ ID NO: 87. In yet other
aspects of this
embodiment, a chromogranin C peptide comprising an altered targeting domain
has, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten contiguous amino acid
additions relative to SEQ ID NO:
87.
[0290] Another example of an altered targeting domain disclosed in the present
specification is, e.g., a
tachykinin peptide, such as, e.g., a Substance P, a neuropeptide K (NPK), a
neuropeptide gamma (NP
gamma), a neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L), a
neurokinin B (NKB), a
hemokinin or a endokinin. Thus, in an embodiment, an altered targeting domain
is derived from a
tachykinin peptide.
[0291] In aspects of this embodiment, a tachykinin peptide comprising an
altered targeting domain is
derived from a Substance P, a neuropeptide K (NPK), a neuropeptide gamma (NP
gamma), a neurokinin
A (NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B (NKB), a
hemokinin or a
endokinin. In other aspects of this embodiment, a tachykinin peptide
comprising an altered targeting
domain is SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID
NO: 92, SEQ ID
NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:
98 or SEQ ID
NO: 99.
[0292] In other aspects of this embodiment, a tachykinin peptide comprising an
altered targeting domain
has, e.g., at least 70% amino acid identity with SEQ ID NO: 88, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID
NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:
96, SEQ ID NO:
97, SEQ ID NO: 98 OR SEQ ID NO: 99, at least 75% amino acid identity with SEQ
ID NO: 88, SEQ ID
NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:
94, SEQ ID NO:
95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99, at least 80%
amino acid identity
with SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO:
92, SEQ ID NO: 93,
SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR
SEQ ID NO: 99, at
least 85% amino acid identity with SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO:
90, SEQ ID NO: 91,
SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ
ID NO: 97, SEQ
ID NO: 98 OR SEQ ID NO: 99, at least 90% amino acid identity with SEQ ID NO:
88, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ
ID NO: 95, SEQ
ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99 or at least 95% amino
acid identity with
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SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ
ID NO: 93, SEQ
ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ
ID NO: 99. In yet
other aspects of this embodiment, a tachykinin peptide comprising an altered
targeting domain has, e.g.,
at most 70% amino acid identity with SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO:
90, SEQ ID NO: 91,
SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ
ID NO: 97, SEQ
ID NO: 98 OR SEQ ID NO: 99, at most 75% amino acid identity with SEQ ID NO:
88, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ
ID NO: 95, SEQ
ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99, at most 80% amino
acid identity with
SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ
ID NO: 93, SEQ
ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ
ID NO: 99, at most
85% amino acid identity with SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ
ID NO: 91, SEQ ID
NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:
97, SEQ ID NO:
98 OR SEQ ID NO: 99, at most 90% amino acid identity with SEQ ID NO: 88, SEQ
ID NO: 89, SEQ ID
NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO:
95, SEQ ID NO:
96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99 or at most 95% amino acid
identity with SEQ ID
NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO:
93, SEQ ID NO:
94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO:
99.
[0293] In other aspects of this embodiment, a tachykinin peptide comprising an
altered targeting domain
has, e.g., at least one, two, three, four or five non-contiguous amino acid
substitutions relative to SEQ ID
NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO:
93, SEQ ID NO:
94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO:
99. In other
aspects of this embodiment, a tachykinin peptide comprising an altered
targeting domain has, e.g., at
most one, two, three, four or five non-contiguous amino acid substitutions
relative to SEQ ID NO: 88,
SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ
ID NO: 94, SEQ
ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99. In
yet other aspects of
this embodiment, a tachykinin peptide comprising an altered targeting domain
has, e.g., at least one, two,
three, four or five non-contiguous amino acid deletions relative to SEQ ID NO:
88, SEQ ID NO: 89, SEQ
ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID
NO: 95, SEQ ID
NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99. In yet other aspects of
this embodiment, a
tachykinin peptide comprising an altered targeting domain has, e.g., at most
one, two, three, four or five
non-contiguous amino acid deletions relative to SEQ ID NO: 88, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID
NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:
96, SEQ ID NO:
97, SEQ ID NO: 98 OR SEQ ID NO: 99. In still other aspects of this embodiment,
a tachykinin peptide
comprising an altered targeting domain has, e.g., at least one, two, three,
four or five non-contiguous
amino acid additions relative to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90,
SEQ ID NO: 91, SEQ
ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID
NO: 97, SEQ ID
NO: 98 OR SEQ ID NO: 99. In yet other aspects of this embodiment, a tachykinin
peptide comprising an
altered targeting domain has, e.g., at most one, two, three, four or five non-
contiguous amino acid
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additions relative to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO:
91, SEQ ID NO: 92,
SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ
ID NO: 98 OR
SEQ ID NO: 99.
[0294] In other aspects of this embodiment, a tachykinin peptide comprising an
altered targeting domain
has, e.g., at least one, two, three, four or five contiguous amino acid
substitutions relative to SEQ ID NO:
88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93,
SEQ ID NO: 94,
SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99.
In other aspects
of this embodiment, a tachykinin peptide comprising an altered targeting
domain has, e.g., at most one,
two, three, four or five contiguous amino acid substitutions relative to SEQ
ID NO: 88, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ
ID NO: 95, SEQ
ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99. In yet other aspects
of this embodiment,
a tachykinin peptide comprising an altered targeting domain has, e.g., at
least one, two, three, four or five
contiguous amino acid deletions relative to SEQ ID NO: 88, SEQ ID NO: 89, SEQ
ID NO: 90, SEQ ID NO:
91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96,
SEQ ID NO: 97,
SEQ ID NO: 98 OR SEQ ID NO: 99. In yet other aspects of this embodiment, a
tachykinin peptide
comprising an altered targeting domain has, e.g., at most one, two, three,
four or five contiguous amino
acid deletions relative to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID
NO: 91, SEQ ID NO:
92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,
SEQ ID NO: 98
OR SEQ ID NO: 99. In still other aspects of this embodiment, a tachykinin
peptide comprising an altered
targeting domain has, e.g., at least one, two, three, four or five contiguous
amino acid additions relative to
SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ
ID NO: 93, SEQ
ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ
ID NO: 99. In yet
other aspects of this embodiment, a tachykinin peptide comprising an altered
targeting domain has, e.g.,
at most one, two, three, four or five contiguous amino acid additions relative
to SEQ ID NO: 88, SEQ ID
NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:
94, SEQ ID NO:
95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 OR SEQ ID NO: 99.
[0295] Another example of an altered targeting domain disclosed in the present
specification is, e.g., a
cholecystokinin peptide, such as, e.g., a cholecystokinin 58, a
cholecystokinin 39, a cholecystokinin 33, a
cholecystokinin 12 or a cholecystokinin 8. Thus, in an embodiment, an altered
targeting domain is
derived from a cholecystokinin peptide.
[0296] In aspects of this embodiment, a cholecystokinin peptide comprising an
altered targeting domain
is derived from a cholecystokinin 58, a cholecystokinin 39, a cholecystokinin
33, a cholecystokinin 12 or a
cholecystokinin 8. In other aspects of this embodiment, a cholecystokinin
peptide comprising an altered
targeting domain is SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:
103, SEQ ID NO:
104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ
ID NO: 115. In
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still other aspects of this embodiment, a cholecystokinin peptide comprising
an altered targeting domain
comprises amino acids 20-58 of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,
SEQ ID NO: 103,
SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:
109, SEQ ID NO:
110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID
NO: 115. In yet
other aspects of this embodiment, a cholecystokinin peptide comprising an
altered targeting domain
comprises amino acids 26-58 of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,
SEQ ID NO: 103,
SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:
109, SEQ ID NO:
110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID
NO: 115. In still
further other aspects of this embodiment, a cholecystokinin peptide comprising
an altered targeting
domain comprises amino acids 47-58 of SEQ ID NO: 100, SEQ ID NO: 110 or SEQ ID
NO: 114. In yet
further aspects of this embodiment, a cholecystokinin peptide comprising an
altered targeting domain
comprises amino acids 51-58 of SEQ ID NO: 100.
[0297] In other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at least 70% amino acid identity with SEQ ID NO: 100, SEQ ID
NO: 101, SEQ ID NO:
102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID
NO: 107, SEQ ID
NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ
ID NO: 113, SEQ
ID NO: 114 or SEQ ID NO: 115, at least 75% amino acid identity with SEQ ID NO:
100, SEQ ID NO: 101,
SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:
106, SEQ ID NO:
107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID
NO: 112, SEQ ID
NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115, at least 80% amino acid identity
with SEQ ID NO: 100,
SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:
105, SEQ ID NO:
106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID
NO: 111, SEQ ID
NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115, at least 85% amino
acid identity with
SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104, SEQ ID NO:
105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID
NO: 110, SEQ ID
NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115, at
least 90% amino
acid identity with SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:
103, SEQ ID NO:
104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ
ID NO: 115 or at
least 95% amino acid identity with SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO:
102, SEQ ID NO: 103,
SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:
108, SEQ ID NO:
109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID
NO: 114 or SEQ ID
NO: 115. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at most 70% amino acid identity with SEQ ID NO:
100, SEQ ID NO: 101, SEQ
ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,
SEQ ID NO: 107,
SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:
112, SEQ ID NO:
113, SEQ ID NO: 114 or SEQ ID NO: 115, at most 75% amino acid identity with
SEQ ID NO: 100, SEQ ID
NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ
ID NO: 106, SEQ
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ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111,
SEQ ID NO: 112,
SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115, at most 80% amino acid
identity with SEQ ID NO:
100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID
NO: 105, SEQ ID
NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ
ID NO: 111, SEQ
ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115, at most 85%
amino acid identity with
SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104, SEQ ID NO:
105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID
NO: 110, SEQ ID
NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115, at
most 90% amino
acid identity with SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:
103, SEQ ID NO:
104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ
ID NO: 115 or at
most 95% amino acid identity with SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO:
102, SEQ ID NO:
103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID
NO: 108, SEQ ID
NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ
ID NO: 114 or
SEQ ID NO: 115.
[0298] In other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten non-contiguous amino
acid substitutions relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,
SEQ ID NO: 103, SEQ
ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108,
SEQ ID NO: 109,
SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114
or SEQ ID NO:
115. In other aspects of this embodiment, a cholecystokinin peptide comprising
an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten non-contiguous amino
acid substitutions relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,
SEQ ID NO: 103, SEQ
ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108,
SEQ ID NO: 109,
SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114
or SEQ ID NO:
115. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten non-contiguous amino
acid deletions relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ
ID NO: 103, SEQ ID
NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ
ID NO: 109, SEQ
ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or
SEQ ID NO: 115.
In yet other aspects of this embodiment, a cholecystokinin peptide comprising
an altered targeting domain
has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid
deletions relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID
NO: 103, SEQ ID NO:
104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ
ID NO: 115. In
still other aspects of this embodiment, a cholecystokinin peptide comprising
an altered targeting domain
has, e.g., at least one, two, three, four, five, six, seven, eight, nine or
ten non-contiguous amino acid
additions relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID
NO: 103, SEQ ID NO:
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104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ
ID NO: 115. In
yet other aspects of this embodiment, a cholecystokinin peptide comprising an
altered targeting domain
has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid
additions relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID
NO: 103, SEQ ID NO:
104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ
ID NO: 115.
[0299] In other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten contiguous amino acid
substitutions relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ
ID NO: 103, SEQ ID
NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ
ID NO: 109, SEQ
ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or
SEQ ID NO: 115.
In other aspects of this embodiment, a cholecystokinin peptide comprising an
altered targeting domain
has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid
substitutions relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ
ID NO: 103, SEQ ID
NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ
ID NO: 109, SEQ
ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or
SEQ ID NO: 115.
In yet other aspects of this embodiment, a cholecystokinin peptide comprising
an altered targeting domain
has, e.g., at least one, two, three, four, five, six, seven, eight, nine or
ten contiguous amino acid deletions
relative to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103,
SEQ ID NO: 104, SEQ
ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109,
SEQ ID NO: 110,
SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO:
115. In yet other
aspects of this embodiment, a cholecystokinin peptide comprising an altered
targeting domain has, e.g.,
at most one, two, three, four, five, six, seven, eight, nine or ten contiguous
amino acid deletions relative to
SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104, SEQ ID NO:
105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID
NO: 110, SEQ ID
NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115. In
still otheraspects
of this embodiment, a cholecystokinin peptide comprising an altered targeting
domain has, e.g., at least
one, two, three, four, five, six, seven, eight, nine or ten contiguous amino
acid additions relative to SEQ
ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104,
SEQ ID NO: 105,
SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:
110, SEQ ID NO:
111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115. In yet
other aspects of
this embodiment, a cholecystokinin peptide comprising an altered targeting
domain has, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten contiguous amino
acid additions relative to SEQ
ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104,
SEQ ID NO: 105,
SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:
110, SEQ ID NO:
111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115.
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[0300] In still other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at least 70% amino acid identity with amino acids
20-58 of SEQ ID NO: 100 or
amino acids 26-58 of SEQ ID NO: 100, at least 75% amino acid identity with
amino acids 20-58 of SEQ
ID NO: 100 or amino acids 26-58 of SEQ ID NO: 100, at least 80% amino acid
identity with amino acids
20-58 of SEQ ID NO: 100 or amino acids 26-58 of SEQ ID NO: 100, at least 85%
amino acid identity with
amino acids 20-58 of SEQ ID NO: 100 or amino acids 26-58 of SEQ ID NO: 100, at
least 90% amino acid
identity with amino acids 20-58 of SEQ ID NO: 100 or amino acids 26-58 of SEQ
ID NO: 100 or at least
95% amino acid identity with amino acids 20-58 of SEQ ID NO: 100 or amino
acids 26-58 of SEQ ID NO:
100. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at most 70% amino acid identity with amino acids 20-58 of
SEQ ID NO: 100 or amino
acids 26-58 of SEQ ID NO: 100, at most 75% amino acid identity with amino
acids 20-58 of SEQ ID NO:
100 or amino acids 26-58 of SEQ ID NO: 100, at most 80% amino acid identity
with amino acids 20-58 of
SEQ ID NO: 100 or amino acids 26-58 of SEQ ID NO: 100, at most 85% amino acid
identity with amino
acids 20-58 of SEQ ID NO: 100 or amino acids 26-58 of SEQ ID NO: 100, at most
90% amino acid
identity with amino acids 20-58 of SEQ ID NO: 100 or amino acids 26-58 of SEQ
ID NO: 100 or at most
95% amino acid identity with amino acids 20-58 of SEQ ID NO: 100 or amino
acids 26-58 of SEQ ID NO:
100.
[0301] In still other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten non-contiguous
amino acid substitutions relative to amino acids 20-58 of SEQ ID NO: 100 or
amino acids 26-58 of SEQ
ID NO: 100. In other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten non-
contiguous amino acid substitutions relative to amino acids 20-58 of SEQ ID
NO: 100 or amino acids 26-
58 of SEQ ID NO: 100. In yet other aspects of this embodiment, a
cholecystokinin peptide comprising an
altered targeting domain has, e.g., at least one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid deletions relative to amino acids 20-58 of SEQ ID NO:
100 or amino acids 26-58
of SEQ ID NO: 100. In yet other aspects of this embodiment, a cholecystokinin
peptide comprising an
altered targeting domain has, e.g., at most one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid deletions relative to amino acids 20-58 of SEQ ID NO:
100 or amino acids 26-58
of SEQ ID NO: 100. In still other aspects of this embodiment, a
cholecystokinin peptide comprising an
altered targeting domain has, e.g., at least one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid additions relative to amino acids 20-58 of SEQ ID NO:
100 or amino acids 26-58
of SEQ ID NO: 100. In yet other aspects of this embodiment, a cholecystokinin
peptide comprising an
altered targeting domain has, e.g., at most one, two, three, four, five, six,
seven, eight, nine or ten non-
contiguous amino acid additions relative to amino acids 20-58 of SEQ ID NO:
100 or amino acids 26-58
of SEQ ID NO: 100.
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[0302] In still other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid substitutions relative to amino acids 20-58 of SEQ ID NO: 100 or
amino acids 26-58 of SEQ
ID NO: 100. In other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid substitutions relative to amino acids 20-58 of SEQ ID NO: 100 or
amino acids 26-58 of SEQ
ID NO: 100. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid deletions relative to amino acids 20-58 of SEQ ID NO: 100 or amino
acids 26-58 of SEQ ID
NO: 100. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid deletions relative to amino acids 20-58 of SEQ ID NO: 100 or amino
acids 26-58 of SEQ ID
NO: 100. In still other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid additions relative to amino acids 20-58 of SEQ ID NO: 100 or amino
acids 26-58 of SEQ ID
NO: 100. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at most one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid additions relative to amino acids 20-58 of SEQ ID NO: 100 or amino
acids 26-58 of SEQ ID
NO: 100.
[0303] In other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at least 70% amino acid identity with amino acids 47-58 of
SEQ ID NO: 100 or amino
acids 51-58 of SEQ ID NO: 100, at least 75% amino acid identity with amino
acids 47-58 of SEQ ID NO:
100 or amino acids 51-58 of SEQ ID NO: 100, at least 80% amino acid identity
with amino acids 47-58 of
SEQ ID NO: 100 or amino acids 51-58 of SEQ ID NO: 100, at least 85% amino acid
identity with amino
acids 47-58 of SEQ ID NO: 100 or amino acids 51-58 of SEQ ID NO: 100, at least
90% amino acid
identity with amino acids 47-58 of SEQ ID NO: 100 or amino acids 51-58 of SEQ
ID NO: 100 or at least
95% amino acid identity with amino acids 47-58 of SEQ ID NO: 100 or amino
acids 51-58 of SEQ ID NO:
100. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at most 70% amino acid identity with amino acids 47-58 of
SEQ ID NO: 100 or amino
acids 51-58 of SEQ ID NO: 100, at most 75% amino acid identity with amino
acids 47-58 of SEQ ID NO:
100 or amino acids 51-58 of SEQ ID NO: 100, at most 80% amino acid identity
with amino acids 47-58 of
SEQ ID NO: 100 or amino acids 51-58 of SEQ ID NO: 100, at most 85% amino acid
identity with amino
acids 47-58 of SEQ ID NO: 100 or amino acids 51-58 of SEQ ID NO: 100, at most
90% amino acid
identity with amino acids 47-58 of SEQ ID NO: 100 or amino acids 51-58 of SEQ
ID NO: 100 or at most
95% amino acid identity with amino acids 47-58 of SEQ ID NO: 100 or amino
acids 51-58 of SEQ ID NO:
100.
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[0304] In other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at least one, two, three or four non-contiguous amino acid
substitutions relative to
amino acids 47-58 of SEQ ID NO: 100 or amino acids 51-58 of SEQ ID NO: 100. In
other aspects of this
embodiment, a cholecystokinin peptide comprising an altered targeting domain
has, e.g., at most one,
two, three or four non-contiguous amino acid substitutions relative to amino
acids 47-58 of SEQ ID NO:
100 or amino acids 51-58 of SEQ ID NO: 100. In yet other aspects of this
embodiment, a cholecystokinin
peptide comprising an altered targeting domain has, e.g., at least one, two,
three or four non-contiguous
amino acid deletions relative to amino acids 47-58 of SEQ ID NO: 100 or amino
acids 51-58 of SEQ ID
NO: 100. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at most one, two, three or four non-contiguous
amino acid deletions relative to
amino acids 47-58 of SEQ ID NO: 100 or amino acids 51-58 of SEQ ID NO: 100. In
still other aspects of
this embodiment, a cholecystokinin peptide comprising an altered targeting
domain has, e.g., at least one,
two, three or four non-contiguous amino acid additions relative to amino acids
47-58 of SEQ ID NO: 100
or amino acids 51-58 of SEQ ID NO: 100. In yet other aspects of this
embodiment, a cholecystokinin
peptide comprising an altered targeting domain has, e.g., at most one, two,
three or four non-contiguous
amino acid additions relative to amino acids 47-58 of SEQ ID NO: 100 or amino
acids 51-58 of SEQ ID
NO: 100.
[0305] In other aspects of this embodiment, a cholecystokinin peptide
comprising an altered targeting
domain has, e.g., at least one, two, three or four contiguous amino acid
substitutions relative to amino
acids 47-58 of SEQ ID NO: 100 or amino acids 51-58 of SEQ ID NO: 100. In other
aspects of this
embodiment, a cholecystokinin peptide comprising an altered targeting domain
has, e.g., at most one,
two, three or four contiguous amino acid substitutions relative to amino acids
47-58 of SEQ ID NO: 100 or
amino acids 51-58 of SEQ ID NO: 100. In yet other aspects of this embodiment,
a cholecystokinin
peptide comprising an altered targeting domain has, e.g., at least one, two,
three or four contiguous
amino acid deletions relative to amino acids 47-58 of SEQ ID NO: 100 or amino
acids 51-58 of SEQ ID
NO: 100. In yet other aspects of this embodiment, a cholecystokinin peptide
comprising an altered
targeting domain has, e.g., at most one, two, three or four contiguous amino
acid deletions relative to
amino acids 47-58 of SEQ ID NO: 100 or amino acids 51-58 of SEQ ID NO: 100. In
still other aspects of
this embodiment, a cholecystokinin peptide comprising an altered targeting
domain has, e.g., at least one,
two, three or four contiguous amino acid additions relative to amino acids 47-
58 of SEQ ID NO: 100 or
amino acids 51-58 of SEQ ID NO: 100. In yet other aspects of this embodiment,
a cholecystokinin
peptide comprising an altered targeting domain has, e.g., at most one, two,
three or four contiguous
amino acid additions relative to amino acids 47-58 of SEQ ID NO: 100 or amino
acids 51-58 of SEQ ID
NO: 100.
[0306] Another example of an altered targeting domain disclosed in the present
specification is, e.g., a
Neuropeptide Y related peptide, such as, e.g., a Neuropeptide Y (NPY), a
Peptide YY (PYY), Pancreatic
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peptide (PP) or a Pancreatic icosapeptide (PIP). Thus, in an embodiment, an
altered targeting domain is
derived from a Neuropeptide Y related peptide.
[0307] In aspects of this embodiment, a Neuropeptide Y related peptide
comprising an altered targeting
domain is derived from a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic
peptide (PP) or a
Pancreatic icosapeptide (PIP). In other aspects of this embodiment, a
Neuropeptide Y related peptide
comprising an altered targeting domain is SEQ ID NO: 116, SEQ ID NO: 117, SEQ
ID NO: 118, SEQ ID
NO: 119 or SEQ ID NO: 120.
[0308] In other aspects of this embodiment, a Neuropeptide Y related peptide
comprising an altered
targeting domain has, e.g., at least 70% amino acid identity with SEQ ID NO:
116, SEQ ID NO: 117, SEQ
ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at least 75% amino acid identity
with SEQ ID NO: 116,
SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at least 80%
amino acid identity
with SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID
NO: 120, at least
85% amino acid identity with SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118,
SEQ ID NO: 119 or
SEQ ID NO: 120, at least 90% amino acid identity with SEQ ID NO: 116, SEQ ID
NO: 117, SEQ ID NO:
118, SEQ ID NO: 119 or SEQ ID NO: 120 or at least 95% amino acid identity with
SEQ ID NO: 116, SEQ
ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other
aspects of this
embodiment, a Neuropeptide Y related peptide comprising an altered targeting
domain has, e.g., at most
70% amino acid identity with SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118,
SEQ ID NO: 119 or
SEQ ID NO: 120, at most 75% amino acid identity with SEQ ID NO: 116, SEQ ID
NO: 117, SEQ ID NO:
118, SEQ ID NO: 119 or SEQ ID NO: 120, at most 80% amino acid identity with
SEQ ID NO: 116, SEQ ID
NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120, at most 85% amino
acid identity with
SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO:
120, at most 90%
amino acid identity with SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ
ID NO: 119 or SEQ ID
NO: 120 or at most 95% amino acid identity with SEQ ID NO: 116, SEQ ID NO:
117, SEQ ID NO: 118,
SEQ ID NO: 119 or SEQ ID NO: 120.
[0309] In other aspects of this embodiment, a Neuropeptide Y related peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten non-contiguous
amino acid substitutions relative to SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID
NO: 118, SEQ ID NO: 119
or SEQ ID NO: 120. In other aspects of this embodiment, a Neuropeptide Y
related peptide comprising
an altered targeting domain has, e.g., at most one, two, three, four, five,
six, seven, eight, nine or ten non-
contiguous amino acid substitutions relative to SEQ ID NO: 116, SEQ ID NO:
117, SEQ ID NO: 118, SEQ
ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, a
Neuropeptide Y related
peptide comprising an altered targeting domain has, e.g., at least one, two,
three, four, five, six, seven,
eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO:
116, SEQ ID NO: 117,
SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this
embodiment, a
Neuropeptide Y related peptide comprising an altered targeting domain has,
e.g., at most one, two, three,
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four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions
relative to SEQ ID NO: 116,
SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In still
other aspects of this
embodiment, a Neuropeptide Y related peptide comprising an altered targeting
domain has, e.g., at least
one, two, three, four, five, six, seven, eight, nine or ten non-contiguous
amino acid additions relative to
SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO:
120. In yet other
aspects of this embodiment, a Neuropeptide Y related peptide comprising an
altered targeting domain
has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid
additions relative to SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID
NO: 119 or SEQ ID
NO: 120.
[0310] In other aspects of this embodiment, a Neuropeptide Y related peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten contiguous
amino acid substitutions relative to SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID
NO: 118, SEQ ID NO: 119
or SEQ ID NO: 120. In other aspects of this embodiment, a Neuropeptide Y
related peptide comprising
an altered targeting domain has, e.g., at most one, two, three, four, five,
six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 116, SEQ ID NO:
117, SEQ ID NO: 118, SEQ
ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this embodiment, a
Neuropeptide Y related
peptide comprising an altered targeting domain has, e.g., at least one, two,
three, four, five, six, seven,
eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 116,
SEQ ID NO: 117, SEQ ID
NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspects of this
embodiment, a Neuropeptide
Y related peptide comprising an altered targeting domain has, e.g., at most
one, two, three, four, five, six,
seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID
NO: 116, SEQ ID NO: 117,
SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In still other aspects of
this embodiment, a
Neuropeptide Y related peptide comprising an altered targeting domain has,
e.g., at least one, two, three,
four, five, six, seven, eight, nine or ten contiguous amino acid additions
relative to SEQ ID NO: 116, SEQ
ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other
aspects of this
embodiment, a Neuropeptide Y related peptide comprising an altered targeting
domain has, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten contiguous amino
acid additions relative to SEQ
ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.
[0311] Another example of an altered targeting domain disclosed in the present
specification is, e.g., a
corticotropin-releasing hormone, a thyrotropin-releasing hormone,
somatostatin, a leukemia inhibitor
factor (LIF) or an interlukin-1 (IL1). Thus, in an embodiment, an altered
targeting domain is derived from
a corticotropin-releasing hormone. In another embodiment, an altered targeting
domain is derived from a
thyrotropin-releasing hormone. In another embodiment, an altered targeting
domain is derived from a
somatostatin. In another embodiment, an altered targeting domain is derived
from a LIF. In another
embodiment, an altered targeting domain is derived from an IL1. In aspects of
this embodiment, a
peptide comprising an altered targeting domain is SEQ ID NO: 121, SEQ ID NO:
122, SEQ ID NO: 123,
SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187.
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[0312] In other aspects of this embodiment, a peptide comprising an altered
targeting domain has, e.g.,
at least 70% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID
NO: 123, SEQ ID NO:
124, SEQ ID NO: 178, SEQ ID NO: 183 or SEQ ID NO: 184, at least 75% amino acid
identity with SEQ ID
NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ
ID NO: 187, at
least 80% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:
123, SEQ ID NO: 124,
SEQ ID NO: 186 or SEQ ID NO: 187, at least 85% amino acid identity with SEQ ID
NO: 121, SEQ ID NO:
122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187, at
least 90% amino acid
identity with SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124,
SEQ ID NO: 186 or
SEQ ID NO: 187 or at least 95% amino acid identity with SEQ ID NO: 121, SEQ ID
NO: 122, SEQ ID NO:
123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187. In yet other aspects of
this embodiment, a
peptide comprising an altered targeting domain has, e.g., at most 70% amino
acid identity with SEQ ID
NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ
ID NO: 187, at
most 75% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:
123, SEQ ID NO:
124, SEQ ID NO: 186 or SEQ ID NO: 187, at most 80% amino acid identity with
SEQ ID NO: 121, SEQ ID
NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187, at
most 85% amino
acid identitywith SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:
124, SEQ ID NO: 186
or SEQ ID NO: 187, at most 90% amino acid identity with SEQ ID NO: 121, SEQ ID
NO: 122, SEQ ID
NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187 or at most 95% amino
acid identity with
SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186
or SEQ ID NO:
187.
[0313] In other aspects of this embodiment, a peptide comprising an altered
targeting domain has, e.g.,
at least one, two, three, four or five non-contiguous amino acid substitutions
relative to SEQ ID NO: 121,
SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO:
187. In other
aspects of this embodiment, a peptide comprising an altered targeting domain
has, e.g., at most one, two,
three, four or five non-contiguous amino acid substitutions relative to SEQ ID
NO: 121, SEQ ID NO: 122,
SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187. In yet other
aspects of this
embodiment, a peptide comprising an altered targeting domain has, e.g., at
least one, two, three, four or
five non-contiguous amino acid deletions relative to SEQ ID NO: 121, SEQ ID
NO: 122, SEQ ID NO: 123,
SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187. In yet other aspects of this
embodiment, a
peptide comprising an altered targeting domain has, e.g., at most one, two,
three, four or five non-
contiguous amino acid deletions relative to SEQ ID NO: 121, SEQ ID NO: 122,
SEQ ID NO: 123, SEQ ID
NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187. In still other aspects of this
embodiment, a peptide
comprising an altered targeting domain has, e.g., at least one, two, three,
four or five non-contiguous
amino acid additions relative to SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:
123, SEQ ID NO: 124,
SEQ ID NO: 186 or SEQ ID NO: 187. In yet other aspects of this embodiment, a
peptide comprising an
altered targeting domain has, e.g., at most one, two, three, four or five non-
contiguous amino acid
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additions relative to SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID
NO: 124, SEQ ID NO:
186 or SEQ ID NO: 187.
[0314] In other aspects of this embodiment, a peptide comprising an altered
targeting domain has, e.g.,
at least one, two, three, four or five contiguous amino acid substitutions
relative to SEQ ID NO: 121, SEQ
ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187.
In other aspects of
this embodiment, a peptide comprising an altered targeting domain has, e.g.,
at most one, two, three, four
or five contiguous amino acid substitutions relative to SEQ ID NO: 121, SEQ ID
NO: 122, SEQ ID NO:
123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187. In yet other aspects of
this embodiment, a
peptide comprising an altered targeting domain has, e.g., at least one, two,
three, four or five contiguous
amino acid deletions relative to SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:
123, SEQ ID NO: 124,
SEQ ID NO: 186 or SEQ ID NO: 187. In yet other aspects of this embodiment, a
peptide comprising an
altered targeting domain has, e.g., at most one, two, three, four or five
contiguous amino acid deletions
relative to SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124,
SEQ ID NO: 186 or
SEQ ID NO: 187. In still other aspects of this embodiment, a peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four or five contiguous amino acid
additions relative to SEQ ID
NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ
ID NO: 187. In
yet other aspects of this embodiment, a peptide comprising an altered
targeting domain has, e.g., at most
one, two, three, four or five contiguous amino acid additions relative to SEQ
ID NO: 121, SEQ ID NO:
122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 186 or SEQ ID NO: 187.
[0315] Another example of an altered targeting domain disclosed in the present
specification is a kinin
peptide, such as, e.g., a bradykinin, a kallidin, a desArg9 bradykinin and a
desArg10 bradykinin. Thus, in
an embodiment, an altered targeting domain is derived from a kinin peptide. In
aspects of this
embodiment, a kinin peptide comprising an altered targeting domain is derived
from a bradykinin, a
kallidin, a desArg9 bradykinin and a desArg10 bradykinin. In other aspects of
this embodiment, a kinin
peptide comprising an altered targeting domain comprises SEQ ID NO: 178, SEQ
ID NO: 179, SEQ ID
NO: 180 or SEQ ID NO: 181.
[0316] In other aspects of this embodiment, a kinin peptide comprising an
altered targeting domain has,
e.g., at least 70% amino acid identity with SEQ ID NO: 178, SEQ ID NO: 179,
SEQ ID NO: 180 or SEQ ID
NO: 181, at least 75% amino acid identity with SEQ ID NO: 178, SEQ ID NO: 179,
SEQ ID NO: 180 or
SEQ ID NO: 181, at least 80% amino acid identity with SEQ ID NO: 178, SEQ ID
NO: 179, SEQ ID NO:
180 or SEQ ID NO: 181, at least 85% amino acid identity with SEQ ID NO: 178,
SEQ ID NO: 179, SEQ ID
NO: 180 or SEQ ID NO: 181, at least 90% amino acid identity with SEQ ID NO:
178, SEQ ID NO: 179,
SEQ ID NO: 180 or SEQ ID NO: 181 or at least 95% amino acid identity with SEQ
ID NO: 178, SEQ ID
NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this
embodiment, a kinin peptide
comprising an altered targeting domain has, e.g., at most 70% amino acid
identity with SEQ ID NO: 178,
SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 75% amino acid
identity with SEQ ID NO:
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178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 80% amino acid
identity with SEQ ID
NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 85% amino
acid identity with
SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 90%
amino acid identity
with SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 or at
most 95% amino acid
identity with SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO:
181.
[0317] In other aspects of this embodiment, a kinin peptide comprising an
altered targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-
contiguous amino acid
substitutions relative to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or
SEQ ID NO: 181. In other
aspects of this embodiment, a kinin peptide comprising an altered targeting
domain has, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten non-contiguous
amino acid substitutions relative to
SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other
aspects of this
embodiment, a kinin peptide comprising an altered targeting domain has, e.g.,
at least one, two, three,
four, five, six, seven, eight, nine or ten non-contiguous amino acid deletions
relative to SEQ ID NO: 178,
SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this
embodiment, a kinin
peptide comprising an altered targeting domain has, e.g., at most one, two,
three, four, five, six, seven,
eight, nine or ten non-contiguous amino acid deletions relative to SEQ ID NO:
178, SEQ ID NO: 179,
SEQ ID NO: 180 or SEQ ID NO: 181. In still other aspects of this embodiment, a
kinin peptide comprising
an altered targeting domain has, e.g., at least one, two, three, four, five,
six, seven, eight, nine or ten non-
contiguous amino acid additions relative to SEQ ID NO: 178, SEQ ID NO: 179,
SEQ ID NO: 180 or SEQ
ID NO: 181. In yet other aspects of this embodiment, a kinin peptide
comprising an altered targeting
domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine
or ten non-contiguous amino
acid additions relative to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or
SEQ ID NO: 181.
[0318] In other aspects of this embodiment, a kinin peptide comprising an
altered targeting domain has,
e.g., at least one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions
relative to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.
In other aspects of
this embodiment, a kinin peptide comprising an altered targeting domain has,
e.g., at most one, two,
three, four, five, six, seven, eight, nine or ten contiguous amino acid
substitutions relative to SEQ ID NO:
178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of
this embodiment, a
kinin peptide comprising an altered targeting domain has, e.g., at least one,
two, three, four, five, six,
seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID
NO: 178, SEQ ID NO: 179,
SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of this embodiment, a
kinin peptide comprising
an altered targeting domain has, e.g., at most one, two, three, four, five,
six, seven, eight, nine or ten
contiguous amino acid deletions relative to SEQ ID NO: 178, SEQ ID NO: 179,
SEQ ID NO: 180 or SEQ
ID NO: 181. In still other aspects of this embodiment, a kinin peptide
comprising an altered targeting
domain has, e.g., at least one, two, three, four, five, six, seven, eight,
nine or ten contiguous amino acid
additions relative to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID
NO: 181. In yet
other aspects of this embodiment, a kinin peptide comprising an altered
targeting domain has, e.g., at
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most one, two, three, four, five, six, seven, eight, nine or ten contiguous
amino acid additions relative to
SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.
[0319] Another example of an altered targeting domain disclosed in the present
specification is a PAR
peptide, such as, e.g., a PAR1 peptide, a PAR2 peptide, a PAR3 peptide and a
PAR4 peptide. Thus, in
an embodiment, an altered targeting domain is derived from a PAR peptide. In
aspects of this
embodiment, a PAR peptide comprising an altered targeting domain is derived
from a PAR1 peptide, a
PAR2 peptide, a PAR3 peptide or a PAR4 peptide. In other aspects of this
embodiment, a PAR peptide
comprising an altered targeting domain comprises amino acids 42-47, amino
acids 42-55, amino acids
29-64 or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-
48, amino acids 24-59
or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52,
amino acids 26-60 or
amino acids 1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61,
amino acids 35-70 or amino
acids 1-70 of SEQ ID NO: 185.
[0320] In other aspects of this embodiment, a PAR peptide comprising an
altered targeting domain has,
e.g., at least 70% amino acid identity with amino acids 42-47, amino acids 42-
55, amino acids 29-64 or
amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48,
amino acids 24-59 or amino
acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino
acids 26-60 or amino acids
1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61, amino acids 35-
70 or amino acids 1-70
of SEQ ID NO: 185, at least 75% amino acid identity with amino acids 42-47,
amino acids 42-55, amino
acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40, amino
acids 35-48, amino acids
24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-
52, amino acids 26-60
or amino acids 1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61,
amino acids 35-70 or
amino acids 1-70 of SEQ ID NO: 185, at least 80% amino acid identity with
amino acids 42-47, amino
acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino
acids 35-40, amino acids
35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-
44, amino acids 39-
52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 184; amino acids 48-
53, amino acids 48-61,
amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 185, at least 85% amino
acid identity with amino
acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ
ID NO: 182; amino acids
35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO:
183; amino acids 39-
44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO:
184; amino acids 48-53,
amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 185, at
least 90% amino acid
identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino
acids 1-64 of SEQ ID
NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino
acids 1-59 of SEQ ID NO:
183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-
60 of SEQ ID NO: 184;
amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of
SEQ ID NO: 185 or at
least 95% amino acid identity with amino acids 42-47, amino acids 42-55, amino
acids 29-64 or amino
acids 1-64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino
acids 24-59 or amino acids
1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-
60 or amino acids 1-60
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of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61, amino acids 35-70 or
amino acids 1-70 of
SEQ ID NO: 185.
[0321] In yet other aspects of this embodiment, a PAR peptide comprising an
altered targeting domain
has, e.g., at most 70% amino acid identity with amino acids 42-47, amino acids
42-55, amino acids 29-64
or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48,
amino acids 24-59 or
amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52,
amino acids 26-60 or amino
acids 1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61, amino
acids 35-70 or amino acids
1-70 of SEQ ID NO: 185, at most 75% amino acid identity with amino acids 42-
47, amino acids 42-55,
amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40,
amino acids 35-48, amino
acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino
acids 39-52, amino acids
26-60 or amino acids 1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-
61, amino acids 35-70
or amino acids 1-70 of SEQ ID NO: 185, at most 80% amino acid identity with
amino acids 42-47, amino
acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino
acids 35-40, amino acids
35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-
44, amino acids 39-
52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 184; amino acids 48-
53, amino acids 48-61,
amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 185, at most 85% amino
acid identity with amino
acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ
ID NO: 182; amino acids
35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO:
183; amino acids 39-
44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO:
184; amino acids 48-53,
amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 185, at
most 90% amino acid
identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino
acids 1-64 of SEQ ID
NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino
acids 1-59 of SEQ ID NO:
183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-
60 of SEQ ID NO: 184;
amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of
SEQ ID NO: 185 or at
most 95% amino acid identity with amino acids 42-47, amino acids 42-55, amino
acids 29-64 or amino
acids 1-64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino
acids 24-59 or amino acids
1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-
60 or amino acids 1-60
of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61, amino acids 35-70 or
amino acids 1-70 of
SEQ ID NO: 185.
[0322] In other aspects of this embodiment, a PAR peptide comprising an
altered targeting domain has,
e.g., at least one, two, three, four or five non-contiguous amino acid
substitutions relative to amino acids
42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO:
182; amino acids 35-
40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO:
183; amino acids 39-44,
amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 184;
amino acids 48-53, amino
acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 185. In other
aspects of this
embodiment, a PAR peptide comprising an altered targeting domain has, e.g., at
most one, two, three,
four or five non-contiguous amino acid substitutions relative to amino acids
42-47, amino acids 42-55,
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amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40,
amino acids 35-48, amino
acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino
acids 39-52, amino acids
26-60 or amino acids 1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-
61, amino acids 35-70
or amino acids 1-70 of SEQ ID NO: 185. In yet other aspects of this
embodiment, a PAR peptide
comprising an altered targeting domain has, e.g., at least one, two, three,
four or five non-contiguous
amino acid deletions relative to amino acids 42-47, amino acids 42-55, amino
acids 29-64 or amino acids
1-64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-
59 or amino acids 1-59
of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or
amino acids 1-60 of
SEQ ID NO: 184; amino acids 48-53, amino acids 48-61, amino acids 35-70 or
amino acids 1-70 of SEQ
ID NO: 185. In yet other aspects of this embodiment, a PAR peptide comprising
an altered targeting
domain has, e.g., at most one, two, three, four or five non-contiguous amino
acid deletions relative to
amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of
SEQ ID NO: 182; amino
acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ
ID NO: 183; amino acids
39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO:
184; amino acids 48-
53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO:
185. In still other aspects
of this embodiment, a PAR peptide comprising an altered targeting domain has,
e.g., at least one, two,
three, four or five non-contiguous amino acid additions relative to amino
acids 42-47, amino acids 42-55,
amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40,
amino acids 35-48, amino
acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino
acids 39-52, amino acids
26-60 or amino acids 1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-
61, amino acids 35-70
or amino acids 1-70 of SEQ ID NO: 185. In yet other aspects of this
embodiment, a PAR peptide
comprising an altered targeting domain has, e.g., at most one, two, three,
four or five non-contiguous
amino acid additions relative to amino acids 42-47, amino acids 42-55, amino
acids 29-64 or amino acids
1-64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-
59 or amino acids 1-59
of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or
amino acids 1-60 of
SEQ ID NO: 184; amino acids 48-53, amino acids 48-61, amino acids 35-70 or
amino acids 1-70 of SEQ
ID NO: 185.
[0323] In other aspects of this embodiment, a PAR peptide comprising an
altered targeting domain has,
e.g., at least one, two, three, four or five contiguous amino acid
substitutions relative to amino acids 42-
47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO:
182; amino acids 35-40,
amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 183;
amino acids 39-44, amino
acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 184; amino
acids 48-53, amino acids
48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 185. In other
aspects of this embodiment,
a PAR peptide comprising an altered targeting domain has, e.g., at most one,
two, three, four or five
contiguous amino acid substitutions relative to amino acids 42-47, amino acids
42-55, amino acids 29-64
or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48,
amino acids 24-59 or
amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52,
amino acids 26-60 or amino
acids 1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61, amino
acids 35-70 or amino acids
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1-70 of SEQ ID NO: 185. In yet other aspects of this embodiment, a PAR peptide
comprising an altered
targeting domain has, e.g., at least one, two, three, four or five contiguous
amino acid deletions relative to
amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of
SEQ ID NO: 182; amino
acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ
ID NO: 183; amino acids
39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO:
184; amino acids 48-
53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO:
185. In yet other aspects
of this embodiment, a PAR peptide comprising an altered targeting domain has,
e.g., at most one, two,
three, four or five contiguous amino acid deletions relative to amino acids 42-
47, amino acids 42-55,
amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40,
amino acids 35-48, amino
acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino
acids 39-52, amino acids
26-60 or amino acids 1-60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-
61, amino acids 35-70
or amino acids 1-70 of SEQ ID NO: 185. In still other aspects of this
embodiment, a PAR peptide
comprising an altered targeting domain has, e.g., at least one, two, three,
four or five contiguous amino
acid additions relative to amino acids 42-47, amino acids 42-55, amino acids
29-64 or amino acids 1-64
of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-59 or
amino acids 1-59 of
SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or
amino acids 1-60 of SEQ
ID NO: 184; amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino
acids 1-70 of SEQ ID
NO: 185. In yet other aspects of this embodiment, a PAR peptide comprising an
altered targeting domain
has, e.g., at most one, two, three, four or five contiguous amino acid
additions relative to amino acids 42-
47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO:
182; amino acids 35-40,
amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 183;
amino acids 39-44, amino
acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 184; amino
acids 48-53, amino acids
48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 185.
[0324] An altered targeting domain disclosed in the present specification
replaces the binding activity of
the Clostridial toxin targeting domain found in naturally occurring
Clostridial toxins. As used herein, the
term "Clostridial toxin targeting domain" is synonymous with "Clostridial
toxin Hcc targeting region" or
"Clostridial toxin Hcc region" and means any naturally occurring Clostridial
toxin polypeptide that can
execute the cell binding step of the intoxication process, including, e.g.,
the binding of the Clostridial toxin
to a Clostridial toxin-specific receptor located on the plasma membrane
surface of a target cell. It is
envisioned that replacement of the binding activity can be achieved by, e.g.,
replacing the entire
Clostridial toxin Hcc targeting domain with an altered targeting domain;
replacing a portion of a Clostridial
toxin Hcc targeting domain with an altered targeting domain, with the proviso
that the portion of a
Clostridial toxin Hcc targeting domain remaining cannot selectively bind to
its Clostridial toxin receptor;
and operably-linking an altered targeting domain to a Clostridial toxin
comprising a Clostridial toxin Hcc
targeting domain, with the proviso that the a Clostridial toxin Hcc targeting
domain is altered so that it
cannot selectively bind to its Clostridial toxin receptor.
[0325] The three-dimensional crystal structures of BoNT/A, BoNT/B and the Hc
domain of TeNT indicate
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that the three functional domains of Clostridial neurotoxins are structurally
distinct. The HEXXH
consensus motif of the light chain forms the tetrahedral zinc binding pocket
of the catalytic site located in
a deep cleft on the protein surface that is accessible by a channel. The
structure of the HN and Hc
domains consists primarily of (3-sheet topologies that are linked by a single
a-helix. The cylindrical-
shaped HN domain comprises two long amphipathic a-helices that resemble the
coiled-coil motif found in
some viral proteins. The HN domain also forms a long unstructured loop called
the `translocation belt,'
which wraps around a large negatively charged cleft of the light chain that
blocks access of the zinc atom
to the catalytic-binding pocket of active site. The Hc domain comprises two
distinct structural features of
roughly equal size that indicate function. The first, designated the HCN
domain, is located in the amino
half of the Hc domain. The HCN domain forms a(3-barrel, jelly-roll fold. The
Hcc domain is the second
domain that comprises the Hc domain. This carboxyl-terminal domain comprises a
modified (3-trefoil
domain which forms three distinct carbohydrate binding regions that resembles
the carbohydrate binding
moiety found in many sugar-binding proteins, such as, e.g., serum amyloid P,
sialidase, cryia, insecticidal
a-endotoxin and lectins. Biochemical studies indicate that the (3-trefoil
domain structure of the Hcc
domain appears to mediate the binding to specific carbohydrate containing
components of the Clostridial
toxin receptor on the cell surface, see, e.g., Krzysztof Ginalski et al.,
Structure-based Sequence
Alignment for the Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family
Provides Residue Level
Information About the Putative Ganglioside Binding Site, 482(1-2) FEBS Lett.
119-124 (2000). The Hc
domain tilts away from the HN domain exposing the surface loops and making
them accessible for
binding. No contacts occur between the light chain and the Hc domain.
[0326] Proteins containing the structural (3-trefoil domain represents a
diverse group of proteins, see,
e.g., C. A. Orengo et al., Protein Superfamilies and Domain Superfolds, 372
Nature 631-634 (1994). The
(3-trefoil domain comprises a six-stranded (3-barrel closed off at one end by
three (3-hairpin structures that
exhibits a characteristic pseudo-threefold axis symmetry. The monomeric
structural unit of this three-fold
symmetry is referred to as the (3-trefoil fold that contains four (3-sheets
organized as a pair of antiparallel
(3-sheets. Dividing each of these (3-trefoil folds is a(3-hairpin turn.
Therefore, in a linear fashion, a(3-
trefoil domain comprises four (3-sheets of the first (3-trefoil fold, a(3-
hairpin turn, four (3-sheets of the
second (3-trefoil fold, a second (3-hairpin turn four (3-sheets of the third
(3-trefoil fold. Because the first
hairpin turn is located between the fourth and fifth (3-sheets of the (3-
trefoil domain, it is designated the
(34/(35 (3-hairpin turn. Likewise, since the second hairpin turn is located
between the eight and ninth (3-
sheets of the (3-trefoil domain, it is designated the (38/(39 (3-hairpin turn.
[0327] As is typical for proteins containing a(3-trefoil fold, the overall
amino acid sequence identity of the
Hcc domain between Clostridial toxins is low. However, key residues essential
for binding activity have
been identified by structural analysis and mutagenesis experiments, see, e.g.,
Krzysztof Ginalski et al.,
Structure-based Sequence Alignment for the Beta-Trefoil Subdomain of the
Clostridial Neurotoxin Family
Provides Residue Level Information About the Putative Ganglioside Binding
Site, 482(1-2) FEBS Lett.
119-124 (2000); and Andreas Rummel et al., The Hcc-Domain of Botulinum
Neurotoxins A and B Exhibits
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a Singular Ganglioside Binding Site Displaying Serotype Specific Carbohydrate
Interaction, 51(3) Mol.
Microbiol. 631-643 (2004). Additionally, research has elucidated that (34/(35
and (38/(39 (3-hairpin turns are
important in conferring the proper pseudo-threefold axis symmetry observed in
the (3-trefoil domain and
that these turns are important for (3-trefoil domain stability, see, e.g.,
Stephen R. Brych et al., Structure
and Stability Effects of Mutations Designed to Increase the Primary Sequence
Symmetry Within the Core
Region of aP-trefoil, 10 Protein Sci. 2587-2599 (2001); Jaewon Kim et al.,
Alternative Type l and 1' Turn
Conformations in the P81P9 P-hairpin of Human Acidic Fibroblast Growth Factor,
11 Protein Sci. 459-466
(2002); Jaewon Kim et al., Sequence swapping Does Not Result in Conformation
Swapping for the P41P5
and P81P9 P-hairpin Turns in Human Acidic Fibroblast Growth Factor, 14 Protein
Sci. 351-359 (2005).
The amino acid sequences comprising the f3-trefoil domains found in various
Clostridial toxins are shown
in Table 2.
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Table 2. {3-trefoil Domains of Clostridial Toxins
Amino Acid Sequence Region of Carbohydrate Binding Moieties
Protein SEQ ID NO:
a-fold -haR~piPn5turn R-fold R-hairpin9turn y-fold
R
BoNT/A 1 1111-1162 1163-1178 1179-1223 1224-1236 1237-1296
BoNT/B 2 1098-1147 1148-1165 1166-1210 1211-1222 1223-1291
BoN T/C 1 3 1112-1150 1151-1166 1167-1218 1219-1229 1230-1291
BoNT/D 4 1099-1137 1138-1153 1154-1207 1208-1218 1219-1276
BoNT/E 5 1086-1129 1130-1146 1147-1190 1191-1198 1199-1252
BoNT/F 6 1106-1152 1153-1171 1172-1213 1214-1221 1222-1274
BoNT/G 7 1106-1153 1154-1172 1173-1218 1219-1230 1231-1297
TeNT 8 1128-1177 1178-1194 1195-1240 1241-1254 1255-1315
[0328] Thus, in an embodiment, a Clostridial toxin targeting domain comprising
an Hcc region can be
replaced with an enhance binding domain disclosed in the present
specification. In aspects of this
embodiment, a BoNT/A Hcc region can be replaced with an altered targeting
domain, a BoNT/B Hcc
region can be replaced with an altered targeting domain, a BoNT/C1 Hcc region
can be replaced with an
altered targeting domain, a BoNT/D Hcc region can be replaced with an altered
targeting domain, a
BoNT/E Hcc region can be replaced with an altered targeting domain, a BoNT/F
Hcc region can be
replaced with an altered targeting domain, a BoNT/G Hcc region can be replaced
with an altered targeting
domain and a TeNT Hcc region can be replaced with an altered targeting domain.
[0329] In aspects of this embodiment, a BoNT/A Hcc region can be replaced with
an altered targeting
domain, a BoNT/B Hcc region can be replaced with an altered targeting domain,
a BoNT/C1 Hcc region
can be replaced with an altered targeting domain, a BoNT/D Hcc region can be
replaced with an altered
targeting domain, a BoNT/E Hcc region can be replaced with an altered
targeting domain, a BoNT/F Hcc
region can be replaced with an altered targeting domain, a BoNT/G Hcc region
can be replaced with an
altered targeting domain and a TeNT Hcc region can be replaced with an altered
targeting domain. In
other aspects of this embodiment, a BoNT/A Hcc region comprising amino acids
1092-1296 of SEQ ID
NO: 1 can be replaced with an altered targeting domain, a BoNT/B Hcc region
comprising amino acids
1079-1291 of SEQ ID NO: 2 can be replaced with an altered targeting domain, a
BoNT/C1 Hcc region
comprising amino acids 1093-1291 of SEQ ID NO: 3 can be replaced with an
altered targeting domain, a
BoNT/D Hcc region comprising amino acids 1080-1276 of SEQ ID NO: 4 can be
replaced with an altered
targeting domain, a BoNT/E Hcc region comprising amino acids 1067-1252 of SEQ
ID NO: 5 can be
replaced with an altered targeting domain, a BoNT/F Hcc region comprising
amino acids 1087-1274 of
SEQ ID NO: 6 can be replaced with an altered targeting domain, a BoNT/G Hcc
region comprising amino
acids 1087-1297 of SEQ ID NO: 7 can be replaced with an altered targeting
domain and a TeNT Hcc
region comprising amino acids 1109-1315 of SEQ ID NO: 8 can be replaced with
an altered targeting
domain.
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[0330] In another embodiment, an altered binding domain disclosed in the
present specification is
operably-linked to a Clostridial toxin comprising a Clostridial toxin
targeting domain altered so that it
cannot selectively bind to its Clostridial toxin receptor. As used herein, the
term "altered," when referring
to a Clostridial toxin targeting domain, means a naturally occurring
Clostridial toxin targeting domain
modified to eliminate or reduce the binding activity of the Clostridial toxin
targeting domain so that the
domain can no longer selectively bind to its Clostridial toxin receptor. By
definition, an altered Clostridial
toxin targeting domain has at least one amino acid change from the
corresponding region of the disclosed
reference sequences (see Table 1) and can be described in percent identity to
the corresponding region
of that reference sequence. As non-limiting examples, a modified BoNT/A Hcc
region comprising amino
acids 1111-1296 of SEQ ID NO: 1 will have at least one amino acid difference,
such as, e.g., an amino
acid substitution, deletion or addition, as compared to the amino acid region
1111-1296 of SEQ ID NO: 1;
a modified BoNT/B Hcc region comprising amino acids 1098-1291 of SEQ ID NO: 2
will have at least one
amino acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the
amino acid region 1098-1291 of SEQ ID NO: 2; a modified BoNT/C1 Hcc region
comprising amino acids
1112-1291 of SEQ ID NO: 3 will have at least one amino acid difference, such
as, e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid region 1112-
1291 of SEQ ID NO: 3; a
modified BoNT/D Hcc region comprising amino acids 1099-1276 of SEQ ID NO: 4
will have at least one
amino acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the
amino acid region 1099-1276 of SEQ ID NO: 4; a modified BoNT/E Hcc region
comprising amino acids
1086-1252 of SEQ ID NO: 5 will have at least one amino acid difference, such
as, e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid region 1086-
1252 of SEQ ID NO: 5; a
modified BoNT/F Hcc region comprising amino acids 1106-1274 of SEQ ID NO: 6
will have at least one
amino acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the
amino acid region 1106-1274 of SEQ ID NO: 6; a modified BoNT/G Hcc region
comprising amino acids
1106-1297 of SEQ ID NO: 7 will have at least one amino acid difference, such
as, e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid region 1106-
1297 of SEQ ID NO: 7; and
a modified TeNT Hcc region comprising amino acids 1128-1315 of SEQ ID NO: 8
will have at least one
amino acid difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the
amino acid region 1128-1315 of SEQ ID NO: 8.
[0331] In aspects of this embodiment, an altered Clostridial toxin Hcc
targeting region comprises a
polypeptide having, e.g., at least 70% amino acid identity with its reference
sequence, at least 75% amino
acid identity with its reference sequence, at least 80% amino acid identity
with its reference sequence, at
least 85% amino acid identity with its reference sequence, at least 90% amino
acid identity with its
reference sequence or at least 95% amino acid identity with its reference
sequence. In yet other aspects
of this embodiment, an altered Clostridial toxin Hcc targeting region
comprises a polypeptide having, e.g.,
at most 70% amino acid identity with its reference sequence, at most 75% amino
acid identity with its
reference sequence, at most 80% amino acid identity with its reference
sequence, at most 85% amino
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acid identity with its reference sequence, at most 90% amino acid identity
with its reference sequence or
at most 95% amino acid identity with its reference sequence.
[0332] In other aspects of this embodiment, an altered Clostridial toxin Hcc
targeting region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50,
100, or 200 non-contiguous amino acid substitutions relative to its reference
sequence. In other aspects
of this embodiment, an altered Clostridial toxin Hcc targeting region
comprises a polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40
, 50, 100 or 200 non-contiguous
amino acid substitutions relative to its reference sequence. In yet other
aspects of this embodiment, an
altered Clostridial toxin Hcc targeting region comprises a polypeptide having,
e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
non-contiguous amino acid
deletions relative to its reference sequence. In other aspects of this
embodiment, an altered Clostridial
toxin Hcc targeting region comprises a polypeptide having, e.g., at least one,
two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino acid
deletions relative to its
reference sequence. In still other aspects of this embodiment, an altered
Clostridial toxin Hcc targeting
region comprises a polypeptide having, e.g., at most one, two, three, four,
five, six, seven, eight, nine, 10,
20, 30, 40 , 50, 100 or 200 non-contiguous amino acid additions relative to
its reference sequence. In
other aspects of this embodiment, an altered Clostridial toxin Hcc targeting
region comprises a
polypeptide having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 non-contiguous amino acid additions relative to its reference sequence.
[0333] In other aspects of this embodiment, an altered Clostridial toxin Hcc
targeting region comprises a
polypeptide having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40 , 50, 100
or 200 contiguous amino acid substitutions relative to its reference sequence.
In other aspects of this
embodiment, an altered Clostridial toxin Hcc targeting region comprises a
polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 ,
50, 100 or 200 contiguous amino
acid substitutions relative to its reference sequence. In yet other aspects of
this embodiment, an altered
Clostridial toxin Hcc targeting region comprises a polypeptide having, e.g.,
at most one, two, three, four,
five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous
amino acid deletions relative to its
reference sequence. In other aspects of this embodiment, an altered
Clostridial toxin Hcc targeting region
comprises a polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30,
40 , 50, 100 or 200 contiguous amino acid deletions relative to its reference
sequence. In still other
aspects of this embodiment, an altered Clostridial toxin Hcc targeting region
comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions relative to its reference sequence. In other
aspects of this embodiment,
an altered Clostridial toxin Hcc targeting region comprises a polypeptide
having, e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200
contiguous amino acid additions
relative to its reference sequence.
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[0334] In another aspect of the invention, a modified Clostridial toxin with
an altered targeting activity
comprises, in part, a protease cleavage site is located within a di-chain loop
region. As used herein, the
term "di-chain loop region" means the amino acid sequence of a Clostridial
toxin containing a protease
cleavage site used to convert the single-chain form of a Clostridial toxin
into the di-chain form. Non-
limiting examples of a Clostridial toxin di-chain loop region, include, a di-
chain loop region of BoNT/A
comprising amino acids 430-454 of SEQ ID NO: 1; a di-chain loop region of
BoNT/B comprising amino
acids 437-446 of SEQ ID NO: 2; a di-chain loop region of BoNT/C1 comprising
amino acids 437-453 of
SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino acids 437-450
of SEQ ID NO: 4; a di-
chain loop region of BoNT/E comprising amino acids 412-426 of SEQ ID NO: 5; a
di-chain loop region of
BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop region
of BoNT/G comprising
amino acids 436-450 of SEQ ID NO: 7; and a di-chain loop region of TeNT
comprising amino acids 439-
467 of SEQ ID NO: 8 (Table 3).
Table 3. Di-chain Loop Region of Clostridial Toxins
SEQ Light Chain Di-chain Loop Region Containing the Heavy Chain
Toxin ID Region Naturally-occurring Protease Cleavage Region
NO: Site
BoNT/A 1 NMNFTKLKNFTGLFEFYKLL CVRGIITSKTKSLDKGYNK*---- ALNDLC IKVNNWDL
BoNT/B 2 KQAYEEISKEHLAVYKIQM CKSVK*-------------------APGIC IDVDNEDL
BoNT/C1 3 PALRKVNPENMLYLFTKF CHKAIDGRSLYNK*------------ TLDC RELLVKNTDL
BoNT/D 4 PALQKLSSESWDLFTKV CLRLTKNSR*---------------DDSTC IKVKNNRL
BoNT/E 5 IITPITGRGLVKKIIRF CKNIVSVKGIR*-------------- KSIC IEINNGEL
BoNT/F 6 IIDSIPDKGLVEKIVKF CKSVIPRKGTK*------------APPRLC IRVNNSEL
BoNT/G 7 KEAYEEISLEHLVIYRIAM CKPVMYKNTGK*--------------SEQC IIVNNEDL
TeNT 8 TNAFRNVDGSGLVSKLIGL CKKIIPPTNIRENLYNRTA*SLTDLGGELC IKIKNEDL
The amino acid sequence displayed are as follows: BoNT/A, residues 325-462 of
SEQ ID No: 1;
BoNT/B, residues 332-454 of SEQ ID No: 2; BoNT/C1, residues 334-463 of SEQ ID
No: 3; BoNT/D,
residues 334-458 of SEQ ID No: 4; BoNT/E, residues 311-434 of SEQ ID No: 5;
BoNT/F, residues 328-
453 of SEQ ID No: 6; BoNT/G, residues 331-458 of SEQ ID No: 7; and TeNT,
residues 334-474 of SEQ
ID No: 8. An asterisks (*) indicates the peptide bond that is cleaved by a
Clostridial toxin protease.
[0335] In is envisioned that any and all protease cleavage sites can be used
to convert the single-chain
polypeptide form of a Clostridial toxin into the di-chain form, including,
without limitation, endogenous di-
chain loop protease cleavage sites and exogenous protease cleavage sites. The
location and kind of
protease cleavage site may be critical because certain targeting domains
require a free amino-terminal or
carboxyl-terminal amino acid. For example, when a targeting domain is placed
between two other
domains, e.g., see FIG. 5, a criteria for selection of a protease cleavage
site could be whether the
protease that cleaves its site leaves a flush cut, exposing the free amino-
terminal or carboxyl-terminal of
the altered targeting domain necessary for selective binding of the targeting
domain to its receptor. The
selection and placement of a protease cleavage site is well known in the art.
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[0336] As used herein, the term "endogenous di-chain loop protease cleavage
site" is synonymous with
a "naturally occurring di-chain loop protease cleavage site" and means a
naturally occurring protease
cleavage site found within the di-chain loop region of a naturally occurring
Clostridial toxin and includes,
without limitation, naturally occurring Clostridial toxin di-chain loop
protease cleavage site variants, such
as, e.g., Clostridial toxin di-chain loop protease cleavage site isoforms and
Clostridial toxin di-chain loop
protease cleavage site subtypes. Non-limiting examples of an endogenous
protease cleavage site,
include, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/B di-
chain loop protease cleavage
site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D di-chain loop
protease cleavage site, a
BoNT/E di-chain loop protease cleavage site, a BoNT/F di-chain loop protease
cleavage site, a BoNT/G
di-chain loop protease cleavage site and a TeNT di-chain loop protease
cleavage site.
[0337] As mentioned above, Clostridial toxins are translated as a single-chain
polypeptide of
approximately 150 kDa that is subsequently cleaved by proteolytic scission
within a disulfide loop by a
naturally-occurring protease. This posttranslational processing yields a di-
chain molecule comprising an
approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain
(HC) held together by
a single disulphide bond and noncovalent interactions. While the identity of
the protease is currently
unknown, the di-chain loop protease cleavage site for many Clostridial toxins
has been proposed. In
BoNTs, cleavage at K448-A449 converts the single polypeptide form of BoNT/A
into the di-chain form;
cleavage at K441-A442 converts the single polypeptide form of BoNT/B into the
di-chain form; cleavage
at K449-T450 converts the single polypeptide form of BoNT/C1 into the di-chain
form; cleavage at R445-
D446 converts the single polypeptide form of BoNT/D into the di-chain form;
cleavage at R422-K423
converts the single polypeptide form of BoNT/E into the di-chain form;
cleavage at K439-A440 converts
the single polypeptide form of BoNT/F into the di-chain form; and cleavage at
K446-S447 converts the
single polypeptide form of BoNT/G into the di-chain form. Proteolytic cleavage
of the single polypeptide
form of TeNT at A457-S458 results in the di-chain form. Such a di-chain loop
protease cleavage site is
operably-linked in-frame to a modified Clostridial toxin as a fusion protein.
[0338] However, it should also be noted that additional cleavage sites within
the di-chain loop also
appear to be cleaved resulting in the generation of a small peptide fragment
being lost. As a non-limiting
example, BoNT/A single-chain polypeptide cleave ultimately results in the loss
of a ten amino acid
fragment within the di-chain loop. Thus, in BoNTs, cleavage at S441-L442
converts the single
polypeptide form of BoNT/A into the di-chain form; cleavage at G444-1445
converts the single polypeptide
form of BoNT/B into the di-chain form; cleavage at S445-L446 converts the
single polypeptide form of
BoNT/C1 into the di-chain form; cleavage at K442-N443 converts the single
polypeptide form of BoNT/D
into the di-chain form; cleavage at K419-G420 converts the single polypeptide
form of BoNT/E into the di-
chain form; cleavage at K423-S424 converts the single polypeptide form of
BoNT/E into the di-chain form;
cleavage at K436-G437 converts the single polypeptide form of BoNT/F into the
di-chain form; cleavage
at T444-G445 converts the single polypeptide form of BoNT/G into the di-chain
form; and cleavage at
E448-Q449 converts the single polypeptide form of BoNT/G into the di-chain
form.
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[0339] Thus, in an embodiment, a protease cleavage site comprising an
endogenous Clostridial toxin di-
chain loop protease cleavage site is used to convert the single-chain toxin
into the di-chain form. In
aspects of this embodiment, conversion into the di-chain form by proteolytic
cleavage occurs from a site
comprising, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/B di-
chain loop protease
cleavage site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D di-
chain loop protease
cleavage site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F di-
chain loop protease cleavage
site, a BoNT/G di-chain loop protease cleavage site or a TeNT di-chain loop
protease cleavage site.
[0340] In other aspects of this embodiment, conversion into the di-chain form
by proteolytic cleavage
occurs from a site comprising, e.g., a di-chain loop region of BoNT/A
comprising amino acids 430-454 of
SEQ ID NO: 1; a di-chain loop region of BoNT/B comprising amino acids 437-446
of SEQ ID NO: 2; a di-
chain loop region of BoNT/C1 comprising amino acids 437-453 of SEQ ID NO: 3; a
di-chain loop region of
BoNT/D comprising amino acids 437-450 of SEQ ID NO: 4; a di-chain loop region
of BoNT/E comprising
amino acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/F
comprising amino acids 429-445
of SEQ ID NO: 6; a di-chain loop region of BoNT/G comprising amino acids 436-
450 of SEQ ID NO: 7; or
a di-chain loop region of TeNT comprising amino acids 439-467 of SEQ ID NO: 8.
[0341] It is also envisioned that an exogenous protease cleavage site can be
used to convert the single-
chain polypeptide form of a modified Clostridial toxin disclosed in the
present specification into the di-
chain form. As used herein, the term "exogenous protease cleavage site" is
synonymous with a "non-
naturally occurring protease cleavage site" and means a protease cleavage site
that is not normally
present in a di-chain loop region from a naturally occurring Clostridial
toxin. Non-limiting examples of
exogenous protease cleavage sites include, e.g., an enterokinase cleavage site
(Table 4); a Thrombin
cleavage site (Table 4); a Factor Xa cleavage site (Table 4); a human
rhinovirus 3C protease cleavage
site (Table 4); a tobacco etch virus (TEV) protease cleavage site (Table 4); a
dipeptidyl aminopeptidase
cleavage site; a small ubiquitin-like modifier (SUMO)/ubiquitin-like protein-
1(ULP-1) protease cleavage
site, such as, e.g.,
MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGK
EMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID. NO: 142); and a Clostridial
toxin
substrate cleavage site.
[0342] As mentioned above, a Clostridial toxin is converted from a single
polypeptide form into a di-chain
molecule by proteolytic cleavage. While the naturally-occurring protease is
currently not known, cleavage
occurs within the di-chain loop region between the two cysteine residues that
form the disulfide bridge
(see Table 3). Replacement of an endogenous protease cleavage site with an
exogenous protease
cleavage site will enable cleavage of a modified Clostridial toxin disclosed
in the present specification
when expressed in an organism that does not produce the naturally-occurring
protease used to cleave
the di-chain loop region of a toxin. Similarly, an addition of an exogenous
protease cleavage site in the
di-chain loop region will also enable cleavage of a modified Clostridial toxin
disclosed in the present
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specification when expressed in an organism that does not produce the
naturally-occurring protease used
to cleave the di-chain loop region of a toxin.
Table 4. Exogenous Protease Cleavage Sites
Protease Cleavage Consensus Sequence Non-limiting SEQ ID
Site Examples NO:
Bovine enterokinase DDDDK* DDDDK* 125
ENLYFQ*G 126
ENLYFQ*S 127
ENIYTQ*G 128
E p5 P4YP2Q*(G/S), ENIYTQ*S 129
Tobacco Etch Virus ENIYLQ*G 130
(TEV) where P2, P4 and P5 can be any amino acid ENIYLQ*S 131
ENVYFQ*G 132
ENVYSQ*S 133
ENVYSQ*G 134
ENVYSQ*S 135
EALFQ*GP 136
P5P4LFQ*GP EVLFQ*GP 137
Human Rhinovirus 3C 4 5 ELLFQ*GP 138
where P is G, A, V, L, I, M, S or T and P can any DALFQ*GP 139
amino acid, with D or E preferred. DVLFQ*GP 140
DLLFQ*GP 141
SUMO/ULP-1 Tertiary structure polypeptide-G* 142
P3P2(R/K)*P' , GVR*G 143
SAR*G 144
Thrombin where P3 is any amino acid and P2 or P' is G with SLR*G 145
the other position being any amino acid DGR*I 146
QGK*I 147
LVPR*GS 148
LVPK*GS 149
P4P3P(R/K)*P' P2 FIPR*TF 150
VLPR*SF 151
where P' and P2 can be any amino acid except for IVPR*SF 152
Thrombin acidic amino acids like D or E; and P3 and P4 are IVPR*GY 153
hydrophobic amino acids like F, L, I, Y, W, V, M, P, WPR*GV 154
C or A VLPR*LI 155
VMPR*SL 156
MFPR*SL 157
Coagulation Factor Xa I(E/D)GR* IDGR* 158
IEGR* 159
An asterisks (*) indicates the peptide bond that is cleaved by the indicated
protease.
[0343] It is envisioned that an exogenous protease cleavage site of any and
all lengths can be useful in
aspects of the present invention with the proviso that the exogenous protease
cleavage site is capable of
being cleaved by its respective protease. Thus, in aspects of this embodiment,
an exogenous protease
cleavage site can be, e.g., at least 6 amino acids in length, at least 7 amino
acids in length, at least 8
amino acids in length, at least 9 amino acids in length, at least 10 amino
acids in length, at least 15 amino
acids in length, at least 20 amino acids in length, at least 25 amino acids in
length, at least 30 amino
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acids in length, at least 40 amino acids in length, at least 50 amino acids in
length or at least 60 amino
acids in length. In other aspects of this embodiment, an exogenous protease
cleavage site can be, e.g.,
at most 6 amino acids in length, at most 7 amino acids in length, at most 8
amino acids in length, at most
9 amino acids in length, at most 10 amino acids in length, at most 15 amino
acids in length, at most 20
amino acids in length, at most 25 amino acids in length, at most 30 amino
acids in length, at most 40
amino acids in length, at most 50 amino acids in length or at most 60 amino
acids in length.
[0344] In aspects of this embodiment, a di-chain loop region can be modified
to substitute a naturally-
occurring protease cleavage site for an exogenous protease cleavage site. In
this type of modification,
the naturally-occurring protease cleavage site is made inoperable and thus can
not be cleaved by its
protease. Only the exogenous protease cleavage site can be cleaved by its
corresponding exogenous
protease. In this type of modification, the exogenous protease site is
operably-linked in-frame to a
modified Clostridial toxin as a fusion protein and the site can be cleaved by
its respective exogenous
protease. As a non-limiting example, a single-chain modified BoNT/A comprising
an exogenous protease
cleavage site in the di-chain loop region can be cleaved by its respective
exogenous protease to produce
the di-chain form of the toxin.
[0345] In other aspects of this embodiment, a di-chain loop region can be
modified to include an
exogenous protease cleavage site in addition to the naturally-occurring
protease cleavage site. In this
type of modification, both cleavage sites are operably-linked in-frame to a
modified Clostridial toxin as a
fusion protein and both sites can be cleaved by their respective proteases. As
a non-limiting example, a
single-chain modified BoNT/A that comprises a di-chain loop containing both
the naturally-occurring
BoNT/A di-chain loop protease cleavage site and an exogenous protease cleavage
site can be cleaved
by either the naturally occurring di-chain loop protease or by the appropriate
exogenous protease to
produce the di-chain form of the toxin.
[0346] A naturally-occurring protease cleavage site can be made inoperable by
altering at least the two
amino acids flanking the peptide bond cleaved by the naturally-occurring di-
chain loop protease. More
extensive alterations can be made, with the proviso that the two cysteine
residues of the di-chain loop
region remain intact and can still form the disulfide bridge. Non-limiting
examples of an amino acid
alteration include deletion of an amino acid or replacement of the original
amino acid with a different
amino acid. Thus, in one embodiment, a naturally-occurring protease cleavage
site is made inoperable
by altering the two amino acids flanking the peptide bond cleaved by a
naturally-occurring protease. In
other aspects of this embodiment, a naturally-occurring protease cleavage site
is made inoperable by
altering, e.g., at least three amino acids including the two amino acids
flanking the peptide bond cleaved
by a naturally-occurring protease; at least four amino acids including the two
amino acids flanking the
peptide bond cleaved by a naturally-occurring protease; at least five amino
acids including the two amino
acids flanking the peptide bond cleaved by a naturally-occurring protease; at
least six amino acids
including the two amino acids flanking the peptide bond cleaved by a naturally-
occurring protease; at
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least seven amino acids including the two amino acids flanking the peptide
bond cleaved by a naturally-
occurring protease; at least eight amino acids including the two amino acids
flanking the peptide bond
cleaved by a naturally-occurring protease; at least nine amino acids including
the two amino acids
flanking the peptide bond cleaved by a naturally-occurring protease; at least
ten amino acids including the
two amino acids flanking the peptide bond cleaved by a naturally-occurring
protease; at least 15 amino
acids including the two amino acids flanking the peptide bond cleaved by a
naturally-occurring protease;
or at least 20 amino acids including the two amino acids flanking the peptide
bond cleaved by a naturally-
occurring protease.
[0347] In still other aspects of this embodiment, a naturally-occurring di-
chain protease cleavage site is
made inoperable by altering, e.g., at most three amino acids including the two
amino acids flanking the
peptide bond cleaved by a naturally-occurring protease; at most four amino
acids including the two amino
acids flanking the peptide bond cleaved by a naturally-occurring protease; at
most five amino acids
including the two amino acids flanking the peptide bond cleaved by a naturally-
occurring protease; at
most six amino acids including the two amino acids flanking the peptide bond
cleaved by a naturally-
occurring protease; at most seven amino acids including the two amino acids
flanking the peptide bond
cleaved by a naturally-occurring protease; at most eight amino acids including
the two amino acids
flanking the peptide bond cleaved by a naturally-occurring protease; at most
nine amino acids including
the two amino acids flanking the peptide bond cleaved by a naturally-occurring
protease; at most ten
amino acids including the two amino acids flanking the peptide bond cleaved by
a naturally-occurring
protease; at most 15 amino acids including the two amino acids flanking the
peptide bond cleaved by a
naturally-occurring protease; or at most 20 amino acids including the two
amino acids flanking the peptide
bond cleaved by a naturally-occurring protease.
[0348] In an embodiment, an exogenous protease cleavage site is located within
the di-chain loop of a
modified Clostridial toxin. In aspects of this embodiment, a modified
Clostridial toxin comprises an
exogenous protease cleavage site comprises, e.g., a bovine enterokinase
protease cleavage site, a
Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C protease
cleavage site, a
SUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site or a
Factor Xa protease
cleavage site. In other aspects of this embodiment, an exogenous protease
cleavage site is located
within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a
modified BoNT/C1, a modified
BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified
TeNT.
[0349] In an aspect of this embodiment, an exogenous protease cleavage site
can be, e.g., a bovine
enterokinase cleavage site is located within the di-chain loop of a modified
Clostridial toxin. In other
aspects of the embodiment, an exogenous protease cleavage site can be, e.g., a
bovine enterokinase
protease cleavage site located within the di-chain loop of a modified
Clostridial toxin comprises SEQ ID
NO: 125. Is still other aspects of this embodiment, a bovine enterokinase
protease cleavage site is
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located within the di-chain loop of, e.g., a modified BoNT/A, a modified
BoNT/B, a modified BoNT/C1, a
modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a
modified TeNT.
[0350] In another aspect of this embodiment, an exogenous protease cleavage
site can be, e.g., a
Tobacco Etch Virus protease cleavage site is located within the di-chain loop
of a modified Clostridial
toxin. In other aspects of the embodiment, an exogenous protease cleavage site
can be, e.g., a Tobacco
Etch Virus protease cleavage site located within the di-chain loop of a
modified Clostridial toxin comprises
SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO:
130, SEQ ID NO:
131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134 or SEQ ID NO: 135. Is
still other aspects of
this embodiment, a Tobacco Etch Virus protease cleavage site is located within
the di-chain loop of, e.g.,
a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a
modified BoNT/E, a
modified BoNT/F, a modified BoNT/G or a modified TeNT.
[0351] In still another aspect of this embodiment, an exogenous protease
cleavage site can be, e.g., a
Human Rhinovirus 3C protease cleavage site is located within the di-chain loop
of a modified Clostridial
toxin. In other aspects of the embodiment, an exogenous protease cleavage site
can be, e.g., a Human
Rhinovirus 3C protease cleavage site located within the di-chain loop of a
modified Clostridial toxin
comprises SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ
ID NO: 140 or
SEQ ID NO: 141. Is still other aspects of this embodiment, a Human Rhinovirus
3C protease cleavage
site is located within the di-chain loop of, e.g., a modified BoNT/A, a
modified BoNT/B, a modified
BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified
BoNT/G or a modified
TeNT.
[0352] In yet another aspect of this embodiment, an exogenous protease
cleavage site can be, e.g., a
SUMO/ULP-1 protease cleavage site is located within the di-chain loop of a
modified Clostridial toxin. In
other aspects of the embodiment, an exogenous protease cleavage site can be,
e.g., a SUMO/ULP-1
protease cleavage site located within the di-chain loop of a modified
Clostridial toxin comprises SEQ ID
NO: 142. Is still other aspects of this embodiment, a SUMO/ULP-1 protease
cleavage site is located
within the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a
modified BoNT/C1, a modified
BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified
TeNT.
[0353] In a further aspect of this embodiment, an exogenous protease cleavage
site can be, e.g., a
Thrombin protease cleavage site is located within the di-chain loop of a
modified Clostridial toxin. In other
aspects of the embodiment, an exogenous protease cleavage site can be, e.g., a
Thrombin protease
cleavage site located within the di-chain loop of a modified Clostridial toxin
comprises SEQ ID NO: 143,
SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO:
148, SEQ ID NO:
149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID
NO: 154, SEQ ID
NO: 155, SEQ ID NO: 156 or SEQ ID NO: 157. Is still other aspects of this
embodiment, a Thrombin
protease cleavage site is located within the di-chain loop of, e.g., a
modified BoNT/A, a modified BoNT/B,
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a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a
modified BoNT/G or
a modified TeNT.
[0354] In another aspect of this embodiment, an exogenous protease cleavage
site can be, e.g., a
Coagulation Factor Xa protease cleavage site is located within the di-chain
loop of a modified Clostridial
toxin. In other aspects of the embodiment, an exogenous protease cleavage site
can be, e.g., a
Coagulation Factor Xa protease cleavage site located within the di-chain loop
of a modified Clostridial
toxin comprises SEQ ID NO: 158 or SEQ ID NO: 159. Is still other aspects of
this embodiment, a
Coagulation Factor Xa protease cleavage site is located within the di-chain
loop of, e.g., a modified
BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified
BoNT/E, a modified
BoNT/F, a modified BoNT/G or a modified TeNT.
[0355] In another embodiment, an exogenous protease site comprises a
Clostridial toxin substrate
cleavage site. As used herein, the term "Clostridial toxin substrate cleavage
site" means a scissile bond
together with adjacent or non-adjacent recognition elements, or both,
sufficient for detectable proteolysis
at the scissile bond by a Clostridial toxin under conditions suitable for
Clostridial toxin protease activity.
By definition, a Clostridial toxin substrate cleavage site is susceptible to
cleavage by at least one
Clostridial toxin under conditions suitable for Clostridial toxin protease
activity. Non-limiting examples of
Clostridial toxin substrate cleavage site are disclosed in, e.g., Lance E.
Steward et al., Self-Activating
Clostridial Toxins, U.S. Patent Application 60/718,616 (Sep, 19, 2005).
[0356] It is understood that a modified Clostridial toxin disclosed in the
present specification can
optionally include one or more additional components. As a non-limiting
example of an optional
component, a modified Clostridial toxin can further comprise a flexible region
comprising a flexible spacer.
Non-limiting examples of a flexible spacer include, e.g., a G-spacer GGGGS
(SEQ ID NO: 160) or an A-
spacer EAAAK (SEQ ID NO: 161). A flexible region comprising flexible spacers
can be used to adjust the
length of a polypeptide region in order to optimize a characteristic,
attribute or property of a polypeptide.
Such a flexible region is operably-linked in-frame to the modified Clostridial
toxin as a fusion protein. As a
non-limiting example, a polypeptide region comprising one or more flexible
spacers in tandem can be use
to better expose a protease cleavage site thereby facilitating cleavage of
that site by a protease. As
another non-limiting example, a polypeptide region comprising one or more
flexible spacers in tandem
can be use to better present an altered targeting domain, thereby facilitating
the binding of that altered
targeting domain to its receptor.
[0357] Thus, in an embodiment, a modified Clostridial toxin disclosed in the
present specification can
further comprise a flexible region comprising a flexible spacer. In another
embodiment, a modified
Clostridial toxin disclosed in the present specification can further comprise
flexible region comprising a
plurality of flexible spacers in tandem. In aspects of this embodiment, a
flexible region can comprise in
tandem, e.g., at least 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers,
at least 4 G-spacers or at
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least 5 G-spacers. In other aspects of this embodiment, a flexible region can
comprise in tandem, e.g., at
most 1 G-spacer, at most 2 G-spacers, at most 3 G-spacers, at most 4 G-spacers
or at most 5 G-
spacers. In still other aspects of this embodiment, a flexible region can
comprise in tandem, e.g., at least
1 A-spacer, at least 2 A-spacers, at least 3 A-spacers, at least 4 A-spacers
or at least 5 A-spacers. In still
other aspects of this embodiment, a flexible region can comprise in tandem,
e.g., at most 1 A-spacer, at
most 2 A-spacers, at most 3 A-spacers, at most 4 A-spacers or at most 5 A-
spacers. In another aspect of
this embodiment, a modified Clostridial toxin can comprise a flexible region
comprising one or more
copies of the same flexible spacers, one or more copies of different flexible-
spacer regions, or any
combination thereof.
[0358] In aspects of this embodiment, a modified Clostridial toxin comprising
a flexible spacer can be,
e.g., a modified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified
BoNT/D, a modified
BoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.
[0359] It is envisioned that a modified Clostridial toxin disclosed in the
present specification can
comprise a flexible spacer in any and all locations with the proviso that
modified Clostridial toxin is
capable of performing the intoxication process. In aspects of this embodiment,
a flexible spacer is
positioned between, e.g., an enzymatic domain and a translocation domain, an
enzymatic domain and an
altered targeting domain, an enzymatic domain and a protease cleavage site. In
other aspects of this
embodiment, a G-spacer is positioned between, e.g., an enzymatic domain and a
translocation domain,
an enzymatic domain and an altered targeting domain, an enzymatic domain and a
protease cleavage
site. In other aspects of this embodiment, a A-spacer is positioned between,
e.g., an enzymatic domain
and a translocation domain, an enzymatic domain and an altered targeting
domain, an enzymatic domain
and a protease cleavage site.
[0360] In other aspects of this embodiment, a flexible spacer is positioned
between, e.g., an altered
targeting domain and a translocation domain, an altered targeting domain and
an enzymatic domain, an
altered targeting domain and a protease cleavage site. In other aspects of
this embodiment, a G-spacer
is positioned between, e.g., an altered targeting domain and a translocation
domain, an altered targeting
domain and an enzymatic domain, an altered targeting domain and a protease
cleavage site. In other
aspects of this embodiment, a A-spacer is positioned between, e.g., an altered
targeting domain and a
translocation domain, an altered targeting domain and an enzymatic domain, an
altered targeting domain
and a protease cleavage site.
[0361] In yet other aspects of this embodiment, a flexible spacer is
positioned between, e.g., a
translocation domain and an enzymatic domain, an translocation domain and an
altered targeting domain,
an translocation domain and a protease cleavage site. In other aspects of this
embodiment, a G-spacer
is positioned between, e.g., a translocation domain and an enzymatic domain,
an translocation domain
and an altered targeting domain, an translocation domain and a protease
cleavage site. In other aspects
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of this embodiment, a A-spacer is positioned between, e.g., a translocation
domain and an enzymatic
domain, an translocation domain and an altered targeting domain, a
translocation domain and a protease
cleavage site.
[0362] As another non-limiting example of an optional component, a modified
Clostridial toxin can further
comprise an epitope-binding region. An epitope-binding region can be used in a
wide variety of
procedures involving, e.g., protein purification and protein visualization.
Such an epitope-binding region
is operably-linked in-frame to a modified Clostridial toxin as a fusion
protein. Non-limiting examples of an
epitope-binding region include, e.g., FLAG, ExpressTM (SEQ ID NO: 162), human
Influenza virus
hemagluttinin (HA) (SEQ ID NO: 163), human p62 -"'y protein (c-MYC) (SEQ ID
NO: 164), Vesicular
Stomatitis Virus Glycoprotein (VSV-G) (SEQ ID NO: 165), Substance P (SEQ ID
NO: 166), glycoprotein-
D precursor of Herpes simplex virus (HSV) (SEQ ID NO: 167), V5 (SEQ ID NO:
168), AU1 (SEQ ID NO:
169) and AU5 (SEQ ID NO: 170); affinity-binding, such as. e.g., polyhistidine
(HIS) (SEQ ID NO: 171),
streptavidin binding peptide (strep), and biotin or a biotinylation sequence;
peptide-binding regions, such
as. e.g., the glutathione binding domain of glutathione-S-transferase, the
calmodulin binding domain of
the calmodulin binding protein, and the maltose binding domain of the maltose
binding protein. Non-
limiting examples of specific protocols for selecting, making and using an
appropriate binding peptide are
described in, e.g., Epitope Tagging, pp. 17.90-17.93 (Sambrook and Russell,
eds., Molecular Cloning A
Laboratory Manual, Vol. 3, 3~d ed. 2001); Antibodies: A Laboratory Manual
(Edward Harlow & David Lane,
eds., Cold Spring Harbor Laboratory Press, 2"d ed. 1998); and Using
Antibodies: A Laboratory Manual:
Portable Protocol No. I (Edward Harlow & David Lane, Cold Spring Harbor
Laboratory Press, 1998). In
addition, non-limiting examples of binding peptides as well as well-
characterized reagents, conditions and
protocols are readily available from commercial vendors that include, without
limitation, BD Biosciences-
Clontech, Palo Alto, CA; BD Biosciences Pharmingen, San Diego, CA; Invitrogen,
Inc, Carlsbad, CA;
QIAGEN, Inc., Valencia, CA; and Stratagene, La Jolla, CA. These protocols are
routine procedures well
within the scope of one skilled in the art and from the teaching herein.
[0363] Thus, in an embodiment, a modified Clostridial toxin disclosed in the
present specification can
further comprise an epitope-binding region. In another embodiment, a modified
Clostridial toxin disclosed
in the present specification can further comprises a plurality of epitope-
binding regions. In aspects of this
embodiment, a modified Clostridial toxin can comprise, e.g., at least 1
epitope-binding region, at least 2
epitope-binding regions, at least 3 epitope-binding regions, at least 4
epitope-binding regions or at least 5
epitope-binding regions. In other aspects of this embodiment, a modified
Clostridial toxin can comprise,
e.g., at most 1 epitope-binding region, at most 2 epitope-binding regions, at
most 3 epitope-binding
regions, at most 4 epitope-binding regions or at most 5 epitope-binding
regions. In another aspect of this
embodiment, a modified Clostridial toxin can comprise one or more copies of
the same epitope-binding
region, one or more copies of different epitope-binding regions, or any
combination thereof.
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[0364] The location of an epitope-binding region can be in various positions,
including, without limitation,
at the amino terminus of a modified Clostridial toxin, within a modified
Clostridial toxin, or at the carboxyl
terminus of a modified Clostridial toxin. Thus, in an embodiment, an epitope-
binding region is located at
the amino-terminus of a modified Clostridial toxin. In such a location, a
start methionine should be placed
in front of the epitope-binding region. In addition, it is known in the art
that when adding a polypeptide
that is operably-linked to the amino terminus of another polypeptide
comprising the start methionine that
the original methionine residue can be deleted. This is due to the fact that
the added polypeptide will
contain a new start methionine and that the original start methionine may
reduce optimal expression of
the fusion protein. In aspects of this embodiment, an epitope-binding region
located at the amino-
terminus of a modified Clostridial toxin disclosed in the present
specification can be, e.g., a FLAG,
ExpressTM epitope-binding region, a human Influenza virus hemagluttinin (HA)
epitope-binding region, a
human p62 -"'y protein (c-MYC) epitope-binding region, a Vesicular Stomatitis
Virus Glycoprotein (VSV-
G) epitope-binding region, a Substance P epitope-binding region, a
glycoprotein-D precursor of Herpes
simplex virus (HSV) epitope-binding region, a V5 epitope-binding region, a AU1
epitope-binding region, a
AU5 epitope-binding region, a polyhistidine epitope-binding region, a
streptavidin binding peptide epitope-
binding region, a biotin epitope-binding region, a biotinylation epitope-
binding region, a glutathione
binding domain of glutathione-S-transferase, a calmodulin binding domain of
the calmodulin binding
protein or a maltose binding domain of the maltose binding protein.
[0365] In another embodiment, an epitope-binding region is located at the
carboxyl-terminus of a
modified Clostridial toxin. In aspects of this embodiment, an epitope-binding
region located at the
carboxyl-terminus of a modified Clostridial toxin disclosed in the present
specification can be, e.g., a
FLAG, ExpressTM epitope-binding region, a human Influenza virus hemagluttinin
(HA) epitope-binding
region, a human p62 -"'y protein (c-MYC) epitope-binding region, a Vesicular
Stomatitis Virus
Glycoprotein (VSV-G) epitope-binding region, a Substance P epitope-binding
region, a glycoprotein-D
precursor of Herpes simplex virus (HSV) epitope-binding region, a V5 epitope-
binding region, a AU1
epitope-binding region, a AU5 epitope-binding region, a polyhistidine epitope-
binding region, a
streptavidin binding peptide epitope-binding region, a biotin epitope-binding
region, a biotinylation
epitope-binding region, a glutathione binding domain of glutathione-S-
transferase, a calmodulin binding
domain of the calmodulin binding protein or a maltose binding domain of the
maltose binding protein.
[0366] It is envisioned that a modified Clostridial toxin disclosed in the
present specification can
comprise an altered targeting domain in any and all locations with the proviso
that modified Clostridial
toxin is capable of performing the intoxication process. Non-limiting examples
include, locating an
enhance targeting domain at the amino terminus of a modified Clostridial toxin
(see FIG. 4); locating an
enhance targeting domain between a Clostridial toxin enzymatic domain and a
Clostridial toxin
translocation domain of a modified Clostridial toxin (see FIG. 5); and
locating an enhance targeting
domain at the carboxyl terminus of a modified Clostridial toxin (see FIG. 6).
The enzymatic domain of
naturally-occurring Clostridial toxins contains the native start methionine.
Thus, in domain organizations
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where the enzymatic domain is not in the amino-terminal location an amino acid
sequence comprising the
start methionine should be placed in front of the amino-terminal domain.
Likewise, where the altered
targeting domain is in the amino-terminal position, an amino acid sequence
comprising a start methionine
and a protease cleavage site may be operably-linked in situations in which the
altered targeting domain
requires a free amino terminus, see, e.g., Shengwen Li et al., Degradable
Clostridial Toxins, International
Patent Application Publication WO 2006/026780 (Mar. 9, 2006). In addition, it
is known in the art that
when adding a polypeptide that is operably-linked to the amino terminus of
another polypeptide
comprising the start methionine that the original methionine residue can be
deleted.
[0367] Thus, in an embodiment, a modified Clostridial toxin can comprise an
amino to carboxyl single
polypeptide linear order comprising a Clostridial toxin enzymatic domain, a
Clostridial toxin translocation
domain, a Clostridial toxin translocation facilitating domain and an altered
targeting domain. In an aspect
of this embodiment, a modified Clostridial toxin can comprise an amino to
carboxyl single polypeptide
linear order comprising a Clostridial toxin enzymatic domain, a protease
cleavage site, a Clostridial toxin
translocation domain, a Clostridial toxin translocation facilitating domain
and an altered targeting domain.
In another aspect of this embodiment, a modified Clostridial toxin can
comprise an amino to carboxyl
single polypeptide linear order comprising a Clostridial toxin enzymatic
domain, an endogenous protease
cleavage site, a Clostridial toxin translocation domain, a Clostridial toxin
translocation facilitating domain
and an altered targeting domain. In another aspect of this embodiment, a
modified Clostridial toxin can
comprise an amino to carboxyl single polypeptide linear order comprising a
Clostridial toxin enzymatic
domain, an exogenous protease cleavage site, a Clostridial toxin translocation
domain, a Clostridial toxin
translocation facilitating domain and an altered targeting domain.
[0368] In another embodiment, a modified Clostridial toxin can comprise an
amino to carboxyl single
polypeptide linear order comprising a Clostridial toxin enzymatic domain, an
altered targeting domain, a
Clostridial toxin translocation domain and a Clostridial toxin translocation
facilitating domain. In an aspect
of this embodiment, a modified Clostridial toxin can comprise an amino to
carboxyl single polypeptide
linear order comprising a Clostridial toxin enzymatic domain, a protease
cleavage site, an altered
targeting domain, a Clostridial toxin translocation domain and a Clostridial
toxin translocation facilitating
domain. In another aspect of this embodiment, a modified Clostridial toxin can
comprise an amino to
carboxyl single polypeptide linear order comprising a Clostridial toxin
enzymatic domain, an endogenous
protease cleavage site, an altered targeting domain, a Clostridial toxin
translocation domain and a
Clostridial toxin translocation facilitating domain. In another aspect of this
embodiment, a modified
Clostridial toxin can comprise an amino to carboxyl single polypeptide linear
order comprising a Clostridial
toxin enzymatic domain, an exogenous protease cleavage site, an altered
targeting domain, a Clostridial
toxin translocation domain and a Clostridial toxin translocation facilitating
domain.
[0369] In another embodiment, a modified Clostridial toxin can comprise an
amino to carboxyl single
polypeptide linear order comprising an altered targeting domain, a Clostridial
toxin translocation domain,
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a Clostridial toxin translocation facilitating domain and a Clostridial toxin
enzymatic domain. In an aspect
of this embodiment, a modified Clostridial toxin can comprise an amino to
carboxyl single polypeptide
linear order comprising an altered targeting domain, a Clostridial toxin
translocation domain, a Clostridial
toxin translocation facilitating domain, a protease cleavage site and a
Clostridial toxin enzymatic domain.
In another aspect of this embodiment, a modified Clostridial toxin can
comprise an amino to carboxyl
single polypeptide linear order comprising an altered targeting domain, a
Clostridial toxin translocation
domain, a Clostridial toxin translocation facilitating domain, an endogenous
protease cleavage site and a
Clostridial toxin enzymatic domain. In another aspect of this embodiment, a
modified Clostridial toxin can
comprise an amino to carboxyl single polypeptide linear order comprising an
altered targeting domain, a
Clostridial toxin translocation domain, a Clostridial toxin translocation
facilitating domain, an exogenous
protease cleavage site and a Clostridial toxin enzymatic domain.
[0370] In another embodiment, a modified Clostridial toxin can comprise an
amino to carboxyl single
polypeptide linear order comprising an altered targeting domain, a Clostridial
toxin enzymatic domain, a
Clostridial toxin translocation domain and a Clostridial toxin translocation
facilitating domain. In an aspect
of this embodiment, a modified Clostridial toxin can comprise an amino to
carboxyl single polypeptide
linear order comprising an altered targeting domain, a Clostridial toxin
enzymatic domain, a protease
cleavage site, a Clostridial toxin translocation domain and a Clostridial
toxin translocation facilitating
domain. In another aspect of this embodiment, a modified Clostridial toxin can
comprise an amino to
carboxyl single polypeptide linear order comprising an altered targeting
domain, a Clostridial toxin
enzymatic domain, an endogenous protease cleavage site, a Clostridial toxin
translocation domain and a
Clostridial toxin translocation facilitating domain. In another aspect of this
embodiment, a modified
Clostridial toxin can comprise an amino to carboxyl single polypeptide linear
order comprising an altered
targeting domain, a Clostridial toxin enzymatic domain, an exogenous protease
cleavage site, a
Clostridial toxin translocation domain and a Clostridial toxin translocation
facilitating domain.
[0371] In another embodiment, a modified Clostridial toxin can comprise an
amino to carboxyl single
polypeptide linear order comprising a Clostridial toxin translocation domain,
a Clostridial toxin
translocation facilitating domain, a Clostridial toxin enzymatic domain and an
altered targeting domain. In
an aspect of this embodiment, a modified Clostridial toxin can comprise an
amino to carboxyl single
polypeptide linear order comprising a Clostridial toxin translocation domain,
a Clostridial toxin
translocation facilitating domain, a protease cleavage site, a Clostridial
toxin enzymatic domain and an
altered targeting domain. In another aspect of this embodiment, a modified
Clostridial toxin can comprise
an amino to carboxyl single polypeptide linear order comprising a Clostridial
toxin translocation domain, a
Clostridial toxin translocation facilitating domain, an endogenous protease
cleavage site, a Clostridial
toxin enzymatic domain and an altered targeting domain. In another aspect of
this embodiment, a
modified Clostridial toxin can comprise an amino to carboxyl single
polypeptide linear order comprising a
Clostridial toxin translocation domain, a Clostridial toxin translocation
facilitating domain, an exogenous
protease cleavage site, a Clostridial toxin enzymatic domain and an altered
targeting domain.
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[0372] Aspects of the present invention provide, in part modified Clostridial
toxins. Non-limiting
examples of Clostridial toxin modifications disclosed in the present
specification include, e.g., addition of
an altered targeting domain, addition of a protease cleavage site,
rearrangement of the enzymatic,
translocation and binding domains and addition of a spacer region. It is
understood that all such
modifications do not substantially affect the ability of a modified
Clostridial toxin to intoxicate a cell. As
used herein, the term "do not substantially affect" means a modified
Clostridial toxin can still execute the
overall cellular mechanism whereby a Clostridial toxin enters a neuron and
inhibits neurotransmitter
release and encompasses the binding of a Clostridial toxin to a low or high
affinity receptor complex, the
internalization of the toxin/receptor complex, the translocation of the
Clostridial toxin light chain into the
cytoplasm and the enzymatic modification of a Clostridial toxin substrate. In
aspects of this embodiment,
the modified Clostridial toxin is, e.g., at least 10% as toxic as a naturally-
occurring Clostridial toxin, at
least 20% as toxic as a naturally-occurring Clostridial toxin, at least 30% as
toxic as a naturally-occurring
Clostridial toxin, at least 40% as toxic as a naturally-occurring Clostridial
toxin, at least 50% as toxic as a
naturally-occurring Clostridial toxin, at least 60% as toxic as a naturally-
occurring Clostridial toxin, at least
70% as toxic as a naturally-occurring Clostridial toxin, at least 80% as toxic
as a naturally-occurring
Clostridial toxin, at least 90% as toxic as a naturally-occurring Clostridial
toxin or at least 95% as toxic as
a naturally-occurring Clostridial toxin. In aspects of this embodiment, the
modified Clostridial toxin is,
e.g., at most 10% as toxic as a naturally-occurring Clostridial toxin, at most
20% as toxic as a naturally-
occurring Clostridial toxin, at most 30% as toxic as a naturally-occurring
Clostridial toxin, at most 40% as
toxic as a naturally-occurring Clostridial toxin, at most 50% as toxic as a
naturally-occurring Clostridial
toxin, at most 60% as toxic as a naturally-occurring Clostridial toxin, at
most 70% as toxic as a naturally-
occurring Clostridial toxin, at most 80% as toxic as a naturally-occurring
Clostridial toxin, at most 90% as
toxic as a naturally-occurring Clostridial toxin or at most 95% as toxic as a
naturally-occurring Clostridial
toxin.
[0373] Another aspect of the present invention provides polynucleotide
molecules encoding modified
Clostridial toxins disclosed in the present specification. It is envisioned
that any and all modified
Clostridial toxin disclosed in the present specification can be encoded by a
polynucleotide molecule.
[0374] Aspects of the present invention provide, in part polynucleotide
molecules. As used herein, the
term "polynucleotide molecule" is synonymous with "nucleic acid molecule" and
means a polymeric form
of nucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides, of
any length. It is envisioned
that any and all polynucleotide molecules that can encode a modified
Clostridial toxin disclosed in the
present specification can be useful, including, without limitation naturally-
occurring and non-naturally-
occurring DNA molecules and naturally-occurring and non-naturally-occurring
RNA molecules. Non-
limiting examples of naturally-occurring and non-naturally-occurring DNA
molecules include single-
stranded DNA molecules, double-stranded DNA molecules, genomic DNA molecules,
cDNA molecules,
vector constructs, such as, e.g., plasmid constructs, phagmid constructs,
bacteriophage constructs,
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retroviral constructs and artificial chromosome constructs. Non-limiting
examples of naturally-occurring
and non-naturally-occurring RNA molecules include single-stranded RNA, double
stranded RNA and
mRNA.
[0375] Thus, in an embodiment, a polynucleotide molecule encodes a modified
Clostridial toxin
disclosed in the present specification.
[0376] In another embodiment, a polynucleotide molecule encodes, in part, a
modified Clostridial toxin
comprising a Clostridial toxin enzymatic domain disclosed in the present
specification. In an aspect of
this embodiment, a polynucleotide molecule encoding a modified Clostridial
toxin enzymatic domain
comprises a naturally occurring Clostridial toxin enzymatic domain variant,
such as, e.g., a Clostridial
toxin enzymatic domain isoform or a Clostridial toxin enzymatic domain
subtype. In another aspect of this
embodiment, a polynucleotide molecule encoding a Clostridial toxin enzymatic
domain comprises a non-
naturally occurring Clostridial toxin enzymatic domain variant, such as, e.g.,
a conservative Clostridial
toxin enzymatic domain variant, a non-conservative Clostridial toxin enzymatic
domain variant or an
active Clostridial toxin enzymatic domain fragment, or any combination
thereof. In other aspects of this
embodiment, a polynucleotide molecule encoding a Clostridial toxin enzymatic
domain comprises a
BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic
domain, a BoNT/D
enzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a
BoNT/G enzymatic
domain, a TeNT enzymatic domain, or active fragment thereof.
[0377] In another embodiment, a polynucleotide molecule encodes, in part, a
modified Clostridial toxin
comprising a Clostridial toxin translocation domain disclosed in the present
specification. In an aspect of
this embodiment, a polynucleotide molecule encoding a modified Clostridial
toxin translocation domain
comprises a naturally occurring Clostridial toxin translocation domain
variant, such as, e.g., a Clostridial
toxin translocation domain isoform or a Clostridial toxin translocation domain
subtype. In another aspect
of this embodiment, a polynucleotide molecule encoding a Clostridial toxin
translocation domain
comprises a non-naturally occurring Clostridial toxin translocation domain
variant, such as, e.g., a
conservative Clostridial toxin translocation domain variant, a non-
conservative Clostridial toxin
translocation domain variant or an active Clostridial toxin translocation
domain fragment, or any
combination thereof. In other aspects of this embodiment, a polynucleotide
molecule encoding a
Clostridial toxin translocation domain comprises a BoNT/A translocation
domain, a BoNT/B translocation
domain, a BoNT/C1 translocation domain, a BoNT/D translocation domain, a
BoNT/E translocation
domain, a BoNT/F translocation domain, a BoNT/G translocation domain, a TeNT
translocation domain,
or active fragment thereof.
[0378] In another embodiment, a polynucleotide molecule encodes, in part, a
modified Clostridial toxin
comprising a translocation facilitating domain disclosed in the present
specification. In an aspect of this
embodiment, a polynucleotide molecule encoding a translocation facilitating
domain comprises a naturally
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occurring Clostridial toxin translocation facilitating domain variant, such
as, e.g., a Clostridial toxin
translocation facilitating domain isoform or a Clostridial toxin translocation
facilitating domain subtype. In
another aspect of this embodiment, a polynucleotide molecule encoding a
translocation facilitating
domain comprises a non-naturally occurring Clostridial toxin translocation
facilitating domain variant, such
as, e.g., a conservative Clostridial toxin translocation facilitating domain
variant, a non-conservative
Clostridial toxin translocation facilitating domain variant or an active
Clostridial toxin translocation
facilitating domain fragment, or any combination thereof. In other aspects of
this embodiment, a
polynucleotide molecule encoding a Clostridial toxin translocation
facilitating domain comprises a BoNT/A
translocation facilitating domain, a BoNT/B translocation facilitating domain,
a BoNT/C1 translocation
facilitating domain, a BoNT/D translocation facilitating domain, a BoNT/E
translocation facilitating domain,
a BoNT/F translocation facilitating domain, a BoNT/G translocation
facilitating domain, a TeNT
translocation facilitating domain, or active fragment thereof. In yet another
aspect of this embodiment, a
polynucleotide molecule encoding a translocation domain comprises a naturally
occurring enveloped virus
fusogenic peptide domain variant, such as, e.g., an enveloped virus fusogenic
peptide domain isoform or
an enveloped virus fusogenic peptide domain subtype. In another aspect of this
embodiment, a
polynucleotide molecule encoding a translocation domain comprises a non-
naturally occurring enveloped
virus fusogenic peptide domain variant, such as, e.g., a conservative
enveloped virus fusogenic peptide
domain variant, a non-conservative enveloped virus fusogenic peptide domain
variant or an active
enveloped virus fusogenic peptide domain fragment, or any combination thereof.
In other aspects of this
embodiment, a polynucleotide molecule encoding an enveloped virus fusogenic
peptide domain
comprisesan influenzavirus fusogenic peptide domain, an alphavirus fusogenic
peptide domain, a
vesiculovirus fusogenic peptide domain, a respirovirus fusogenic peptide
domain, a morbillivirus
fusogenic peptide domain, an avulavirus fusogenic peptide domain, a
henipavirus fusogenic peptide
domain, a metapneumovirus fusogenic peptide domain, a foamy virus fusogenic
peptide domain, or
active fragment thereof.
[0379] In another embodiment, a polynucleotide molecule encodes, in part, a
modified Clostridial toxin
comprising an altered targeting domain disclosed in the present specification.
In an aspect of this
embodiment, a polynucleotide molecule encoding an altered targeting domain
comprises a polypeptide
that selectively binds to a non-Clostridial toxin receptor present on a non-
Clostridial toxin target cell. In an
aspect of this embodiment, a polynucleotide molecule encoding a polypeptide
that selectively binds to a
non-Clostridial toxin receptor present on a non-Clostridial toxin target cell
comprises a naturally occurring
variant, such as, e.g., an isoform or a subtype. In another aspect of this
embodiment, a polynucleotide
molecule encoding a polypeptide that selectively binds to a non-Clostridial
toxin receptor present on a
non-Clostridial toxin target cell comprises a non-naturally occurring variant,
such as, e.g., a conservative
variant, a non-conservative variant or an active fragment, or any combination
thereof. In other aspects of
this embodiment, a polynucleotide molecule encoding a polypeptide that
selectively binds to a non-
Clostridial toxin receptor present on a non-Clostridial toxin target cell
comprises an opioid peptide, such
as, e.g., an enkephalin, a bovine adrenomedullary-22 (BAM22) peptide, an
endomorphin, an endorphin, a
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dynorphin, a nociceptin or a hemorphin; a melanocortin peptide, such as, e.g.,
an a-melanocyte
stimulating hormones (a-MSH), a(3-melanocyte stimulating hormones ((3-MSH), a
y-melanocyte
stimulating hormones (y-MSH), an adrenocorticotropin (ACTH), a Corticotropin-
like intermediary peptide
(CLIP), a(3-lipotropin ((3-LPH) and a y-lipotropin (y-LPH); a galanin, such
as, e.g., a galanin and a galanin
message-associated peptide (GMAP); a granin, such as, e.g., a chromogranin A
peptide like a(3-granin, a
vasostatin, a chromostatin, a pancreastatin, a WE-14, a catestatin, a
parastatin and a GE-25, a
chromogranin B (secretogranin I) peptide like a GAWK peptide, a
adrenomedullary peptide and a
secretolytin and a chromogranin C (secretogranin II) peptide like
secretoneurin, EM66 and manserin; a
tachykinin peptide, such as, e.g., Substance P, neuropeptide K (NPK),
neuropeptide gamma (NP
gamma), neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L),
neurokinin B (NKB), a
hemokinin and a endokinin; a cholecystokinin, such as, e.g., a cholecystokinin
58, a cholecystokinin 39,
a cholecystokinin 33, a cholecystokinin 12 and a cholecystokinin 8; a
Neuropeptide Y related peptide,
such as, e.g., a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide
(PP) and a Pancreatic
icosapeptide (PIP); , a kinin peptide, such as, e.g., a bradykinin, a
kallidan, a desArg9 bradykinin and a
desArg10 bradykinin; a protease activated receptor (PAR) peptide, such as,
e.g., a PAR1 peptide, a PAR2
peptide, a PAR3 peptide and a PAR4 peptide; a corticotropin-releasing hormone;
a thyrotropin-releasing
hormone; a somatostatin; a leukemia inhibitor factor (LIF); and an interleukin-
1 ( IL1).
[0380] In another embodiment, a polynucleotide molecule encodes, in part, a
modified Clostridial toxin
comprising a protease cleavage site disclosed in the present specification. In
an aspect of this
embodiment, a polynucleotide molecule encoding a protease cleavage site
comprises an endogenous
Clostridial toxin protease site. In aspects of this embodiment, a
polynucleotide molecule encoding an
endogenous Clostridial toxin protease site can be, e.g., a BoNT/A di-chain
loop protease cleavage site, a
BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop protease
cleavage site, a BoNT/D
di-chain loop protease cleavage site, a BoNT/E di-chain loop protease cleavage
site, a BoNT/F di-chain
loop protease cleavage site, a BoNT/G di-chain loop protease cleavage site or
a TeNT di-chain loop
protease cleavage site. In another aspect of this embodiment, a polynucleotide
molecule encoding a
protease cleavage site comprises an exogenous Clostridial toxin protease site.
In aspects of this
embodiment, a polynucleotide molecule encoding an exogenous Clostridial toxin
protease site can be,
e.g., a bovine enterokinase protease cleavage site, a Tobacco Etch Virus
protease cleavage site, a
Human Rhinovirus 3C protease cleavage site, a SUMO/ULP-1 protease cleavage
site, a Thrombin
protease cleavage site, a Coagulation Factor Xa protease cleavage site or a
Clostridial toxin substrate
cleavage site, such as, e.g., a BoNT/A substrate cleavage site, a BoNT/B
substrate cleavage site, a
BoNT/C1 substrate cleavage site, a BoNT/D substrate cleavage site, a BoNT/E
substrate cleavage site, a
BoNT/F substrate cleavage site, a BoNT/G substrate cleavage site or a TeNT
substrate cleavage site.
[0381] In another embodiment, a polynucleotide molecule encodes, in part, a
modified Clostridial toxin
comprising a flexible spacer disclosed in the present specification. In an
aspect of this embodiment, a
polynucleotide molecule encoding a flexible spacer a G-spacer, a A-spacer of
any combination thereof.
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[0382] Well-established molecular biology techniques that may be necessary to
make a polynucleotide
molecule encoding a modified Clostridial toxin disclosed in the present
specification including, but not
limited to, procedures involving polymerase chain reaction (PCR)
amplification, restriction enzyme
reactions, agarose gel electrophoresis, nucleic acid ligation, bacterial
transformation, nucleic acid
purification, nucleic acid sequencing and recombination-based techniques are
routine procedures well
within the scope of one skilled in the art and from the teaching herein. Non-
limiting examples of specific
protocols necessary to make a polynucleotide molecule encoding a modified
Clostridial toxin are
described in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); and
CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons,
2004). Additionally, a
variety of commercially available products useful for making a polynucleotide
molecule encoding a
modified Clostridial toxin are widely available. These protocols are routine
procedures well within the
scope of one skilled in the art and from the teaching herein.
[0383] Another aspect of the present invention provides a method of producing
a modified Clostridial
toxin disclosed in the present specification, such method comprising the step
of expressing a
polynucleotide molecule encoding a modified Clostridial toxin in a cell.
Another aspect of the present
invention provides a method of producing a modified Clostridial toxin
disclosed in the present
specification, such method comprising the steps of introducing an expression
construct comprising a
polynucleotide molecule encoding a modified Clostridial toxin into a cell and
expressing the expression
construct in the cell.
[0384] The methods disclosed in the present specification include, in part, a
modified Clostridial toxin. It
is envisioned that any and all modified Clostridial toxins disclosed in the
present specification can be
produced using the methods disclosed in the present specification. It is also
envisioned that any and all
polynucleotide molecules encoding a modified Clostridial toxins disclosed in
the present specification can
be useful in producing a modified Clostridial toxins disclosed in the present
specification using the
methods disclosed in the present specification.
[0385] The methods disclosed in the present specification include, in part, an
expression construct. An
expression construct comprises a polynucleotide molecule disclosed in the
present specification
operably-linked to an expression vector useful for expressing the
polynucleotide molecule in a cell or cell-
free extract. A wide variety of expression vectors can be employed for
expressing a polynucleotide
molecule encoding a modified Clostridial toxin, including, without limitation,
a viral expression vector; a
prokaryotic expression vector; eukaryotic expression vectors, such as, e.g., a
yeast expression vector, an
insect expression vector and a mammalian expression vector; and a cell-free
extract expression vector. It
is further understood that expression vectors useful to practice aspects of
these methods may include
those which express a modified Clostridial toxin under control of a
constitutive, tissue-specific, cell-
specific or inducible promoter element, enhancer element or both. Non-limiting
examples of expression
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vectors, along with well-established reagents and conditions for making and
using an expression
construct from such expression vectors are readily available from commercial
vendors that include,
without limitation, BD Biosciences-Clontech, Palo Alto, CA; BD Biosciences
Pharmingen, San Diego, CA;
Invitrogen, Inc, Carlsbad, CA; EMD Biosciences-Novagen, Madison, WI; QIAGEN,
Inc., Valencia, CA;
and Stratagene, La Jolla, CA. The selection, making and use of an appropriate
expression vector are
routine procedures well within the scope of one skilled in the art and from
the teachings herein.
[0386] Thus, aspects of this embodiment include, without limitation, a viral
expression vector operably-
linked to a polynucleotide molecule encoding a modified Clostridial toxin; a
prokaryotic expression vector
operably-linked to a polynucleotide molecule encoding a modified Clostridial
toxin; a yeast expression
vector operably-linked to a polynucleotide molecule encoding a modified
Clostridial toxin; an insect
expression vector operably-linked to a polynucleotide molecule encoding a
modified Clostridial toxin; and
a mammalian expression vector operably-linked to a polynucleotide molecule
encoding a modified
Clostridial toxin. Other aspects of this embodiment include, without
limitation, expression constructs
suitable for expressing a modified Clostridial toxin disclosed in the present
specification using a cell-free
extract comprising a cell-free extract expression vector operably linked to a
polynucleotide molecule
encoding a modified Clostridial toxin.
[0387] The methods disclosed in the present specification include, in part, a
cell. It is envisioned that
any and all cells can be used. Thus, aspects of this embodiment include,
without limitation, prokaryotic
cells including, without limitation, strains of aerobic, microaerophilic,
capnophilic, facultative, anaerobic,
gram-negative and gram-positive bacterial cells such as those derived from,
e.g., Escherichia coli,
Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis, Clostridia
perfringens, Clostridia difficile,
Caulobacter crescentus, Lactococcus lactis, Methylobacterium extorquens,
Neisseria meningirulls,
Neisseria meningitidis, Pseudomonas fluorescens and Salmonella typhimurium;
and eukaryotic cells
including, without limitation, yeast strains, such as, e.g., those derived
from Pichia pastoris, Pichia
methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces
cerevisiae and Yarrowia
lipolytica; insect cells and cell lines derived from insects, such as, e.g.,
those derived from Spodoptera
frugiperda, Trichoplusia ni, Drosophila melanogaster and Manduca sexta; and
mammalian cells and cell
lines derived from mammalian cells, such as, e.g., those derived from mouse,
rat, hamster, porcine,
bovine, equine, primate and human. Cell lines may be obtained from the
American Type Culture
Collection, European Collection of Cell Cultures and the German Collection of
Microorganisms and Cell
Cultures. Non-limiting examples of specific protocols for selecting, making
and using an appropriate cell
line are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F. A.
Goosen et al. eds., Marcel
Dekker, 1993); INSECT CELL CULTURES: FUNDAMENTAL AND APPLIED ASPECTS (J. M.
Vlak et al. eds., Kluwer
Academic Publishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL
TECHNIQUES OF CELL CULTURE
(Cambridge University Press, 1997); CELL AND TISSUE CULTURE: LABORATORY
PROCEDURES (Alan Doyle et
al eds., John Wiley and Sons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS:
A MANUAL OF BASIC
TECHNIQUE (Wiley-Liss, 4th ed. 2000); ANIMAL CELL CULTURE: A PRACTICAL
APPROACH (John R. W. Masters
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ed., Oxford University Press, 3~d ed. 2000); MOLECULAR CLONING A LABORATORY
MANUAL, supra, (2001);
BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press, 2"d ed.
2002); and CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004). These protocols are routine
procedures within the
scope of one skilled in the art and from the teaching herein.
[0388] The methods disclosed in the present specification include, in part,
introducing into a cell a
polynucleotide molecule. A polynucleotide molecule introduced into a cell can
be transiently or stably
maintained by that cell. Stably-maintained polynucleotide molecules may be
extra-chromosomal and
replicate autonomously, or they may be integrated into the chromosomal
material of the cell and replicate
non-autonomously. It is envisioned that any and all methods for introducing a
polynucleotide molecule
disclosed in the present specification into a cell can be used. Methods useful
for introducing a nucleic
acid molecule into a cell include, without limitation, chemical-mediated
transfection such as, e.g., calcium
phosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated, lipid-
mediated, polyethyleneimine
(PEI)-mediated, polylysine-mediated and polybrene-mediated; physical-mediated
tranfection, such as,
e.g., biolistic particle delivery, microinjection, protoplast fusion and
electroporation; and viral-mediated
transfection, such as, e.g., retroviral-mediated transfection, see, e.g.,
Introducing Cloned Genes into
Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds., Molecular
Cloning A Laboratory
Manual, Vol. 3, 3~d ed. 2001). One skilled in the art understands that
selection of a specific method to
introduce an expression construct into a cell will depend, in part, on whether
the cell will transiently
contain an expression construct or whether the cell will stably contain an
expression construct. These
protocols are routine procedures within the scope of one skilled in the art
and from the teaching herein.
[0389] In an aspect of this embodiment, a chemical-mediated method, termed
transfection, is used to
introduce a polynucleotide molecule encoding a modified Clostridial toxin into
a cell. In chemical-
mediated methods of transfection the chemical reagent forms a complex with the
nucleic acid that
facilitates its uptake into the cells. Such chemical reagents include, without
limitation, calcium phosphate-
mediated, see, e.g., Martin Jordan & Florian Worm, Transfection of adherent
and suspended cells by
calcium phosphate, 33(2) Methods 136-143 (2004); diethyl-aminoethyl (DEAE)
dextran-mediated, lipid-
mediated, cationic polymer-mediated like polyethyleneimine (PEI)-mediated and
polylysine-mediated and
polybrene-mediated, see, e.g., Chun Zhang et al., Polyethylenimine strategies
for plasmid delivery to
brain-derived cells, 33(2) Methods 144-150 (2004). Such chemical-mediated
delivery systems can be
prepared by standard methods and are commercially available, see, e.g.,
CellPhect Transfection Kit
(Amersham Biosciences, Piscataway, NJ); Mammalian Transfection Kit, Calcium
phosphate and DEAE
Dextran, (Stratagene, Inc., La Jolla, CA); LipofectamineTM Transfection
Reagent (Invitrogen, Inc.,
Carlsbad, CA); ExGen 500 Transfection kit (Fermentas, Inc., Hanover, MD), and
SuperFect and
Effectene Transfection Kits (Qiagen, Inc., Valencia, CA).
[0390] In another aspect of this embodiment, a physical-mediated method is
used to introduce a
polynucleotide molecule encoding a modified Clostridial toxin into a cell.
Physical techniques include,
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without limitation, electroporation, biolistic and microinjection. Biolistics
and microinjection techniques
perforate the cell wall in order to introduce the nucleic acid molecule into
the cell, see, e.g., Jeike E.
Biewenga et al., Plasmid-mediated gene transfer in neurons using the
biolistics technique, 71(1) J.
Neurosci. Methods. 67-75 (1997); and John O'Brien & Sarah C. R. Lummis,
Biolistic and diolistic
transfection: using the gene gun to deliver DNA and lipophilic dyes into
mammalian cells, 33(2) Methods
121-125 (2004). Electroporation, also termed electropermeabilization, uses
brief, high-voltage, electrical
pulses to create transient pores in the membrane through which the nucleic
acid molecules enter and can
be used effectively for stable and transient transfections of all cell types,
see, e.g., M. Golzio et al., In vitro
and in vivo electric field-mediated permeabilization, gene transfer, and
expression, 33(2) Methods 126-
135 (2004); and Oliver Greschet al., New non-viral method for gene transfer
into primary cells, 33(2)
Methods 151-163 (2004).
[0391] In another aspect of this embodiment, a viral-mediated method, termed
transduction, is used to
introduce a polynucleotide molecule encoding a modified Clostridial toxin into
a cell. In viral-mediated
methods of transient transduction, the process by which viral particles infect
and replicate in a host cell
has been manipulated in order to use this mechanism to introduce a nucleic
acid molecule into the cell.
Viral-mediated methods have been developed from a wide variety of viruses
including, without limitation,
retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses,
picornaviruses,
alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral and MLV
based retroviral vectors for
ex vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); and Maurizio
Federico, From
lentiviruses to lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M.
Poeschla, Non-primate
lentiviral vectors, 5(5) Curr. Opin. Mol. Ther. 529-540 (2003); Karim Benihoud
et al, Adenovirus vectors
for gene delivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler,
Adeno-associated viral vectors
for gene transfer and gene therapy, 380(6) Biol. Chem. 613-622 (1999); Chooi
M. Lai et al., Adenovirus
and adeno-associated virus vectors, 21(12) DNA Cell Biol. 895-913 (2002);
Edward A. Burton et al., Gene
delivery using herpes simplex virus vectors, 21(12) DNA Cell Biol. 915-936
(2002); Paola Grandi et al.,
Targeting HSV amplicon vectors, 33(2) Methods 179-186 (2004); Ilya Frolov et
al., Alphavirus-based
expression vectors: strategies and applications, 93(21) Proc. Natl. Acad. Sci.
U. S. A. 11371-11377
(1996); Markus U. Ehrengruber, Alphaviral gene transfer in neurobiology, 59(1)
Brain Res. Bull. 13-22
(2002); Thomas A. Kost & J. Patrick Condreay, Recombinant baculoviruses as
mammalian cell gene-
delivery vectors, 20(4) Trends Biotechnol. 173-180 (2002); and A. Huser & C.
Hofmann, Baculovirus
vectors: novel mammalian cell gene-delivery vehicles and their applications,
3(1) Am. J.
Pharmacogenomics 53-63 (2003).
[0392] Adenoviruses, which are non-enveloped, double-stranded DNA viruses, are
often selected for
mammalian cell transduction because adenoviruses handle relatively large
polynucleotide molecules of
about 36 kb, are produced at high titer, and can efficiently infect a wide
variety of both dividing and non-
dividing cells, see, e.g., Wim T. J. M. C. Hermens et al., Transient gene
transfer to neurons and glia:
analysis of adenoviral vector performance in the CNS and PNS, 71(1) J.
Neurosci. Methods 85-98 (1997);
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and Hiroyuki Mizuguchi et al., Approaches for generating recombinant
adenovirus vectors, 52(3) Adv.
Drug Deliv. Rev. 165-176 (2001). Transduction using adenoviral-based system do
not support prolonged
protein expression because the nucleic acid molecule is carried from an
episome in the cell nucleus,
rather than being integrated into the host cell chromosome. Adenoviral vector
systems and specific
protocols for how to use such vectors are disclosed in, e.g., ViraPowerTM
Adenoviral Expression System
(Invitrogen, Inc., Carlsbad, CA) and ViraPowerTM Adenoviral Expression System
Instruction Manual 25-
0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and AdEasyTM Adenoviral
Vector System (Stratagene,
Inc., La Jolla, CA) and AdEasyTM Adenoviral Vector System Instruction Manual
064004f, Stratagene, Inc..
[0393] Nucleic acid molecule delivery can also use single-stranded RNA
retroviruses, such as, e.g.,
oncoretroviruses and lentiviruses. Retroviral-mediated transduction often
produce transduction
efficiencies close to 100%, can easily control the proviral copy number by
varying the multiplicity of
infection (MOI), and can be used to either transiently or stably transduce
cells, see, e.g., Tiziana Tonini et
al., Transient production of retroviral- and lentiviral-based vectors for the
transduction of Mammalian cells,
285 Methods Mol. Biol. 141-148 (2004); Armin Blesch, Lentiviral and MLV based
retroviral vectors for ex
vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); Felix Recillas-
Targa, Gene transfer and
expression in mammalian cell lines and transgenic animals, 267 Methods Mol.
Biol. 417-433 (2004); and
Roland Wolkowicz et al., Lentiviral vectors for the delivery of DNA into
mammalian cells, 246 Methods
Mol. Biol. 391-411 (2004). Retroviral particles consist of an RNA genome
packaged in a protein capsid,
surrounded by a lipid envelope. The retrovirus infects a host cell by
injecting its RNA into the cytoplasm
along with the reverse transcriptase enzyme. The RNA template is then reverse
transcribed into a linear,
double stranded cDNA that replicates itself by integrating into the host cell
genome. Viral particles are
spread both vertically (from parent cell to daughter cells via the provirus)
as well as horizontally (from cell
to cell via virions). This replication strategy enables long-term persistent
expression since the nucleic
acid molecules of interest are stably integrated into a chromosome of the host
cell, thereby enabling long-
term expression of the protein. For instance, animal studies have shown that
lentiviral vectors injected
into a variety of tissues produced sustained protein expression for more than
1 year, see, e.g., Luigi
Naldini et al., In vivo gene delivery and stable transduction of non-dividing
cells by a lentiviral vector,
272(5259) Science 263-267 (1996). The Oncoretroviruses-derived vector systems,
such as, e.g.,
Moloney murine leukemia virus (MoMLV), are widely used and infect many
different non-dividing cells.
Lentiviruses can also infect many different cell types, including dividing and
non-dividing cells and
possess complex envelope proteins, which allows for highly specific cellular
targeting.
[0394] Retroviral vectors and specific protocols for how to use such vectors
are disclosed in, e.g., U.S.
Patent Nos. Manfred Gossen & Hermann Bujard, Tight control of gene expression
in eukaryotic cells by
tetracycline-responsive promoters, U.S. Patent No. 5,464,758 (Nov. 7, 1995)
and Hermann Bujard &
Manfred Gossen, Methods for regulating gene expression, U.S. Patent No.
5,814,618 (Sep. 29, 1998)
David S. Hogness, Polynucleotides encoding insect steroid hormone receptor
polypeptides and cells
transformed with same, U.S. Patent No. 5,514,578 (May 7, 1996) and David S.
Hogness, Polynucleotide
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encoding insect ecdysone receptor, U.S. Patent 6,245,531 (Jun. 12, 2001);
Elisabetta Vegeto et al.,
Progesterone receptor having C. terminal hormone binding domain truncations,
U.S. Patent No.
5,364,791 (Nov. 15, 1994), Elisabetta Vegeto et al., Mutated steroid hormone
receptors, methods for their
use and molecular switch for gene therapy, U.S. Patent No. 5,874,534 (Feb. 23,
1999) and Elisabetta
Vegeto et al., Mutated steroid hormone receptors, methods for their use and
molecular switch for gene
therapy, U.S. Patent No. 5,935,934 (Aug. 10, 1999). Furthermore, such viral
delivery systems can be
prepared by standard methods and are commercially available, see, e.g., BDT"'
Tet-Off and Tet-On Gene
Expression Systems (BD Biosciences-Clonetech, Palo Alto, CA) and BDT"' Tet-Off
and Tet-On Gene
Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech, (Mar. 14,
2003), GeneSwitchr"'
System (Invitrogen, Inc., Carlsbad, CA) and GeneSwitchT"' System A
Mifepristone-Regulated Expression
System for Mammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4,
2002); ViraPowerTM Lentiviral
Expression System (Invitrogen, Inc., Carlsbad, CA) and ViraPowerTM Lentiviral
Expression System
Instruction Manual 25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and
Complete Control Retroviral
Inducible Mammalian Expression System (Stratagene, La Jolla, CA) and Complete
Control Retroviral
Inducible Mammalian Expression System Instruction Manual, 064005e.
[0395] The methods disclosed in the present specification include, in part,
expressing a modified
Clostridial toxin from a polynucleotide molecule. It is envisioned that any of
a variety of expression
systems may be useful for expressing a modified Clostridial toxin from a
polynucleotide molecule
disclosed in the present specification, including, without limitation, cell-
based systems and cell-free
expression systems. Cell-based systems include, without limitation, viral
expression systems, prokaryotic
expression systems, yeast expression systems, baculoviral expression systems,
insect expression
systems and mammalian expression systems. Cell-free systems include, without
limitation, wheat germ
extracts, rabbit reticulocyte extracts and E. coli extracts and generally are
equivalent to the method
disclosed herein. Expression of a polynucleotide molecule using an expression
system can include any
of a variety of characteristics including, without limitation, inducible
expression, non-inducible expression,
constitutive expression, viral-mediated expression, stably-integrated
expression, and transient
expression. Expression systems that include well-characterized vectors,
reagents, conditions and cells
are well-established and are readily available from commercial vendors that
include, without limitation,
Ambion, Inc. Austin, TX; BD Biosciences-Clontech, Palo Alto, CA; BD
Biosciences Pharmingen, San
Diego, CA; Invitrogen, Inc, Carlsbad, CA; QIAGEN, Inc., Valencia, CA; Roche
Applied Science,
Indianapolis, IN; and Stratagene, La Jolla, CA. Non-limiting examples on the
selection and use of
appropriate heterologous expression systems are described in e.g., PROTEIN
EXPRESSION. A PRACTICAL
APPROACH (S. J. Higgins and B. David Hames eds., Oxford University Press,
1999); Joseph M.
Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE
ART OF EXPRESSION
(Academic Press, 1999); and Meena Rai & Harish Padh, Expression Systems for
Production of
Heterologous Proteins, 80(9) CURRENT SCIENCE 1121-1128, (2001). These
protocols are routine
procedures well within the scope of one skilled in the art and from the
teaching herein.
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[0396] A variety of cell-based expression procedures are useful for expressing
a modified Clostridial
toxin encoded by polynucleotide molecule disclosed in the present
specification. Examples included,
without limitation, viral expression systems, prokaryotic expression systems,
yeast expression systems,
baculoviral expression systems, insect expression systems and mammalian
expression systems. Viral
expression systems include, without limitation, the ViraPowerTM Lentiviral
(Invitrogen, Inc., Carlsbad, CA),
the Adenoviral Expression Systems (Invitrogen, Inc., Carlsbad, CA), the
AdEasyTM XL Adenoviral Vector
System (Stratagene, La Jolla, CA) and the ViraPort Retroviral Gene Expression
System (Stratagene, La
Jolla, CA). Non-limiting examples of prokaryotic expression systems include
the ChampionTM pET
Expression System (EMD Biosciences-Novagen, Madison, WI), the TriExTM
Bacterial Expression System
(EMD Biosciences-Novagen, Madison, WI), the QlAexpress Expression System
(QIAGEN, Inc.), and the
Affinity Protein Expression and Purification System (Stratagene, La Jolla,
CA). Yeast expression
systems include, without limitation, the EasySelectT"' Pichia Expression Kit
(Invitrogen, Inc., Carlsbad,
CA), the YES-EchoTM Expression Vector Kits (Invitrogen, Inc., Carlsbad, CA )
and the SpECTRA TM S.
pombe Expression System (Invitrogen, Inc., Carlsbad, CA). Non-limiting
examples of baculoviral
expression systems include the BaculoDirectT"' (Invitrogen, Inc., Carlsbad,
CA), the Bac-to-Bac
(Invitrogen, Inc., Carlsbad, CA), and the BD BaculoGoldT"' (BD Biosciences-
Pharmigen, San Diego, CA).
Insect expression systems include, without limitation, the Drosophila
Expression System (DES )
(Invitrogen, Inc., Carlsbad, CA), InsectSelectT"' System (Invitrogen, Inc.,
Carlsbad, CA) and
InsectDirectT"' System (EMD Biosciences-Novagen, Madison, WI). Non-limiting
examples of mammalian
expression systems include the T-RExTM (Tetracycline-Regulated Expression)
System (Invitrogen, Inc.,
Carlsbad, CA), the Flp-InTM T-RExTM System (Invitrogen, Inc., Carlsbad, CA),
the pcDNA TM system
(Invitrogen, Inc., Carlsbad, CA), the pSecTag2 system (Invitrogen, Inc.,
Carlsbad, CA), the Exchanger
System, InterPlayTM Mammalian TAP System (Stratagene, La Jolla, CA), Complete
Control Inducible
Mammalian Expression System (Stratagene, La Jolla, CA) and LacSwitch II
Inducible Mammalian
Expression System (Stratagene, La Jolla, CA).
[0397] Another procedure of expressing a modified Clostridial toxin encoded by
polynucleotide molecule
disclosed in the present specification employs a cell-free expression system
such as, without limitation,
prokaryotic extracts and eukaryotic extracts. Non-limiting examples of
prokaryotic cell extracts include
the RTS 100 E. coli HY Kit (Roche Applied Science, Indianapolis, IN), the
ActivePro In Vitro Translation
Kit (Ambion, Inc., Austin, TX), the EcoProTM System (EMD Biosciences-Novagen,
Madison, WI) and the
ExpresswayTM Plus Expression System (Invitrogen, Inc., Carlsbad, CA).
Eukaryotic cell extract include,
without limitation, the RTS 100 Wheat Germ CECF Kit (Roche Applied Science,
Indianapolis, IN), the
TnT Coupled Wheat Germ Extract Systems (Promega Corp., Madison, WI), the
Wheat Germ IVTT"' Kit
(Ambion, Inc., Austin, TX), the Retic Lysate IVTT"' Kit (Ambion, Inc., Austin,
TX), the PROTEINscript II
System (Ambion, Inc., Austin, TX) and the TnT Coupled Reticulocyte Lysate
Systems (Promega Corp.,
Madison, WI).
[0398] Other aspects of this invention can be described as follows:
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1. A modified Clostridial toxin comprising:
a) a Clostridial toxin enzymatic domain capable of executing an enzymatic
target modification step
of a Clostridial toxin intoxication process;
b) a Clostridial toxin translocation domain capable of executing a
translocation step of a Clostridial
toxin intoxication process;
c) a translocation facilitating domain capable of facilitating a translocation
step of a Clostridial toxin
intoxication process;
d) an altered targeting domain capable of selectively binding a non-
Clostridial toxin receptor present
on a non-Clostridial toxin target cell and executing a cell binding step of a
Clostridial toxin
intoxication process; and
e) a protease cleavage site
wherein cleavage of the protease cleavage site converts the single-chain form
of the modified
Clostridial toxin into the di-chain form.
2. The modified Clostridial toxin according to 1, wherein the modified
Clostridial toxin comprises, in a
linear amino-to-carboxyl single polypeptide order, the Clostridial toxin
enzymatic domain, the
protease cleavage site, the Clostridial toxin translocation domain, the
translocation facilitating domain
and the altered targeting domain.
3. The modified Clostridial toxin according to 1, wherein the modified
Clostridial toxin comprises, in a
linear amino-to-carboxyl single polypeptide order, the Clostridial toxin
enzymatic domain, the
protease cleavage site, the altered targeting domain, the Clostridial toxin
translocation domain and
the translocation facilitating domain.
4. The modified Clostridial toxin according to 1, wherein the modified
Clostridial toxin comprises, in a
linear amino-to-carboxyl single polypeptide order, the altered targeting
domain, the Clostridial toxin
translocation domain, the translocation facilitating domain, the protease
cleavage site and the
Clostridial toxin enzymatic domain.
5. The modified Clostridial toxin according to 1, wherein the modified
Clostridial toxin comprises, in a
linear amino-to-carboxyl single polypeptide order, the altered targeting
domain, the Clostridial toxin
enzymatic domain, the protease cleavage site, the Clostridial toxin
translocation domain and the
translocation facilitating domain.
6. The modified Clostridial toxin according to 1, wherein the modified
Clostridial toxin comprises, in a
linear amino-to-carboxyl single polypeptide order, the Clostridial toxin
translocation domain, the
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translocation facilitating domain, the protease cleavage site, the Clostridial
toxin enzymatic domain
and the altered targeting domain.
7. The modified Clostridial toxin according to 1, wherein the modified
Clostridial toxin comprises, in a
linear amino-to-carboxyl single polypeptide order, the Clostridial toxin
translocation domain, the
translocation facilitating domain, the protease cleavage site, the altered
targeting domain and the
Clostridial toxin enzymatic domain.
8. The modified Clostridial toxin according to 1, wherein the Clostridial
toxin enzymatic domain is
selected from the group consisting of a BoNT/A enzymatic domain, a BoNT/B
enzymatic domain, a
BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic
domain, a BoNT/F
enzymatic domain, a BoNT/G enzymatic domain and a TeNT enzymatic domain.
9. The modified Clostridial toxin according to 1, wherein the Clostridial
toxin translocation domain is
selected from the group consisting of a BoNT/A translocation domain, a BoNT/B
translocation
domain, a BoNT/C1 translocation domain, a BoNT/D translocation domain, a
BoNT/E translocation
domain, a BoNT/F translocation domain, a BoNT/G translocation domain and a
TeNT translocation
domain.
10. The modified Clostridial toxin according to 1, wherein the translocation
facilitating domain is a
Clostridial toxin translocation facilitating domain.
11. The modified Clostridial toxin according to 10, wherein the Clostridial
toxin translocation facilitating
domain is selected from the group consisting of a BoNT/A translocation
facilitating domain, a BoNT/B
translocation facilitating domain, a BoNT/C1 translocation facilitating
domain, a BoNT/D translocation
domain, a BoNT/E translocation facilitating domain, a BoNT/F translocation
facilitating domain, a
BoNT/G translocation facilitating domain and a TeNT translocation facilitating
domain.
12. The modified Clostridial toxin according to 10, wherein the Clostridial
toxin translocation facilitating
domain comprises an amino acid sequence selected from the group consisting of
amino acids 874-
1110 of SEQ ID NO: 1, amino acids 861-1097of SEQ ID NO: 2, amino acids 869-
1111 of SEQ ID NO:
3, amino acids 865-1098 of SEQ ID NO: 4, amino acids 848-1085 of SEQ ID NO: 5,
amino acids 867-
1105 of SEQ ID NO: 6, amino acids 866-1105 of SEQ ID NO: 7 and amino acid 882-
1127 of SEQ ID
NO: 8.
13. The modified Clostridial toxin according to 10, wherein the Clostridial
toxin translocation facilitating
domain is selected from the group consisting of a BoNT/A translocation
facilitating domain, a BoNT/B
translocation facilitating domain, a BoNT/C1 translocation facilitating
domain, a BoNT/D translocation
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domain, a BoNT/E translocation facilitating domain, a BoNT/F translocation
facilitating domain, a
BoNT/G translocation facilitating domain and a TeNT translocation facilitating
domain.
14. The modified Clostridial toxin according to 1, wherein the translocation
facilitating domain is an
enveloped virus fusogenic peptide domain.
15. The modified Clostridial toxin according to 14, wherein the enveloped
virus fusogenic peptide domain
is selected from the group consisting of an influenzavirus fusogenic peptide
domain, an alphavirus
fusogenic peptide domain, a vesiculovirus fusogenic peptide domain, a
respirovirus fusogenic peptide
domain, a morbillivirus fusogenic peptide domain, an avulavirus fusogenic
peptide domain, a
henipavirus fusogenic peptide domain, a metapneumovirus fusogenic peptide
domain and a foamy
virus fusogenic peptide domain.
16. The modified Clostridial toxin according to 15, wherein the influenzavirus
fusogenic peptide domain is
selected from the group consisting of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID
NO: 196, SEQ ID
NO: 197 and SEQ ID NO: 198.
17. The modified Clostridial toxin according to 15, wherein the alphavirus
fusogenic peptide domain is
selected from the group consisting of SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID
NO: 201, SEQ ID
NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ
ID NO: 207,
SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO:
212, SEQ ID
NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ
ID NO: 218,
SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO:
223, SEQ ID
NO: 224 and SEQ ID NO: 225.
18. The modified Clostridial toxin according to 15, wherein the vesiculovirus
fusogenic peptide domain is
selected from the group consisting of SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID
NO: 228, SEQ ID
NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ
ID NO: 234,
SEQ ID NO: 235 and SEQ ID NO: 236.
19. The modified Clostridial toxin according to 15, wherein the respirovirus
fusogenic peptide domain is
selected from the group consisting of SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID
NO: 239, SEQ ID
NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 243.
20. The modified Clostridial toxin according to 15, wherein the morbillivirus
fusogenic peptide domain is
selected from the group consisting of SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID
NO: 246, SEQ ID
NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ
ID NO: 252
and SEQ ID NO: 253.
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21. The modified Clostridial toxin according to 15, wherein the avulavirus
fusogenic peptide domain is
selected from the group consisting of SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID
NO: 256, SEQ ID
NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ
ID NO: 262,
SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 and SEQ ID NO: 266.
22. The modified Clostridial toxin according to 15, wherein the henipavirus
fusogenic peptide domain is
selected from the group consisting of SEQ ID NO: 267 and SEQ ID NO: 268.
23. The modified Clostridial toxin according to 15, wherein the
metapneumovirus fusogenic peptide
domain is SEQ ID NO: 269.
24. The modified Clostridial toxin according to 1, wherein the altered
targeting domain is selected from
the group consisting of an opioid, a melanocortin, a galanin, a granin, a
tachykinin, a cholecystokinin,
a Neuropeptide Y related, a kinin peptide, a PAR peptide, a corticotropin-
releasing hormone, a
thyrotropin-releasing hormone and a somatostatin.
25. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises an
opioid selected from the group consisting of an enkephalin, a BAM22 peptide,
an endomorphin, an
endorphin, a dynorphin, a nociceptin and a hemorphin.
26. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises an
enkephalin selected from the group consisting of a Leu-enkephalin, a Met-
enkephalin, a Met-
enkephalin MRGL and a Met-enkephalin MRF
27. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises an
enkephalin selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11 and
SEQ ID NO: 12.
28. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises a
BAM22 peptide selected from the group consisting of a BAM22 peptide (1-12), a
BAM22 peptide (6-
22), a BAM22 peptide (8-22) or a BAM22 peptide (1-22).
29. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises a
BAM22 peptide selected from the group consisting of amino acids 1-12 of SEQ ID
NO: 172, amino
acids 6-22 of SEQ ID NO: 172, amino acids 8-22 of SEQ ID NO: 172, amino acids
1-22 of SEQ ID
NO: 172, amino acids 1-12 of SEQ ID NO: 173, amino acids 6-22 of SEQ ID NO:
173, amino acids 8-
22 of SEQ ID NO: 173, amino acids 1-22 of SEQ ID NO: 173, amino acids 1-12 of
SEQ ID NO: 174,
amino acids 6-22 of SEQ ID NO: 174, amino acids 8-22 of SEQ ID NO: 174, amino
acids 1-22 of
SEQ ID NO: 174, amino acids 1-12 of SEQ ID NO: 175, amino acids 6-22 of SEQ ID
NO: 175, amino
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acids 8-22 of SEQ ID NO: 175, amino acids 1-22 of SEQ ID NO: 175, amino acids
1-12 of SEQ ID
NO: 176, amino acids 6-22 of SEQ ID NO: 176, amino acids 8-22 of SEQ ID NO:
176, amino acids 1-
22 of SEQ ID NO: 176, amino acids 1-12 of SEQ ID NO: 177, amino acids 6-22 of
SEQ ID NO: 177,
amino acids 8-22 of SEQ ID NO: 177 and amino acids 1-22 of SEQ ID NO: 177.
30. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises an
endomorphin selected from the group consisting of an endomorphin-1 and an
endomorphin-2.
31. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises an
endomorphin selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO:
14.
32. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises an
endorphin selected from the group consisting of an endorphin-a, a neoendorphin-
a, an endorphin-(3, a
neoendorphin-(3 or an endorphin-y.
33. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises an
endorphin selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
34. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises a
dynorphin selected from the group consisting of a dynorphin A, a dynorphin B
and a rimorphin.
35. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises a
dynorphin selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22,
SEQ ID NO: 23,
SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ
ID NO: 29,
SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ
ID NO: 35,
SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ
ID NO: 41,
SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ
ID NO: 47,
SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51.
36. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises a
nociceptin selected from the group consisting of a nociceptin RK, a
nociceptin, a neuropeptide 1, a
neuropeptide 2 or a neuropeptide 3.
37. The modified Clostridial toxin according to 25, wherein the altered
targeting domain comprises a
nociceptin selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 53,
SEQ ID NO: 54,
SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ
ID NO: 60
and SEQ ID NO: 61.
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38. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
melanocortin selected from the group consisting of an a-melanocyte stimulating
hormones (a-MSH),
a(3-melanocyte stimulating hormones ((3-MSH), a y-melanocyte stimulating
hormones (y-MSH), an
adrenocorticotropin (ACTH), a Corticotropin-like intermediary peptide (CLIP),
a(3-lipotropin ((3-LPH)
and a y-lipotropin (y-LPH).
39. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
melanocortin selected from the group consisting SEQ ID NO: 62, SEQ ID NO: 63,
SEQ ID NO: 64,
SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ
ID NO: 70
and SEQ ID NO: 71.
40. The modified Clostridial toxin according to 11, wherein the altered
targeting domain comprises a
galanin selected from the group consisting of a galanin and a galanin message-
associated peptide
(GMAP).
41. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
galanin selected from the group consisting of SEQ ID NO: 72 and SEQ ID NO: 73.
42. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
grainin selected from the group consisting of a chromogranin A peptide, a
chromogranin B peptide
and a chromogranin C peptide.
43. The modified Clostridial toxin according to 42, wherein the altered
targeting domain comprises a
chromogranin A peptide selected from the group consisting of a(3-granin, a
vasostatin, a
chromostatin, a pancreastatin, a WE-14, a catestatin, a parastatin and a GE-
25.
44. The modified Clostridial toxin according to 42, wherein the altered
targeting domain comprises a
chromogranin A peptide selected from the group consisting of SEQ ID NO: 74,
SEQ ID NO: 75, SEQ
ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 and SEQ
ID NO: 81
45. The modified Clostridial toxin according to 42, wherein the altered
targeting domain comprises a
chromogranin B peptide selected from the group consisting of a GAWK peptide,
an adrenomedullary
peptide and a secretolytin.
46. The modified Clostridial toxin according to 42, wherein the altered
targeting domain comprises a
chromogranin B peptide selected from the group consisting of SEQ ID NO: 82,
SEQ ID NO: 83, SEQ
ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86
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47. The modified Clostridial toxin according to 42, wherein the altered
targeting domain comprises a
chromogranin C peptide is a secretoneurin, a EM66 and a manserin.
48. The modified Clostridial toxin according to 42, wherein the altered
targeting domain comprises a
chromogranin C peptide is SEQ ID NO: 87.
49. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
tachykinin peptide selected from the group consisting of a Substance P, a
neuropeptide K (NPK), a
neuropeptide gamma, a neurokinin A, a neurokinin B, a hemokinin and a
endokinin.
50. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
tachykinin peptide selected from the group consisting of SEQ ID NO: 88, SEQ ID
NO: 89, SEQ ID
NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO:
95, SEQ ID
NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 and SEQ ID NO: 99.
51. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
cholecystokinin peptide selected from the group consisting of a
cholecystokinin 58, a cholecystokinin
39, a cholecystokinin 33, a cholecystokinin 12 and a cholecystokinin 8.
52. The modified Clostridial toxin according to 51, wherein the altered
targeting domain comprises a
cholecystokinin 58 peptide selected from the group consisting of SEQ ID NO:
100, SEQ ID NO: 101,
SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:
106, SEQ ID
NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ
ID NO: 112,
SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115.
53. The modified Clostridial toxin according to 51, wherein the altered
targeting domain comprises a
cholecystokinin 39 peptide selected from the group consisting of amino acids
20-58 of SEQ ID NO:
100, amino acids 20-58 of SEQ ID NO: 101, amino acids 20-58 of SEQ ID NO: 102,
amino acids 20-
58 of SEQ ID NO: 103, amino acids 20-58 of SEQ ID NO: 104, amino acids 20-58
of SEQ ID NO:
105, amino acids 20-58 of SEQ ID NO: 107, amino acids 20-58 of SEQ ID NO: 108,
amino acids 20-
58 of SEQ ID NO: 109, amino acids 20-58 of SEQ ID NO: 110, amino acids 20-58
of SEQ ID NO:
111, amino acids 20-58 of SEQ ID NO: 112, SEQ ID NO: 113, amino acids 20-58 of
SEQ ID NO: 114
or amino acids 20-58 of SEQ ID NO: 115.
54. The modified Clostridial toxin according to 51, wherein the altered
targeting domain comprises a
cholecystokinin 33 peptide selected from the group consisting of amino acids
26-58 of SEQ ID NO:
100, amino acids 26-58 of SEQ ID NO: 101, amino acids 26-58 of SEQ ID NO: 102,
amino acids 26-
58 of SEQ ID NO: 103, amino acids 26-58 of SEQ ID NO: 104, amino acids 26-58
of SEQ ID NO:
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105, amino acids 26-58 of SEQ ID NO: 107, amino acids 26-58 of SEQ ID NO: 108,
amino acids 26-
58 of SEQ ID NO: 109, amino acids 26-58 of SEQ ID NO: 110, amino acids 26-58
of SEQ ID NO:
111, amino acids 26-58 of SEQ ID NO: 112, amino acids 26-58 of SEQ ID NO: 113,
amino acids 26-
58 of SEQ ID NO: 114 and amino acids 26-58 of SEQ ID NO: 115.
55. The modified Clostridial toxin according to 51, wherein the altered
targeting domain comprises a
cholecystokinin 12 peptide selected from the group consisting of amino acids
47-58 of SEQ ID NO:
100, amino acids 47-58 of SEQ ID NO: 110 and amino acids 47-58 of SEQ ID NO:
114.
56. The modified Clostridial toxin according to 51, wherein the altered
targeting domain comprises a
cholecystokinin 8 peptide is amino acids 51-58 of SEQ ID NO: 100.
57. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
Neuropeptide Y related peptide selected from the group consisting of a
Neuropeptide Y (NPY), a
Peptide YY (PYY), a Pancreatic peptide (PP) and a Pancreatic icosapeptide
(PIP).
58. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a
Neuropeptide Y related peptide selected from the group consisting of SEQ ID
NO: 116, SEQ ID NO:
117, SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120.
59. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a kinin
peptide selected from the group consisting of a bradykinin, a kallidin, a
desArg9 bradykinin and a
desArg10 bradykinin.
60. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a kinin
peptide selected from the group consisting of SEQ ID NO: 178, SEQ ID NO: 179,
SEQ ID NO: 180
and SEQ ID NO: 181.
61. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a PAR
peptide selected from the group consisting of a PAR1 peptide, a PAR2 peptide,
a PAR3 peptide and
a PAR4 peptide.
62. The modified Clostridial toxin according to 24, wherein the altered
targeting domain comprises a PAR
peptide selected from the group consisting of SEQ ID NO: 182, SEQ ID NO: 283,
SEQ ID NO: 184
and SEQ ID NO: 185. amino acids 42-47 of SEQ ID NO: 182, amino acids 42-55 of
SEQ ID NO: 182,
amino acids 29-64 of SEQ ID NO: 182, amino acids 1-64 of SEQ ID NO: 182, amino
acids 35-40 of
SEQ ID NO: 183, amino acids 35-48 of SEQ ID NO: 183, amino acids 24-59 of SEQ
ID NO: 183,
amino acids 1-59 of SEQ ID NO: 183, amino acids 39-44 of SEQ ID NO: 184, amino
acids 39-52 of
SEQ ID NO: 184, amino acids 26-60 of SEQ ID NO: 184, amino acids 1-60 of SEQ
ID NO: 184,
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amino acids 48-53 of SEQ ID NO: 185, amino acids 48-61 of SEQ ID NO: 185,
amino acids 35-70 of
SEQ ID NO: 185 and amino acids 1-70 of SEQ ID NO: 185.
63. The modified Clostridial toxin according to 24, wherein the altered
targeting domain is selected from
the group consisting of SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID
NO: 124, SEQ
ID NO: 186 and SEQ ID NO: 187.
64. The modified Clostridial toxin according to 1, wherein the protease
cleavage site is an endogenous
Clostridial toxin di-chain loop protease cleavage site or an exogenous
cleavage site.
65. The modified Clostridial toxin according to 64, wherein the endogenous
Clostridial toxin di-chain loop
protease cleavage site is selected from the group consisting of a BoNT/A di-
chain loop protease
cleavage site, a BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-
chain loop protease
cleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/E di-
chain loop protease
cleavage site, a BoNT/F di-chain loop protease cleavage site, a BoNT/G di-
chain loop protease
cleavage site and a TeNT di-chain loop protease cleavage site.
66. The modified Clostridial toxin according to 64, wherein the exogenous
protease cleavage site is
selected from the group consisting of an enterokinase cleavage site, a
Thrombin cleavage site, a
Factor Xa cleavage site, a human rhinovirus 3C protease cleavage site, a
tobacco etch virus protease
cleavage site, a dipeptidyl aminopeptidase cleavage site, a small ubiquitin-
like modifier
(SUMO)/ubiquitin-like protein-1(ULP-1) protease cleavage site, and a
Clostridial toxin substrate
cleavage site.
67. The modified Clostridial toxin according to 66, wherein the Clostridial
toxin substrate cleavage site is
selected from the group consisting of a BoNT/A substrate cleavage site, a
BoNT/B substrate
cleavage site, a BoNT/C1 substrate cleavage site, a BoNT/D substrate cleavage
site, a BoNT/E
substrate cleavage site, a BoNT/F substrate cleavage site, a BoNT/G substrate
cleavage site and a
TeNT substrate cleavage site.
68. A polynucleotide molecule encoding a modified Clostridial toxin, the
polynucleotide molecule
comprising:
a) a polynucleotide molecule encoding a Clostridial toxin enzymatic domain
capable of executing an
enzymatic target modification step of a Clostridial toxin intoxication
process;
b) a polynucleotide molecule encoding a Clostridial toxin translocation domain
capable of executing
a translocation step of a Clostridial toxin intoxication process;
c) a polynucleotide molecule encoding a translocation facilitating domain
capable of facilitating a
translocation step of a Clostridial toxin intoxication process;
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d) a polynucleotide molecule encoding an altered targeting domain capable of
selectively binding a
non-Clostridial toxin receptor present on a Clostridial toxin target cell and
executing a cell binding
step of a Clostridial toxin intoxication process; and
e) a polynucleotide molecule encoding a protease cleavage site
wherein cleavage of the protease cleavage site converts the single-chain form
of the modified
Clostridial toxin into the di-chain form.
69. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encodes the
modified Clostridial toxin of any one of Claims 1-7.
70. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding a
Clostridial toxin enzymatic domain selected from the group consisting of a
polynucleotide molecule
encoding a BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1
enzymatic domain,
a BoNT/D enzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic
domain, a BoNT/G
enzymatic domain and a TeNT enzymatic domain.
71. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding a
Clostridial toxin translocation domain selected from the group consisting of a
polynucleotide molecule
encoding a BoNT/A translocation domain, a BoNT/B translocation domain, a
BoNT/C1 translocation
domain, a BoNT/D translocation domain, a BoNT/E translocation domain, a BoNT/F
translocation
domain, a BoNT/G translocation domain and a TeNT translocation domain.
72. The polynucleotide molecule according to 68, wherein the translocation
facilitating domain is a
Clostridial toxin translocation facilitating domain.
73. The polynucleotide molecule according to 72, wherein the Clostridial toxin
translocation facilitating
domain is selected from the group consisting of a BoNT/A translocation
facilitating domain, a BoNT/B
translocation facilitating domain, a BoNT/C1 translocation facilitating
domain, a BoNT/D translocation
domain, a BoNT/E translocation facilitating domain, a BoNT/F translocation
facilitating domain, a
BoNT/G translocation facilitating domain and a TeNT translocation facilitating
domain.
74. The polynucleotide molecule according to 68, wherein the translocation
facilitating domain is an
enveloped virus fusogenic peptide domain.
75. The polynucleotide molecule according to 74, wherein the enveloped virus
fusogenic peptide domain
is selected from the group consisting of an influenzavirus fusogenic peptide
domain, an alphavirus
fusogenic peptide domain, a vesiculovirus fusogenic peptide domain, a
respirovirus fusogenic peptide
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domain, a morbillivirus fusogenic peptide domain, an avulavirus fusogenic
peptide domain, a
henipavirus fusogenic peptide domain, a metapneumovirus fusogenic peptide
domain and a foamy
virus fusogenic peptide domain.
76. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain is selected from the group consisting of a
polynucleotide molecule encoding
an opioid, a melanocortin, a galanin, a granin, a tachykinin, a
cholecystokinin, a Neuropeptide Y
related, a kinin peptide, a PAR peptide, a corticotropin-releasing hormone, a
thyrotropin-releasing
hormone and a somatostatin.
77. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprising an opioid selected from the group
consisting of an enkephalin, a
BAM22 peptide, an endomorphin, an endorphin, a dynorphin, a nociceptin and a
hemorphin.
78. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a melanocortin selected from the group
consisting of a
polynucleotide molecule encoding an a-melanocyte stimulating hormones (a-MSH),
a(3-melanocyte
stimulating hormones ((3-MSH), a y-melanocyte stimulating hormones (y-MSH), an
adrenocorticotropin (ACTH), a Corticotropin-like intermediary peptide (CLIP),
a(3-lipotropin ((3-LPH)
and a y-lipotropin (y-LPH).
79. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a galanin selected from the group
consisting of a polynucleotide
molecule encoding a galanin and a galanin message-associated peptide (GMAP).
80. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a grainin selected from the group
consisting of a polynucleotide
molecule encoding a chromogranin A peptide, a chromogranin B peptide and a
chromogranin C
peptide.
81. The polynucleotide molecule according to 80, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a chromogranin A peptide selected from the
group consisting of a
polynucleotide molecule encoding a(3-granin, a vasostatin, a chromostatin, a
pancreastatin, a WE-14,
a catestatin, a parastatin and a GE-25.
82. The polynucleotide molecule according to 80, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a chromogranin B peptide selected from the
group consisting of a
polynucleotide molecule encoding a GAWK peptide, an adrenomedullary peptide
and a secretolytin.
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83. The polynucleotide molecule according to 80, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a chromogranin C peptide is a
polynucleotide molecule encoding
a secretoneurin.
84. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a tachykinin peptide selected from the
group consisting of a
polynucleotide molecule encoding a Substance P, a neuropeptide K (NPK), a
neuropeptide gamma, a
neurokinin A, a neurokinin B, a hemokinin and a endokinin.
85. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a cholecystokinin peptide selected from the
group consisting of a
polynucleotide molecule encoding a cholecystokinin 58, a cholecystokinin 39, a
cholecystokinin 33, a
cholecystokinin 12 and a cholecystokinin 8.
86. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a Neuropeptide Y related peptide selected
from the group
consisting of a polynucleotide molecule encoding a Neuropeptide Y (NPY), a
Peptide YY (PYY), a
Pancreatic peptide (PP) and a Pancreatic icosapeptide (PIP).
87. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a kinin peptide selected from the group
consisting of a
polynucleotide molecule encoding a bradykinin, a kallidin, a desArg9
bradykinin and a desArg'o
bradykinin.
88. The polynucleotide molecule according to 68, wherein the polynucleotide
molecule encoding the
altered targeting domain comprises a PAR peptide selected from the group
consisting of a
polynucleotide molecule encoding a PAR1, a PAR2, a PAR3 and a PAR4.
89.A method of producing a modified Clostridial toxin comprising the step of
expressing a polynucleotide
molecule encoding a modified Clostridial toxin in a cell, the polynucleotide
molecule comprising:
a) a polynucleotide molecule encoding a Clostridial toxin enzymatic domain
capable of executing an
enzymatic target modification step of a Clostridial toxin intoxication
process;
b) a polynucleotide molecule encoding a Clostridial toxin translocation domain
capable of executing
a translocation step of a Clostridial toxin intoxication process;
c) a polynucleotide molecule encoding a translocation facilitating domain
capable of facilitating a
translocation step of a Clostridial toxin intoxication process;
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d) a polynucleotide molecule encoding an altered targeting domain capable of
selectively binding a
non-Clostridial toxin receptor present on a Clostridial toxin target cell and
executing a cell binding
step of a Clostridial toxin intoxication process; and
e) a polynucleotide molecule encoding a protease cleavage site
wherein cleavage of the protease cleavage site converts the single-chain form
of the modified
Clostridial toxin into the di-chain form.
90. The method according to 89, wherein the polynucleotide molecule is any one
of the polynucleotide
molecules of 69.
91. A method of producing a modified Clostridial toxin comprising the steps
of:
a) introducing into a cell a polynucleotide molecule encoding a modified
Clostridial toxin, the
polynucleotide molecule comprising:
i) a polynucleotide molecule encoding a Clostridial toxin enzymatic domain
capable of
executing an enzymatic target modification step of a Clostridial toxin
intoxication process;
ii) a polynucleotide molecule encoding a Clostridial toxin translocation
domain capable of
executing a translocation step of a Clostridial toxin intoxication process;
iii) a polynucleotide molecule encoding a translocation facilitating domain
capable of facilitating a
translocation step of a Clostridial toxin intoxication process;
iv) a polynucleotide molecule encoding an altered targeting domain capable of
selectively
binding a non-Clostridial toxin receptor present on a Clostridial toxin target
cell and executing
a cell binding step of a Clostridial toxin intoxication process; and
v) a polynucleotide molecule encoding a protease cleavage site
wherein cleavage of the protease cleavage site converts the single-chain form
of the modified
Clostridial toxin into the di-chain form.
b) expressing the modified Clostridial toxin encoded by the polynucleotide
molecule.
92. The method according to 91, wherein the polynucleotide molecule is any one
of the polynucleotide
molecules of 69.
EXAMPLES
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[0399] The following non-limiting examples are provided for illustrative
purposes only in order to facilitate
a more complete understanding of disclosed embodiments and are in no way
intended to limit any of the
embodiments disclosed in the present specification.
Example 1
Construction of a modified Clostridial toxin comprising a translocation
facilitating domain
and an amino-terminally presented altered targeting domain
[0400] This example illustrates how to make a modified Clostridial toxin
disclosed in the present
specification comprising a translocation facilitating domain and an altered
targeting domain located at the
amino terminus of the modified toxin.
Ia. A targeting-translocation-translocation facilitating-enzymatic domain
organization.
[0401] A polynucleotide molecule based on BoNT/A-AP4A-Nociceptin (SEQ ID NO:
188) will be
synthesized using standard procedures (BlueHeron Biotechnology, Bothell, WA).
This polynucleotide
molecule encodes a BoNT/A modified to replace amino acids 1111-1296 of SEQ ID
NO: 1, a BoNT/A Hcc
targeting domain, with SEQ ID NO: 52, a nociceptin-RK targeting domain, and
has the general domain
arrangement of FIG. 4A. In addition to the nociceptin-RK targeting domain, the
altered targeting domain
further comprises at its amino terminus, a PAR 1 leader sequence ending in an
enterokinse cleavage site.
Cleavage of this site results in exposing the first amino acid of the
nociceptin-RK targeting domain.
Oligonucleotides of 20 to 50 bases in length are synthesized using standard
phosphoramidite synthesis.
These oligonucleotides will be hybridized into double stranded duplexes that
are ligated together to
assemble the full-length polynucleotide molecule. This polynucleotide molecule
will be cloned using
standard molecular biology methods into a pUCBHB1 vector at the Smal site to
generate
pUCBHBI/BoNT/A-AP4A-Nociceptin. The synthesized polynucleotide molecule is
verified by sequencing
using Big Dye TerminatorTM Chemistry 3.1 (Applied Biosystems, Foster City, CA)
and an ABI 3100
sequencer (Applied Biosystems, Foster City, CA).
[0402] If desired, an expression optimized polynucleotide molecule based on
BoNT/A-AP4A-Nociceptin
(SEQ ID NO: 188) can be synthesized in order to improve expression in an
Escherichia coli strain. The
polynucleotide molecule encoding the BoNT/A-AP4A-Nociceptin will be modified
to 1) contain
synonymous codons typically present in native polynucleotide molecules of an
Escherichia coli strain; 2)
contain a G+C content that more closely matches the average G+C content of
native polynucleotide
molecules found in an Escherichia coli strain; 3) reduce polymononucleotide
regions found within the
polynucleotide molecule; and/or 4) eliminate internal regulatory or structural
sites found within the
polynucleotide molecule, see, e.g., Lance E. Steward et al., Optimizing
Expression of Active Botulinum
Toxin Type E, International Patent Publication WO 2006/011966 (Feb. 2, 2006);
Lance E. Steward et al.,
Optimizing Expression of Active Botulinum Toxin Type A, International Patent
Publication WO
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2006/017749 (Feb. 16, 2006). Once sequence optimization is complete,
oligonucleotides of 20 to 50
bases in length are synthesized using standard phosphoramidite synthesis.
These oligonucleotides are
hybridized into double stranded duplexes that are ligated together to assemble
the full-length
polynucleotide molecule. This polynucleotide molecule is cloned using standard
molecular biology
methods into a pUCBHB1 vector at the Smal site to generate pUCBHBI/BoNT/A-AP4A-
Nociceptin. The
synthesized polynucleotide molecule is verified by sequencing using Big Dye
TerminatorTM Chemistry 3.1
(Applied Biosystems, Foster City, CA) and an ABI 3100 sequencer (Applied
Biosystems, Foster City, CA).
If so desired, expression optimization to a different organism, such as, e.g.,
a yeast strain, an insect cell-
line or a mammalian cell line, can be done, see, e.g., Steward, supra, (Feb.
2, 2006); and Steward, supra,
(Feb. 16, 2006).
[0403] A similar cloning strategy will be used to make pUCBHB1 cloning
constructs for BoNT/B-AP4A-
Nociceptin, a modified BoNT/B where amino acids 1098-1291 of SEQ ID NO: 2 are
replaced with SEQ ID
NO: 52; BoNT/C1-AP4A-Nociceptin, a modified BoNT/C1 where amino acids 1112-
1291 of SEQ ID NO: 3
are replaced with SEQ ID NO: 52; BoNT/D-AP4A-Nociceptin, a modified BoNT/D
where amino acids
1099-1276 of SEQ ID NO: 4 are replaced with SEQ ID NO: 52; BoNT/E-AP4A-
Nociceptin, a modified
BoNT/E where amino acids 1086-1252 of SEQ ID NO: 5 are replaced with SEQ ID
NO: 52; BoNT/F-
AP4A-Nociceptin, a modified BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6
are replaced with
SEQ ID NO: 52; BoNT/G-AP4A-Nociceptin, a modified BoNT/G where amino acids
1106-1297 of SEQ ID
NO: 7 are replaced with SEQ ID NO: 52; and TeNT-AP4A-Nociceptin, a modified
TeNT where amino
acids 1128-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 52.
[0404] Likewise, a similar cloning strategy will be used to make pUCBHB1
cloning constructs comprising
a polynucleotide molecule encoding a modified Clostridial toxin-AP4A that will
replace the Hcc targeting
domain from a Clostridial toxin the with an altered targeting domain
comprising, e.g, altered targeting
domains SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID NO: 183 or SEQ ID NO: 184; and
altered targeting
domains requiring a free amino-terminal amino acid like SEQ ID NO: 9 to SEQ ID
NO: 51 or SEQ ID NO:
53 to SEQ ID NO: 61, as well as, amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino acids 1-
22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-
22 of SEQ ID NO: 175;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 176 or amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 177; amino acids 42-
47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO:
182; amino acids 35-40,
amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 183;
amino acids 39-44, amino
acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 184; or amino
acids 48-53, amino
acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 185.
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[0405] To construct pET29/BoNT/A-AP4A-Nociceptin, a pUCBHBI/BoNT/A-AP4A-
Nociceptin construct
will be digested with restriction endonucleases that 1) will excise the
polynucleotide molecule encoding
the open reading frame of BoNT/A-AP4A-Nociceptin; and 2) will enable this
polynucleotide molecule to be
operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, WI). This
insert will be
subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested
with appropriate
restriction endonucleases to yield pET29/BoNT/A-AP4A-Nociceptin. The ligation
mixture will be
transformed into chemically competent E. coli DH5a cells (Invitrogen, Inc,
Carlsbad, CA) using a heat
shock method, will be plated on 1.5% Luria-Bertani agar plates (pH 7.0)
containing 50 pg/mL of
Kanamycin, and will be placed in a 37 C incubator for overnight growth.
Bacteria containing expression
constructs will be identified as Kanamycin resistant colonies. Candidate
constructs will be isolated using
an alkaline lysis plasmid mini-preparation procedure and will be analyzed by
restriction endonuclease
digest mapping to determine the presence and orientation of the insert. This
cloning strategy will yield a
pET29 expression construct comprising the polynucleotide molecule encoding the
BoNT/A-AP4A-
Nociceptin operably-linked to a carboxyl terminal polyhistidine affinity
binding peptide.
[0406] A similar cloning strategy will be used to make pET29 expression
constructs for other modified
Clostridial toxin-AP4A-Nociceptin toxins, such as, e.g., BoNT/B-AP4A-
Nociceptin, BoNT/C1-AP4A-
Nociceptin, BoNT/D-AP4A-Nociceptin, BoNT/E-AP4A-Nociceptin, BoNT/F-AP4A-
Nociceptin, BoNT/G-
AP4A-Nociceptin or TeNT-AP4A-Nociceptin. Likewise, a similar cloning strategy
will be used to make
pET29 expression constructs comprising a polynucleotide molecule encoding a
modified Clostridial toxin-
AP4A comprising an altered targeting domain such as, e.g, altered targeting
domains SEQ ID NO: 62 to
SEQ ID NO: 71, SEQ ID NO: 183 or SEQ ID NO: 184; and altered targeting domains
requiring a free
amino-terminal amino acid like SEQ ID NO: 9 to SEQ ID NO: 51 or SEQ ID NO: 53
to SEQ ID NO: 61, as
well as, amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-
22 of SEQ ID NO: 172;
amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of
SEQ ID NO: 173; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 174; amino acids 1-
12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 175;
amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 176 or amino
acids 1-12, amino acids 6-
22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 177; amino acids 42-47,
amino acids 42-55,
amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40,
amino acids 35-48, amino
acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino
acids 39-52, amino acids
26-60 or amino acids 1-60 of SEQ ID NO: 184; or amino acids 48-53, amino acids
48-61, amino acids 35-
70 or amino acids 1-70 of SEQ ID NO: 185.
[0407] To construct a BoNT/A-AP4A-Nociceptin that will replace the BoNT/A
translocation facilitating
domain with another Clostridial toxin translocation facilitating domain, a
translocation facilitating domain of
BoNT/B will be introduced into the BoNT/A-AP4A-Nociceptin as described above
using a Splicing by
Overlapping ends polymerase chain reaction (SOE-PCR) procedure, see, e.g., R.
M. Horton et al.,
Engineering hybrid genes without the use of restriction enzymes: gene splicing
by overlapping extension,
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77(1) Gene 61-68 (1989); and R. M. Horton, PCR-mediated recombination and
mutagenesis. SOEing
together tailor-made genes, 3(2) Mol. Biotechnol. 93-99 (1995). A nucleic acid
fragment comprising a
region encoding amino acids 859 to 1097 of BoNT/B (SEQ ID NO: 2) will be
operably-linked by SOE-PCR
to replace the region corresponding to the BoNT/A translocation facilitating
domain comprising amino
acids 874-1110 of SEQ ID NO: 1 of the BoNT/A-AP4A-Nociceptin and will be
subcloned into a pCR2.1
vector using the TOPO TA cloning method (Invitrogen, Inc, Carlsbad, CA). The
forward and reverse
oligonucleotide primers used for these reactions are designed to include
unique restriction enzyme sites
useful for subsequent subcloning steps. The resulting construct will be
digested with restriction enzymes
that 1) will excise the polynucleotide molecule containing the entire open
reading frame encoding the
modified BoNT/A-AP4A-Nociceptin; and 2) will enable this polynucleotide
molecule to be operably-linked
to a pET29 vector (EMD Biosciences-Novagen, Madison, WI). The resulting
restriction fragment will be
purified by the QlAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, CA), and
will be subcloned using a
T4 DNA ligase procedure into a pET29 vector. This cloning strategy yielded a
pET29 expression
construct encoding a BoNT/A-AP4A-Nociceptin comprising a BoNT/B translocation
facilitating domain.
[0408] A similar cloning strategy will be used to make pET29 expression
constructs comprising a
polynucleotide molecule encoding a BoNT/A-AP4A-Nociceptin that replaces the
region corresponding to
the BoNT/A translocation facilitating domain comprising amino acids 874-1110
of SEQ ID NO: 1 with,
e.g., a translocation facilitating domain comprising amino acids 869-1111 of
BoNT/C1 of SEQ ID NO: 3; a
translocation facilitating domain comprising amino acids 865-1098 of BoNT/D of
SEQ ID NO: 4; a
translocation facilitating domain comprising amino acids 846-1058 of BoNT/E of
SEQ ID NO: 5; a
translocation facilitating domain comprising amino acids 867-1105 of BoNT/F of
SEQ ID NO: 6; a
translocation facilitating domain comprising amino acids 866-1105 of BoNT/G of
SEQ ID NO: 7; or a
translocation facilitating domain comprising amino acids 882-1127 of TeNT of
SEQ ID NO: 8. Likewise, a
similar cloning strategy will be used to make pET29 expression constructs
comprising a polynucleotide
molecule encoding a BoNT/A-AP4A-Nociceptin that replaces the region
corresponding to the BoNT/A
translocation facilitating domain comprising amino acids 874-1110 of SEQ ID
NO: 1 with a translocation
facilitating domain comprising an enveloped virus fusogenic peptide domain,
such as, e,g, SEQ ID NO:
194 to SEQ ID NO: 269.
[0409] Likewise, a polynucleotide molecule encoding a Clostridial
translocation facilitating domain as
described above can be introduced into a polynucleotide molecule encoding
BoNT/B-AP4A-Nociceptin,
BoNT/C1-AP4A-Nociceptin, BoNT/D-AP4A-Nociceptin, BoNT/E-AP4A-Nociceptin,
BoNT/F-AP4A-
Nociceptin, BoNT/G-AP4A-Nociceptin, TeNT-AP4A-Nociceptin, as well as the
modified Clostridial toxin-
AP4A indicated above comprising SEQ ID NO: 9 to SEQ ID NO: 51, SEQ ID NO: 53
to SEQ ID NO: 71,
SEQ ID NO: 183 or SEQ ID NO: 184, as well as, amino acids 1-12, amino acids 6-
22, amino acids 8-22
or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
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SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-
64 of SEQ ID NO: 182;
amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of
SEQ ID NO: 183; amino
acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ
ID NO: 184; or amino
acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ
ID NO: 185.
lb. A targeting-enzymatic-translocation-translocation facilitating domain
organization.
[0410] A polynucleotide molecule based on BoNT/A-AP4B-Nociceptin (SEQ ID NO:
189) will be
synthesized and cloned into a pUCBHB1 vector as described in Example la. This
polynucleotide
molecule encodes a BoNT/A modified to replace amino acids 1111-1296 of SEQ ID
NO: 1, a BoNT/A Hcc
targeting domain, with SEQ ID NO: 52, a nociceptin-RK targeting domain, and
has the general domain
arrangement of FIG. 4B. In addition to the nociceptin-RK targeting domain, the
altered targeting domain
further comprises at its amino terminus, a PAR 1 leader sequence ending in an
enterokinse cleavage site.
Cleavage of this site results in exposing the first amino acid of the
nociceptin-RK targeting domain. If so
desired, expression optimization to a different organism, such as, e.g., a
bacteria, a yeast strain, an insect
cell-line or a mammalian cell line, can be done as described above, see, e.g.,
Steward, supra, (Feb. 2,
2006); and Steward, supra, (Feb. 16, 2006).
[0411] Likewise, a similar cloning strategy will be used to make pUCBHB1
cloning constructs comprising
a polynucleotide molecule encoding a modified BoNT/A-AP4B with an altered
targeting domain such as,
e.g, altered targeting domains SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID NO: 183
or SEQ ID NO: 184;
and altered targeting domains requiring a free amino-terminal amino acid like
SEQ ID NO: 9 to SEQ ID
NO: 51 or SEQ ID NO: 53 to SEQ ID NO: 61, as well as, amino acids 1-12, amino
acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino
acids 6-22, amino acids 8-
22 or amino acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22
or amino acids 1-22 of
SEQ ID NO: 177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or
amino acids 1-64 of SEQ
ID NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino
acids 1-59 of SEQ ID
NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino
acids 1-60 of SEQ ID NO:
184; or amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids
1-70 of SEQ ID NO:
185. In addition, similar cloning strategy will be used to produce a modified
Clostridial toxin-AP4B, such
as, e.g., BoNT/B-AP4B, BoNT/C1-AP4B, BoNT/D-AP4B, BoNT/E-AP4B, BoNT/F-AP4B,
BoNT/G-AP4B
or TeNT-AP4B, to comprise an altered targeting domain comprising any one of
SEQ ID NO: 9 to SEQ ID
NO: 71, SEQ ID NO: 183 or SEQ ID NO: 184, or amino acids 1-12, amino acids 6-
22, amino acids 8-22
or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or amino
189
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acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-
64 of SEQ ID NO: 182;
amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of
SEQ ID NO: 183; amino
acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ
ID NO: 184; or amino
acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ
ID NO: 185.
[0412] To construct pET29/BoNT/A-AP4B-Nociceptin, a similar cloning strategy
will be used as
described in Example 1a. This cloning strategy will yield a pET29 expression
construct comprising the
polynucleotide molecule encoding the BoNT/A-AP4B-Nociceptin operably-linked to
a carboxyl terminal
polyhistidine affinity binding peptide. A similar cloning strategy will be
used to make pET29 expression
constructs for other modified Clostridial toxin-AP4B-Nociceptin toxins, such
as, e.g., BoNT/B-AP4B-
Nociceptin, BoNT/C1-AP4B-Nociceptin, BoNT/D-AP4B-Nociceptin, BoNT/E-AP4B-
Nociceptin, BoNT/F-
AP4B-Nociceptin, BoNT/G-AP4B-Nociceptin or TeNT-AP4B-Nociceptin. Likewise, a
similar cloning
strategy will be used to make pET29 expression constructs comprising a
polynucleotide molecule
encoding a modified Clostridial toxin-AP4B with an altered targeting domain
such as, e.g, altered
targeting domains SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID NO: 183 or SEQ ID NO:
184; and altered
targeting domains requiring a free amino-terminal amino acid like SEQ ID NO: 9
to SEQ ID NO: 51 or
SEQ ID NO: 53 to SEQ ID NO: 61, as well as, amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-
64 of SEQ ID NO: 182;
amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of
SEQ ID NO: 183; amino
acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ
ID NO: 184; or amino
acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ
ID NO: 185.
[0413] To construct a BoNT/A-AP4B-Nociceptin that will replace the BoNT/A
translocation facilitating
domain with another Clostridial toxin translocation facilitating domain, a
similar cloning strategy using
SOE-PCR will be used as described in Example 1a. This cloning strategy yielded
a pET29 expression
construct encoding a BoNT/A-AP4B-Nociceptin comprising a BoNT/B translocation
facilitating domain, a
BoNT/C1 translocation facilitating domain, a BoNT/D translocation facilitating
domain, a BoNT/E
translocation facilitating domain, a BoNT/F translocation facilitating domain,
a BoNT/G translocation
facilitating domain and a TeNT translocation facilitating domain, as well as,
a translocation facilitating
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domain comprising an enveloped virus fusogenic peptide domain, such as, e,g,
SEQ ID NO: 194 to SEQ
ID NO: 269.
[0414] Likewise, a polynucleotide molecule encoding a Clostridial
translocation facilitating domain as
described above can be introduced into a polynucleotide molecule encoding
BoNT/B-AP4B-Nociceptin,
BoNT/C1-AP4B-Nociceptin, BoNT/D-AP4B-Nociceptin, BoNT/E-AP4B-Nociceptin,
BoNT/F-AP4B-
Nociceptin, BoNT/G-AP4B-Nociceptin, TeNT-AP4B-Nociceptin, as well as the
modified Clostridial toxin-
AP4B indicated above comprising SEQ ID NO: 9 to SEQ ID NO: 51 or SEQ ID NO: 53
to SEQ ID NO: 71,
SEQ ID NO: 183 or SEQ ID NO: 184, as well as, amino acids 1-12, amino acids 6-
22, amino acids 8-22
or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-
64 of SEQ ID NO: 182;
amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of
SEQ ID NO: 183; amino
acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ
ID NO: 184; or amino
acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ
ID NO: 185.
Example 2
Construction of a modified Clostridial toxin comprising a translocation
facilitating domain
and a centrally presented altered targeting domain
[0415] This example illustrates how to make a modified Clostridial toxin
disclosed in the present
specification comprising a translocation facilitating domain and an altered
targeting domain located
between two other domains of the modified toxin.
2a. An enzymatic-targeting-translocation-translocation facilitating domain
organization.
[0416] A polynucleotide molecule based on BoNT/A-CP5A-Nociceptin (SEQ ID NO:
190) will be
synthesized using standard procedures (BlueHeron Biotechnology, Bothell, WA).
This polynucleotide
molecule encodes a BoNT/A modified to replace amino acids 1111-1296 of SEQ ID
NO: 1, a BoNT/A Hcc
targeting domain, with SEQ ID NO: 52, a nociceptin-RK targeting domain, and
has the general domain
arrangement of FIG. 5A. Cleavage of an enterokinse cleavage site used to form
the di-chain toxin also
exposes the first amino acid of the nociceptin-RK targeting domain.
Oligonucleotides of 20 to 50 bases in
length are synthesized using standard phosphoramidite synthesis. These
oligonucleotides will be
hybridized into double stranded duplexes that are ligated together to assemble
the full-length
polynucleotide molecule. This polynucleotide molecule will be cloned using
standard molecular biology
methods into a pUCBHB1 vector at the Smal site to generate pUCBHBI/BoNT/A-CP5A-
Nociceptin. The
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synthesized polynucleotide molecule is verified by sequencing using Big Dye
TerminatorTM Chemistry 3.1
(Applied Biosystems, Foster City, CA) and an ABI 3100 sequencer (Applied
Biosystems, Foster City, CA).
[0417] If desired, an expression optimized polynucleotide molecule based on
BoNT/A-CP5A-Nociceptin
(SEQ ID NO: 190) can be synthesized in order to improve expression in an
Escherichia coli strain. The
polynucleotide molecule encoding the BoNT/A-CP5A-Nociceptin will be modified
to 1) contain
synonymous codons typically present in native polynucleotide molecules of an
Escherichia coli strain; 2)
contain a G+C content that more closely matches the average G+C content of
native polynucleotide
molecules found in an Escherichia coli strain; 3) reduce polymononucleotide
regions found within the
polynucleotide molecule; and/or 4) eliminate internal regulatory or structural
sites found within the
polynucleotide molecule, see, e.g., Lance E. Steward et al., Optimizing
Expression of Active Botulinum
Toxin Type E, International Patent Publication WO 2006/011966 (Feb. 2, 2006);
Lance E. Steward et al.,
Optimizing Expression of Active Botulinum Toxin Type A, International Patent
Publication WO
2006/017749 (Feb. 16, 2006). Once sequence optimization is complete,
oligonucleotides of 20 to 50
bases in length are synthesized using standard phosphoramidite synthesis.
These oligonucleotides are
hybridized into double stranded duplexes that are ligated together to assemble
the full-length
polynucleotide molecule. This polynucleotide molecule is cloned using standard
molecular biology
methods into a pUCBHB1 vector at the Smal site to generate pUCBHBI/BoNT/A-CP5A-
Nociceptin. The
synthesized polynucleotide molecule is verified by sequencing using Big Dye
TerminatorTM Chemistry 3.1
(CP5Aplied Biosystems, Foster City, CA) and an ABI 3100 sequencer (CP5Aplied
Biosystems, Foster
City, CA). If so desired, expression optimization to a different organism,
such as, e.g., a yeast strain, an
insect cell-line or a mammalian cell line, can be done, see, e.g., Steward,
supra, (Feb. 2, 2006); and
Steward, supra, (Feb. 16, 2006).
[0418] A similar cloning strategy will be used to make pUCBHB1 cloning
constructs for BoNT/B-CP5A-
Nociceptin, a modified BoNT/B where amino acids 1098-1291 of SEQ ID NO: 2 are
replaced with SEQ ID
NO: 52; BoNT/C1-CP5A-Nociceptin, a modified BoNT/C1 where amino acids 1112-
1291 of SEQ ID NO: 3
are replaced with SEQ ID NO: 52; BoNT/D-CP5A-Nociceptin, a modified BoNT/D
where amino acids
1099-1276 of SEQ ID NO: 4 are replaced with SEQ ID NO: 52; BoNT/E-CP5A-
Nociceptin, a modified
BoNT/E where amino acids 1086-1252 of SEQ ID NO: 5 are replaced with SEQ ID
NO: 52; BoNT/F-
CP5A-Nociceptin, a modified BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6
are replaced with
SEQ ID NO: 52; BoNT/G-CP5A-Nociceptin, a modified BoNT/G where amino acids
1106-1297 of SEQ ID
NO: 7 are replaced with SEQ ID NO: 52; and TeNT-CP5A-Nociceptin, a modified
TeNT where amino
acids 1128-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 52.
[0419] Likewise, a similar cloning strategy will be used to make pUCBHB1
cloning constructs comprising
a polynucleotide molecule encoding a modified Clostridial toxin-CP5A with an
altered targeting domain
such as, e.g, altered targeting domains SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID
NO: 183 or SEQ ID
NO: 184; and altered targeting domains requiring a free amino-terminal amino
acid like SEQ ID NO: 9 to
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CA 02657521 2009-01-12
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SEQ ID NO: 51 or SEQ ID NO: 53 to SEQ ID NO: 61, as well as, amino acids 1-12,
amino acids 6-22,
amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12,
amino acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino
acids 6-22, amino acids 8-
22 or amino acids 1-22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 176 or amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino acids 1-
22 of SEQ ID NO: 177; amino acids 42-47, amino acids 42-55, amino acids 29-64
or amino acids 1-64 of
SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-59 or
amino acids 1-59 of SEQ
ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino
acids 1-60 of SEQ ID
NO: 184; or amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino
acids 1-70 of SEQ ID
NO: 185.
[0420] To construct pET29/BoNT/A-CP5A-Nociceptin, a pUCBHBI/BoNT/A-CP5A-
Nociceptin construct
will be digested with restriction endonucleases that 1) will excise the
polynucleotide molecule encoding
the open reading frame of BoNT/A-CP5A-Nociceptin; and 2) will enable this
polynucleotide molecule to
be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, WI).
This insert will be
subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested
with appropriate
restriction endonucleases to yield pET29/BoNT/A-CP5A-Nociceptin. The ligation
mixture will be
transformed into chemically competent E. coli DH5a cells (Invitrogen, Inc,
Carlsbad, CA) using a heat
shock method, will be plated on 1.5% Luria-Bertani agar plates (pH 7.0)
containing 50 pg/mL of
Kanamycin, and will be placed in a 37 C incubator for overnight growth.
Bacteria containing expression
constructs will be identified as Kanamycin resistant colonies. Candidate
constructs will be isolated using
an alkaline lysis plasmid mini-preparation procedure and will be analyzed by
restriction endonuclease
digest mapping to determine the presence and orientation of the insert. This
cloning strategy will yield a
pET29 expression construct comprising the polynucleotide molecule encoding the
BoNT/A-CP5A-
Nociceptin operably-linked to a carboxyl terminal polyhistidine affinity
binding peptide.
[0421] A similar cloning strategy will be used to make pET29 expression
constructs for other modified
Clostridial toxin-CP5A-Nociceptin toxins, such as, e.g., BoNT/B-CP5A-
Nociceptin, BoNT/C1-CP5A-
Nociceptin, BoNT/D-CP5A-Nociceptin, BoNT/E-CP5A-Nociceptin, BoNT/F-CP5A-
Nociceptin, BoNT/G-
CP5A-Nociceptin or TeNT-CP5A-Nociceptin. Likewise, a similar cloning strategy
will be used to make
pET29 expression constructs comprising a polynucleotide molecule encoding a
modified Clostridial toxin-
CP5A with an altered targeting domain such as, e.g, altered targeting domains
SEQ ID NO: 62 to SEQ ID
NO: 71, SEQ ID NO: 183 or SEQ ID NO: 184; and altered targeting domains
requiring a free amino-
terminal amino acid like SEQ ID NO: 9 to SEQ ID NO: 51 or SEQ ID NO: 53 to SEQ
ID NO: 61, as well
as, amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22
of SEQ ID NO: 172; amino
acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID
NO: 173; amino acids 1-
12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 174;
amino acids 1-12, amino
acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 175; amino
acids 1-12, amino acids 6-
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22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 176 or amino acids 1-
12, amino acids 6-22,
amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 177; amino acids 42-47,
amino acids 42-55, amino
acids 29-64 or amino acids 1-64 of SEQ ID NO: 182; amino acids 35-40, amino
acids 35-48, amino acids
24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-
52, amino acids 26-60
or amino acids 1-60 of SEQ ID NO: 184; or amino acids 48-53, amino acids 48-
61, amino acids 35-70 or
amino acids 1-70 of SEQ ID NO: 185.
[0422] To construct a BoNT/A-CP5A-Nociceptin that will replace the BoNT/A
translocation facilitating
domain with another Clostridial toxin translocation facilitating domain, a
translocation facilitating domain of
BoNT/B will be introduced into the BoNT/A-CP5A-Nociceptin as described above
using a Splicing by
Overlapping ends polymerase chain reaction (SOE-PCR) procedure, see, e.g., R.
M. Horton et al.,
Engineering hybrid genes without the use of restriction enzymes: gene splicing
by overlapping extension,
77(1) Gene 61-68 (1989); and R. M. Horton, PCR-mediated recombination and
mutagenesis. SOEing
together tailor-made genes, 3(2) Mol. Biotechnol. 93-99 (1995). A nucleic acid
fragment comprising a
region encoding amino acids 859 to 1097 of BoNT/B (SEQ ID NO: 2) will be
operably-linked by SOE-PCR
to replace the region corresponding to the BoNT/A translocation facilitating
domain comprising amino
acids 874-1110 of SEQ ID NO: 1 of the BoNT/A-CP5A-Nociceptin and will be
subcloned into a pCR2.1
vector using the TOPO TA cloning method (Invitrogen, Inc, Carlsbad, CA). The
forward and reverse
oligonucleotide primers used for these reactions are designed to include
unique restriction enzyme sites
useful for subsequent subcloning steps. The resulting construct will be
digested with restriction enzymes
that 1) will excise the polynucleotide molecule containing the entire open
reading frame encoding the
modified BoNT/A-CP5A-Nociceptin; and 2) will enable this polynucleotide
molecule to be operably-linked
to a pET29 vector (EMD Biosciences-Novagen, Madison, WI). The resulting
restriction fragment will be
purified by the QlAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, CA), and
will be subcloned using a
T4 DNA ligase procedure into a pET29 vector. This cloning strategy yielded a
pET29 expression
construct encoding a BoNT/A-CP5A-Nociceptin comprising a BoNT/B translocation
facilitating domain.
[0423] A similar cloning strategy will be used to make pET29 expression
constructs comprising a
polynucleotide molecule encoding a BoNT/A-CP5A-Nociceptin that replaces the
region corresponding to
the BoNT/A translocation facilitating domain comprising amino acids 874-1110
of SEQ ID NO: 1 with,
e.g., a translocation facilitating domain comprising amino acids 869-1111 of
BoNT/C1 of SEQ ID NO: 3; a
translocation facilitating domain comprising amino acids 865-1098 of BoNT/D of
SEQ ID NO: 4; a
translocation facilitating domain comprising amino acids 846-1058 of BoNT/E of
SEQ ID NO: 5; a
translocation facilitating domain comprising amino acids 867-1105 of BoNT/F of
SEQ ID NO: 6; a
translocation facilitating domain comprising amino acids 866-1105 of BoNT/G of
SEQ ID NO: 7; or a
translocation facilitating domain comprising amino acids 882-1127 of TeNT of
SEQ ID NO: 8. Likewise, a
similar cloning strategy will be used to make pET29 expression constructs
comprising a polynucleotide
molecule encoding a BoNT/A-CP5A-Nociceptin that replaces the region
corresponding to the BoNT/A
translocation facilitating domain comprising amino acids 874-1110 of SEQ ID
NO: 1 with a translocation
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facilitating domain comprising an enveloped virus fusogenic peptide domain,
such as, e,g, SEQ ID NO:
194 to SEQ ID NO: 269.
[0424] Likewise, a polynucleotide molecule encoding a Clostridial
translocation facilitating domain as
described above can be introduced into a polynucleotide molecule encoding
BoNT/B-CP5A-Nociceptin,
BoNT/C1-CP5A-Nociceptin, BoNT/D-CP5A-Nociceptin, BoNT/E-CP5A-Nociceptin,
BoNT/F-CP5A-
Nociceptin, BoNT/G-CP5A-Nociceptin, TeNT-CP5A-Nociceptin, as well as the
modified Clostridial toxin-
CP5A indicated above comprising SEQ ID NO: 9 to SEQ ID NO: 51 or SEQ ID NO: 53
to SEQ ID NO: 71,
SEQ ID NO: 183 or SEQ ID NO: 184, as well as, amino acids 1-12, amino acids 6-
22, amino acids 8-22
or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-
64 of SEQ ID NO: 182;
amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of
SEQ ID NO: 183; amino
acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ
ID NO: 184; or amino
acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ
ID NO: 185.
2b. A translocation-translocation facilitating-targeting-enzymatic domain
organization.
[0425] A polynucleotide molecule based on BoNT/A-CP5B-Nociceptin (SEQ ID NO:
191) will be
synthesized and cloned into a pUCBHB1 vector as described in Example la. This
polynucleotide
molecule encodes a BoNT/A modified to replace amino acids 1111-1296 of SEQ ID
NO: 1, a BoNT/A Hcc
targeting domain, with SEQ ID NO: 52, a nociceptin-RK targeting domain, and
has the general domain
arrangement of FIG. 5B. Cleavage of an enterokinse cleavage site used to form
the di-chain toxin also
exposes the first amino acid of the nociceptin-RK targeting domain. If so
desired, expression optimization
to a different organism, such as, e.g., a bacteria, a yeast strain, an insect
cell-line or a mammalian cell
line, can be done as described above, see, e.g., Steward, supra, (Feb. 2,
2006); and Steward, supra,
(Feb. 16, 2006).
[0426] Likewise, a similar cloning strategy will be used to make pUCBHB1
cloning constructs comprising
a polynucleotide molecule encoding a modified BoNT/A-CP5B with an altered
targeting domain such as,
e.g, altered targeting domains SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID NO: 183
or SEQ ID NO: 184;
and altered targeting domains requiring a free amino-terminal amino acid like
SEQ ID NO: 9 to SEQ ID
NO: 51 or SEQ ID NO: 53 to SEQ ID NO: 61, as well as, amino acids 1-12, amino
acids 6-22, amino
acids 8-22 or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino
acids 6-22, amino acids 8-
22 or amino acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
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acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22
or amino acids 1-22 of
SEQ ID NO: 177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or
amino acids 1-64 of SEQ
ID NO: 179; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino
acids 1-59 of SEQ ID
NO: 180; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino
acids 1-60 of SEQ ID NO:
181; or amino acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids
1-70 of SEQ ID NO:
182.. In addition, similar cloning strategy will be used to produce a modified
Clostridial toxin-CP5B, such
as, e.g., BoNT/B-CP5B, BoNT/C1-CP5B, BoNT/D-CP5B, BoNT/E-CP5B, BoNT/F-CP5B,
BoNT/G-CP5B
or TeNT-CP5B, to comprise an altered targeting domain comprising any one of
SEQ ID NO: 9 to SEQ ID
NO: 71, SEQ ID NO: 183 or SEQ ID NO: 184, or amino acids 1-12, amino acids 6-
22, amino acids 8-22
or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-
64 of SEQ ID NO: 182;
amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of
SEQ ID NO: 183; amino
acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ
ID NO: 184; or amino
acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ
ID NO: 185.
[0427] To construct pET29/BoNT/A-CP5B-Nociceptin, a similar cloning strategy
will be used as
described in Example 1a. This cloning strategy will yield a pET29 expression
construct comprising the
polynucleotide molecule encoding the BoNT/A-CP5B-Nociceptin operably-linked to
a carboxyl terminal
polyhistidine affinity binding peptide. A similar cloning strategy will be
used to make pET29 expression
constructs for other modified Clostridial toxin-CP5B-Nociceptin toxins, such
as, e.g., BoNT/B-CP5B-
Nociceptin, BoNT/C1-CP5B-Nociceptin, BoNT/D-CP5B-Nociceptin, BoNT/E-CP5B-
Nociceptin, BoNT/F-
CP5B-Nociceptin, BoNT/G-CP5B-Nociceptin or TeNT-CP5B-Nociceptin. Likewise, a
similar cloning
strategy will be used to make pET29 expression constructs comprising a
polynucleotide molecule
encoding a modified Clostridial toxin-CP5B with an altered targeting domain
such as, e.g, altered
targeting domains SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID NO: 183 or SEQ ID NO:
184; and altered
targeting domains requiring a free amino-terminal amino acid like SEQ ID NO: 9
to SEQ ID NO: 51 or
SEQ ID NO: 53 to SEQ ID NO: 61, as well as, amino acids 1-12, amino acids 6-
22, amino acids 8-22 or
amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino
acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-
64 of SEQ ID NO: 182;
amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of
SEQ ID NO: 183; amino
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acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ
ID NO: 184; or amino
acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ
ID NO: 185.
[0428] To construct a BoNT/A-CP5B-Nociceptin that will replace the BoNT/A
translocation facilitating
domain with another Clostridial toxin translocation facilitating domain, a
similar cloning strategy using
SOE-PCR will be used as described in Example 1a. This cloning strategy yielded
a pET29 expression
construct encoding a BoNT/A-CP5B-Nociceptin comprising a BoNT/B translocation
facilitating domain, a
BoNT/C1 translocation facilitating domain, a BoNT/D translocation facilitating
domain, a BoNT/E
translocation facilitating domain, a BoNT/F translocation facilitating domain,
a BoNT/G translocation
facilitating domain and a TeNT translocation facilitating domain, as well as,
a translocation facilitating
domain comprising an enveloped virus fusogenic peptide domain, such as, e,g,
SEQ ID NO: 194 to SEQ
ID NO: 269.
[0429] Likewise, a polynucleotide molecule encoding a Clostridial
translocation facilitating domain as
described above can be introduced into a polynucleotide molecule encoding
BoNT/B-CP5B-Nociceptin,
BoNT/C1-CP5B-Nociceptin, BoNT/D-CP5B-Nociceptin, BoNT/E-CP5B-Nociceptin,
BoNT/F-CP5B-
Nociceptin, BoNT/G-CP5B-Nociceptin, TeNT-CP5B-Nociceptin, as well as the
modified Clostridial toxin-
CP5B indicated above comprising SEQ ID NO: 9 to SEQ ID NO: 51 or SEQ ID NO: 53
to SEQ ID NO: 71,
SEQ ID NO: 183 or SEQ ID NO: 184, as well as, amino acids 1-12, amino acids 6-
22, amino acids 8-22
or amino acids 1-22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22,
amino acids 8-22 or amino
acids 1-22 of SEQ ID NO: 173; amino acids 1-12, amino acids 6-22, amino acids
8-22 or amino acids 1-
22 of SEQ ID NO: 174; amino acids 1-12, amino acids 6-22, amino acids 8-22 or
amino acids 1-22 of
SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino
acids 1-22 of SEQ ID
NO: 176 or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids
1-22 of SEQ ID NO:
177; amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-
64 of SEQ ID NO: 182;
amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of
SEQ ID NO: 183; amino
acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ
ID NO: 184; or amino
acids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ
ID NO: 185.
Example 3
Construction of a modified Clostridial toxin comprising a translocation
facilitating domain
and a carboxyl-terminally presented altered targeting domain
[0430] This example illustrates how to make a modified Clostridial toxin
disclosed in the present
specification comprising a translocation facilitating domain and an altered
targeting domain located at the
carboxyl terminus of the modified toxin.
3a. An enzymatic-translocation-translocation facilitating-targeting domain
organization.
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[0431] A polynucleotide molecule based on BoNT/A-XP6A-Galanin (SEQ ID NO: 192)
will be
synthesized using standard procedures (BlueHeron Biotechnology, Bothell, WA).
This polynucleotide
molecule encodes a BoNT/A modified to replace amino acids 1111-1296 of SEQ ID
NO: 1, a BoNT/A Hcc
targeting domain, with SEQ ID NO: 72, a Galanin targeting domain, and has the
general domain
arrangement of FIG. 6A. Oligonucleotides of 20 to 50 bases in length are
synthesized using standard
phosphoramidite synthesis. These oligonucleotides will be hybridized into
double stranded duplexes that
are ligated together to assemble the full-length polynucleotide molecule. This
polynucleotide molecule
will be cloned using standard molecular biology methods into a pUCBHB1 vector
at the Smal site to
generate pUCBHBI/BoNT/A-XP6A-Galanin. The synthesized polynucleotide molecule
is verified by
sequencing using Big Dye TerminatorTM Chemistry 3.1 (Applied Biosystems,
Foster City, CA) and an ABI
3100 sequencer (Applied Biosystems, Foster City, CA).
[0432] If desired, an expression optimized polynucleotide molecule based on
BoNT/A-XP6A-Galanin
(SEQ ID NO: 192) can be synthesized in order to improve expression in an
Escherichia coli strain. The
polynucleotide molecule encoding the BoNT/A-XP6A-Galanin will be modified to
1) contain synonymous
codons typically present in native polynucleotide molecules of an Escherichia
coli strain; 2) contain a G+C
content that more closely matches the average G+C content of native
polynucleotide molecules found in
an Escherichia coli strain; 3) reduce polymononucleotide regions found within
the polynucleotide
molecule; and/or 4) eliminate internal regulatory or structural sites found
within the polynucleotide
molecule, see, e.g., Lance E. Steward et al., Optimizing Expression of Active
Botulinum Toxin Type E,
International Patent Publication WO 2006/011966 (Feb. 2, 2006); Lance E.
Steward et al., Optimizing
Expression of Active Botulinum Toxin Type A, International Patent Publication
WO 2006/017749 (Feb. 16,
2006). Once sequence optimization is complete, oligonucleotides of 20 to 50
bases in length are
synthesized using standard phosphoramidite synthesis. These oligonucleotides
are hybridized into
double stranded duplexes that are ligated together to assemble the full-length
polynucleotide molecule.
This polynucleotide molecule is cloned using standard molecular biology
methods into a pUCBHB1 vector
at the Smal site to generate pUCBHBI/BoNT/A-XP6A-Galanin. The synthesized
polynucleotide molecule
is verified by sequencing using Big Dye TerminatorTM Chemistry 3.1 (Applied
Biosystems, Foster City,
CA) and an ABI 3100 sequencer (Applied Biosystems, Foster City, CA). If so
desired, expression
optimization to a different organism, such as, e.g., a yeast strain, an insect
cell-line or a mammalian cell
line, can be done, see, e.g., Steward, supra, (Feb. 2, 2006); and Steward,
supra, (Feb. 16, 2006).
[0433] A similar cloning strategy will be used to make pUCBHB1 cloning
constructs for BoNT/B-XP6A-
Galanin, a modified BoNT/B where amino acids 1098-1291 of SEQ ID NO: 2 are
replaced with SEQ ID
NO: 72; BoNT/C1-XP6A-Galanin, a modified BoNT/C1 where amino acids 1112-1291
of SEQ ID NO: 3
are replaced with SEQ ID NO: 72; BoNT/D-XP6A-Galanin, a modified BoNT/D where
amino acids 1099-
1276 of SEQ ID NO: 4 are replaced with SEQ ID NO: 72; BoNT/E-XP6A-Galanin, a
modified BoNT/E
where amino acids 1086-1252 of SEQ ID NO: 5 are replaced with SEQ ID NO: 72;
BoNT/F-XP6A-
Galanin, a modified BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6 are
replaced with SEQ ID
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NO: 72; BoNT/G-XP6A-Galanin, a modified BoNT/G where amino acids 1106-1297 of
SEQ ID NO: 7 are
replaced with SEQ ID NO: 72; and TeNT-XP6A-Galanin, a modified TeNT where
amino acids 1128-1315
of SEQ ID NO: 8 are replaced with SEQ ID NO: 72. Likewise, a similar cloning
strategy will be used to
make pUCBHB1 cloning constructs comprising a polynucleotide molecule encoding
a modified Clostridial
toxin-XP6A with an altered targeting domain such as, e.g, altered targeting
domains SEQ ID NO: 62 to
SEQ ID NO: 71, SEQ ID NO: 183 or SEQ ID NO: 184; and altered targeting domains
requiring a free
carboxyl-terminal amino acid like SEQ ID NO: 88 to SEQ ID NO: 115 and SEQ ID
NO: 178 to SEQ ID
NO: 181 and SEQ ID NO: 178 to SEQ ID NO: 181, as well as, amino acids 26-58 of
SEQ ID NO: 100,
SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:
105, SEQ ID NO:
107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID
NO: 112, SEQ ID
NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115; amino acids 47-58 of SEQ ID NO:
100, SEQ ID NO: 110
or SEQ ID NO: 114; or amino acids 51-58 of SEQ ID NO: 100.
[0434] To construct pET29/BoNT/A-XP6A-Galanin, a pUCBHBI/BoNT/A-XP6A-Galanin
construct will be
digested with restriction endonucleases that 1) will excise the polynucleotide
molecule encoding the open
reading frame of BoNT/A-XP6A-Galanin; and 2) will enable this polynucleotide
molecule to be operably-
linked to a pET29 vector (EMD Biosciences-Novagen, Madison, WI). This insert
will be subcloned using
a T4 DNA ligase procedure into a pET29 vector that is digested with
appropriate restriction
endonucleases to yield pET29/BoNT/A-XP6A-Galanin. The ligation mixture will be
transformed into
chemically competent E. coli DH5a cells (Invitrogen, Inc, Carlsbad, CA) using
a heat shock method, will
be plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 pg/mL of
Kanamycin, and will be
placed in a 37 C incubator for overnight growth. Bacteria containing
expression constructs will be
identified as Kanamycin resistant colonies. Candidate constructs will be
isolated using an alkaline lysis
plasmid mini-preparation procedure and will be analyzed by restriction
endonuclease digest mapping to
determine the presence and orientation of the insert. This cloning strategy
will yield a pET29 expression
construct comprising the polynucleotide molecule encoding the BoNT/A-XP6A-
Galanin operably-linked to
a carboxyl terminal polyhistidine affinity binding peptide.
[0435] A similar cloning strategy will be used to make pET29 expression
constructs for other modified
Clostridial toxin-XP6A-Galanin toxins, such as, e.g., BoNT/B-XP6A-Galanin,
BoNT/C1-XP6A-Galanin,
BoNT/D-XP6A-Galanin, BoNT/E-XP6A-Galanin, BoNT/F-XP6A-Galanin, BoNT/G-XP6A-
Galanin or TeNT-
XP6A-Galanin. Likewise, a similar cloning strategy will be used to make pET29
expression constructs
comprising a polynucleotide molecule encoding a modified Clostridial toxin-
XP6A with an altered targeting
domain such as, e.g, altered targeting domains SEQ ID NO: 62 to SEQ ID NO: 71,
SEQ ID NO: 183 or
SEQ ID NO: 184; and altered targeting domains requiring a free amino-terminal
amino acid like SEQ ID
NO: 88 to SEQ ID NO: 115 and SEQ ID NO: 178 to SEQ ID NO: 181, as well as,
amino acids 26-58 of
SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104, SEQ ID NO:
105, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID
NO: 111, SEQ ID
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NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115; amino acids 47-58
of SEQ ID NO: 100,
SEQ ID NO: 110 or SEQ ID NO: 114; or amino acids 51-58 of SEQ ID NO: 100.
[0436] To construct a BoNT/A-XP6A-Galanin that will replace the BoNT/A
translocation facilitating
domain with another Clostridial toxin translocation facilitating domain, a
translocation facilitating domain of
BoNT/B will be introduced into the BoNT/A-XP6A-Galanin as described above
using a Splicing by
Overlapping ends polymerase chain reaction (SOE-PCR) procedure, see, e.g., R.
M. Horton et al.,
Engineering hybrid genes without the use of restriction enzymes: gene splicing
by overlapping extension,
77(1) Gene 61-68 (1989); and R. M. Horton, PCR-mediated recombination and
mutagenesis. SOEing
together tailor-made genes, 3(2) Mol. Biotechnol. 93-99 (1995). A nucleic acid
fragment comprising a
region encoding amino acids 859 to 1097 of BoNT/B (SEQ ID NO: 2) will be
operably-linked by SOE-PCR
to replace the region corresponding to the BoNT/A translocation facilitating
domain comprising amino
acids 874-1110 of SEQ ID NO: 1 of the BoNT/A-XP6A-Galanin and will be
subcloned into a pCR2.1
vector using the TOPO TA cloning method (Invitrogen, Inc, Carlsbad, CA). The
forward and reverse
oligonucleotide primers used for these reactions are designed to include
unique restriction enzyme sites
useful for subsequent subcloning steps. The resulting construct will be
digested with restriction enzymes
that 1) will excise the polynucleotide molecule containing the entire open
reading frame encoding the
modified BoNT/A-XP6A-Galanin; and 2) will enable this polynucleotide molecule
to be operably-linked to
a pET29 vector (EMD Biosciences-Novagen, Madison, WI). The resulting
restriction fragment will be
purified by the QlAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, CA), and
will be subcloned using a
T4 DNA ligase procedure into a pET29 vector. This cloning strategy yielded a
pET29 expression
construct encoding a BoNT/A-XP6A-Galanin comprising a BoNT/B translocation
facilitating domain.
[0437] A similar cloning strategy will be used to make pET29 expression
constructs comprising a
polynucleotide molecule encoding a BoNT/A-XP6A-Galanin that replaces the
region corresponding to the
BoNT/A translocation facilitating domain comprising amino acids 874-1110 of
SEQ ID NO: 1 with, e.g., a
translocation facilitating domain comprising amino acids 869-1111 of BoNT/C1
of SEQ ID NO: 3; a
translocation facilitating domain comprising amino acids 865-1098 of BoNT/D of
SEQ ID NO: 4; a
translocation facilitating domain comprising amino acids 846-1058 of BoNT/E of
SEQ ID NO: 5; a
translocation facilitating domain comprising amino acids 867-1105 of BoNT/F of
SEQ ID NO: 6; a
translocation facilitating domain comprising amino acids 866-1105 of BoNT/G of
SEQ ID NO: 7; or a
translocation facilitating domain comprising amino acids 882-1127 of TeNT of
SEQ ID NO: 8. Likewise, a
similar cloning strategy will be used to make pET29 expression constructs
comprising a polynucleotide
molecule encoding a BoNT/A-XP6A-Galanin that replaces the region corresponding
to the BoNT/A
translocation facilitating domain comprising amino acids 874-1110 of SEQ ID
NO: 1 with a translocation
facilitating domain comprising an enveloped virus fusogenic peptide domain,
such as, e,g, SEQ ID NO:
194 to SEQ ID NO: 269.
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[0438] Likewise, a polynucleotide molecule encoding a Clostridial
translocation facilitating domain as
described above can be introduced into a polynucleotide molecule encoding
BoNT/B-XP6A-Galanin,
BoNT/C1-XP6A-Galanin, BoNT/D-XP6A-Galanin, BoNT/E-XP6A-Galanin, BoNT/F-XP6A-
Galanin,
BoNT/G-XP6A-Galanin, TeNT-XP6A-Galanin, as well as the modified Clostridial
toxin-XP6A indicated
above comprising SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID NO: 88 to SEQ ID NO:
115, SEQ ID NO:
178 to SEQ ID NO: 181, SEQ ID NO: 183 or SEQ ID NO: 184, as well as, amino
acids 26-58 of SEQ ID
NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ
ID NO: 105, SEQ
ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111,
SEQ ID NO: 112,
SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115; amino acids 47-58 of SEQ ID
NO: 100, SEQ ID
NO: 110 or SEQ ID NO: 114; or amino acids 51-58 of SEQ ID NO: 100.
3b. A translocation-translocation facilitating-enzymatic-targeting domain
organization.
[0439] A polynucleotide molecule based on BoNT/A-XP6B-Galanin (SEQ ID NO: 193)
will be
synthesized and cloned into a pUCBHB1 vector as described in Example la. This
polynucleotide
molecule encodes a BoNT/A modified to replace amino acids 1111-1296 of SEQ ID
NO: 1, a BoNT/A Hcc
targeting domain, with SEQ ID NO: 72, a Galanin targeting domain, and has the
general domain
arrangement of FIG. 6B. If so desired, expression optimization to a different
organism, such as, e.g., a
bacteria, a yeast strain, an insect cell-line or a mammalian cell line, can be
done as described above,
see, e.g., Steward, supra, (Feb. 2, 2006); and Steward, supra, (Feb. 16,
2006).
[0440] Likewise, a similar cloning strategy will be used to make pUCBHB1
cloning constructs comprising
a polynucleotide molecule encoding a modified BoNT/A-XP6B with an altered
targeting domain such as,
e.g, altered targeting domains SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID NO: 183
or SEQ ID NO: 184;
and altered targeting domains requiring a free amino-terminal amino acid like
SEQ ID NO: 88 to SEQ ID
NO: 115 and SEQ ID NO: 178 to SEQ ID NO: 181, as well as, amino acids 26-58 of
SEQ ID NO: 100,
SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:
105, SEQ ID NO:
107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID
NO: 112, SEQ ID
NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115; amino acids 47-58 of SEQ ID NO:
100, SEQ ID NO: 110
or SEQ ID NO: 114; or amino acids 51-58 of SEQ ID NO: 100. In addition,
similar cloning strategy will be
used to produce a modified Clostridial toxin-XP6B, such as, e.g., BoNT/B-XP6B,
BoNT/C1-XP6B,
BoNT/D-XP6B, BoNT/E-XP6B, BoNT/F-XP6B, BoNT/G-XP6B or TeNT-XP6B, to comprise
an altered
targeting domain comprising any one of SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID
NO: 88 to SEQ ID
NO: 115, SEQ ID NO: 183 or SEQ ID NO: 184, or amino acids 26-58 of SEQ ID NO:
100, SEQ ID NO:
101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID
NO: 107, SEQ ID
NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ
ID NO: 113, SEQ
ID NO: 114 or SEQ ID NO: 115; amino acids 47-58 of SEQ ID NO: 100, SEQ ID NO:
110 or SEQ ID NO:
114; or amino acids 51-58 of SEQ ID NO: 100.
201
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Non-Provisional Patent Application 18049 (BOT)
Steward, L.E. et al., Modified Clostridial Toxins with Enhanced Translocation
Capabilities and
Altered Targeting Activity For Non-Clostridial Toxin Target Cells
[0441] To construct pET29/BoNT/A-XP6B-Galanin, a similar cloning strategy will
be used as described
in Example la. This cloning strategy will yield a pET29 expression construct
comprising the
polynucleotide molecule encoding the BoNT/A-XP6B-Galanin operably-linked to a
carboxyl terminal
polyhistidine affinity binding peptide. A similar cloning strategy will be
used to make pET29 expression
constructs for other modified Clostridial toxin-XP6B-Galanin toxins, such as,
e.g., BoNT/B-XP6B-Galanin,
BoNT/C1-XP6B-Galanin, BoNT/D-XP6B-Galanin, BoNT/E-XP6B-Galanin, BoNT/F-XP6B-
Galanin,
BoNT/G-XP6B-Galanin or TeNT-XP6B-Galanin. Likewise, a similar cloning strategy
will be used to make
pET29 expression constructs comprising a polynucleotide molecule encoding a
modified Clostridial toxin-
XP6B with an altered targeting domain such as, e.g, altered targeting domains
SEQ ID NO: 62 to SEQ ID
NO: 71, SEQ ID NO: 183 or SEQ ID NO: 184; and altered targeting domains
requiring a free amino-
terminal amino acid like SEQ ID NO: 88 to SEQ ID NO: 115 and SEQ ID NO: 178 to
SEQ ID NO: 181, as
well as, amino acids 26-58 of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,
SEQ ID NO: 103,
SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:
109, SEQ ID NO:
110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID
NO: 115; amino
acids 47-58 of SEQ ID NO: 100, SEQ ID NO: 110 or SEQ ID NO: 114; or amino
acids 51-58 of SEQ ID
NO: 100.
[0442] To construct a BoNT/A-XP6B-Galanin that will replace the BoNT/A
translocation facilitating
domain with another Clostridial toxin translocation facilitating domain, a
similar cloning strategy using
SOE-PCR will be used as described in Example la. This cloning strategy yielded
a pET29 expression
construct encoding a BoNT/A-XP6B-Galanin comprising a BoNT/B translocation
facilitating domain, a
BoNT/C1 translocation facilitating domain, a BoNT/D translocation facilitating
domain, a BoNT/E
translocation facilitating domain, a BoNT/F translocation facilitating domain,
a BoNT/G translocation
facilitating domain and a TeNT translocation facilitating domain, as well as,
a translocation facilitating
domain comprising an enveloped virus fusogenic peptide domain, such as, e,g,
SEQ ID NO: 194 to SEQ
ID NO: 269.
[0443] Likewise, a polynucleotide molecule encoding a Clostridial
translocation facilitating domain as
described above can be introduced into a polynucleotide molecule encoding
BoNT/B-XP6B-Galanin,
BoNT/C1-XP6B-Galanin, BoNT/D-XP6B-Galanin, BoNT/E-XP6B-Galanin, BoNT/F-XP6B-
Galanin,
BoNT/G-XP6B-Galanin, TeNT-XP6B-Galanin, as well as the modified Clostridial
toxin-XP6B indicated
above comprising SEQ ID NO: 62 to SEQ ID NO: 71, SEQ ID NO: 88 to SEQ ID NO:
115, SEQ ID NO:
178 to SEQ ID NO: 181, SEQ ID NO: 183 or SEQ ID NO: 184, as well as, amino
acids 26-58 of SEQ ID
NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ
ID NO: 105, SEQ
ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111,
SEQ ID NO: 112,
SEQ ID NO: 113, SEQ ID NO: 114 or SEQ ID NO: 115; amino acids 47-58 of SEQ ID
NO: 100, SEQ ID
NO: 110 or SEQ ID NO: 114; or amino acids 51-58 of SEQ ID NO: 100.
Example 4
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Expression of Modified Clostridial Toxins in a Bacterial Cell
[0444] The following example illustrates a procedure useful for expressing any
of the modified Clostridial
toxins disclosed in the present specification in a bacterial cell.
[0445] An expression construct, such as, e.g., any of the expression
constructs in Examples 1-3, will be
introduced into chemically competent E. coli BL21 (DE3) cells (Invitrogen,
Inc, Carlsbad, CA) using a
heat-shock transformation protocol. The heat-shock reaction will be plated
onto 1.5% Luria-Bertani agar
plates (pH 7.0) containing 50 pg/mL of Kanamycin and will be placed in a 37 C
incubator for overnight
growth. Kanamycin-resistant colonies of transformed E. coli containing the
expression construct will be
used to inoculate a baffled flask containing 3.0 mL of PA-0.5G media
containing 50 pg/mL of Kanamycin
which will then placed in a 37 C incubator, shaking at 250 rpm, for overnight
growth. The resulting
overnight starter culture will be used to inoculate a 3 L baffled flask
containing ZYP-5052 autoinducing
media containing 50 pg/mL of Kanamycin at a dilution of 1:1000. Culture
volumes will ranged from about
600 mL (20% flask volume) to about 750 mL (25% flask volume). These cultures
will be grown in a 37 C
incubator shaking at 250 rpm for approximately 5.5 hours and will be then
transferred to a 16 C incubator
shaking at 250 rpm for overnight expression. Cells will be harvested by
centrifugation (4,000 rpm at 4 C
for 20-30 minutes) and will be used immediately, or will be stored dry at -80
C until needed.
Example 5
Purification and Quantification of Modified Clostridial Toxins
[0446] The following example illustrates methods useful for purification and
quantification of any
modified Clostridial toxins disclosed in the present specification.
[0447] For immobilized metal affinity chromatography (IMAC) protein
purification, E. coli BL21 (DE3) cell
pellets used to express a modified Clostridial toxin, as described in Example
4, will be resuspended in
Column Binding Buffer (25 mM N-(2-hydroxyethyl) piperazine-N' (2-
ethanesulfonic acid) (HEPES), pH
7.8; 500 mM sodium chloride; 10 mM imidazole; 2x Protease Inhibitor Cocktail
Set III (EMD Biosciences-
Calbiochem, San Diego CA); 5 units/mL of Benzonase (EMD Biosciences-Novagen,
Madison, WI); 0.1%
(v/v) Triton-X 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol), and
will then be transferred to a
cold Oakridge centrifuge tube. The cell suspension will be sonicated on ice
(10-12 pulses of 10 seconds
at 40% amplitude with 60 seconds cooling intervals on a Branson Digital
Sonifier) in order to lyse the cells
and then is centrifuged (16,000 rpm at 4 C for 20 minutes) to clarify the
lysate. An immobilized metal
affinity chromatography column will be prepared using a 20 mL Econo-Pac column
support (Bio-Rad
Laboratories, Hercules, CA) packed with 2.5-5.0 mL of TALONT"' SuperFlow Co2+
affinity resin (BD
Biosciences-Clontech, Palo Alto, CA), which will then be equilibrated by
rinsing with 5 column volumes of
deionized, distilled water, followed by 5 column volumes of Column Binding
Buffer. The clarified lysate
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will be applied slowly to the equilibrated column by gravity flow
(approximately 0.25-0.3 mL/minute). The
column will then be washed with 5 column volumes of Column Wash Buffer (N-(2-
hydroxyethyl)
piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodium chloride;
10 mM imidazole; 0.1%
(v/v) Triton-X 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol). The
modified Clostridial toxin will
be eluted with 20-30 mL of Column Elution Buffer (25 mM N-(2-hydroxyethyl)
piperazine-N' (2-
ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodium chloride; 500 mM
imidazole; 0.1% (v/v) Triton-X
100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol) and will be collected
in approximately twelve 1 mL
fractions. The amount of modified Clostridial toxin contained in each elution
fraction will be determined by
a Bradford dye assay. In this procedure, 20 pL aliquots of each 1.0 mL
fraction will be combined with 200
pL of Bio-Rad Protein Reagent (Bio-Rad Laboratories, Hercules, CA), diluted 1
to 4 with deionized,
distilled water, and then the intensity of the colorimetric signal will be
measured using a
spectrophotometer. The five fractions with the strongest signal will be
considered the elution peak and
will be combined together. Total protein yield will be determined by
estimating the total protein
concentration of the pooled peak elution fractions using bovine gamma globulin
as a standard (Bio-Rad
Laboratories, Hercules, CA).
[0448] For purification of a modified Clostridial toxin using a FPLC desalting
column, a HiPrepTM 26/10
size exclusion column (Amersham Biosciences, Piscataway, NJ) will be pre-
equilibrated with 80 mL of
4 C Column Buffer (50 mM sodium phosphate, pH 6.5). After the column is
equilibrated, a modified
Clostridial toxin sample will be applied to the size exclusion column with an
isocratic mobile phase of 4 C
Column Buffer and at a flow rate of 10 mL/minute using a BioLogic DuoFlow
chromatography system
(Bio-Rad Laboratories, Hercules, CA). The desalted modified Clostridial toxin
sample will be collected as
a single fraction of approximately 7-12 mL.
[0449] For purification of a modified Clostridial toxin using a FPLC ion
exchange column, a modified
Clostridial toxin sample that has been desalted following elution from an IMAC
column will be applied to a
1 mL Q1 T"' anion exchange column (Bio-Rad Laboratories, Hercules, CA) using a
BioLogic DuoFlow
chromatography system (Bio-Rad Laboratories, Hercules, CA). The sample will be
applied to the column
in 4 C Column Buffer (50 mM sodium phosphate, pH 6.5) and will be eluted by
linear gradient with 4 C
Elution Buffer (50 mM sodium phosphate, 1 M sodium chloride, pH 6.5) as
follows: step 1, 5.0 mL of 5%
Elution Buffer at a flow rate of 1 mL/minute; step 2, 20.0 mL of 5-30% Elution
Buffer at a flow rate of 1
mL/minute; step 3, 2.0 mL of 50% Elution Buffer at a flow rate of 1.0
mL/minute; step 4, 4.0 mL of 100%
Elution Buffer at a flow rate of 1.0 mL/minute; and step 5, 5.0 mL of 0%
Elution Buffer at a flow rate of 1.0
mL/minute. Elution of modified Clostridial toxin from the column will be
monitored at 280, 260, and 214
nm, and peaks absorbing above a minimum threshold (0.01 au) at 280 nm will be
collected. Most of the
modified Clostridial toxin will be eluted at a sodium chloride concentration
of approximately 100 to 200
mM. Average total yields of modified Clostridial toxin will be determined by a
Bradford assay.
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[0450] Expression of a modified Clostridial toxin will be analyzed by
polyacrylamide gel electrophoresis.
Samples purified using the procedure described above are added to 2x LDS
Sample Buffer (Invitrogen,
Inc, Carlsbad, CA) and will be separated by MOPS polyacrylamide gel
electrophoresis using NuPAGE
Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad,
CA) under denaturing,
reducing conditions. Gels will be stained with SYPRO Ruby (Bio-Rad
Laboratories, Hercules, CA) and
the separated polypeptides will be imaged using a Fluor-S MAX Multilmager (Bio-
Rad Laboratories,
Hercules, CA) for quantification of modified Clostridial toxin expression
levels. The size and amount of
modified Clostridial toxin will be determined by comparison to MagicMarkT"'
protein molecular weight
standards (Invitrogen, Inc, Carlsbad, CA).
[0451] Expression of modified Clostridial toxin will also be analyzed by
Western blot analysis. Protein
samples purified using the procedure described above will be added to 2x LDS
Sample Buffer (Invitrogen,
Inc, Carlsbad, CA) and will be separated by MOPS polyacrylamide gel
electrophoresis using NuPAGE
Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad,
CA) under denaturing,
reducing conditions. Separated polypeptides will be transferred from the gel
onto polyvinylidene fluoride
(PVDF) membranes (Invitrogen, Inc, Carlsbad, CA) by Western blotting using a
Trans-Blot SD semi-dry
electrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules, CA).
PVDF membranes will be
blocked by incubating at room temperature for 2 hours in a solution containing
25 mM Tris-Buffered
Saline (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-
HCI)(pH 7.4), 137 mM
sodium chloride, 2.7 mM potassium chloride), 0.1% TWEEN-20 , polyoxyethylene
(20) sorbitan
monolaureate, 2% bovine serum albumin, 5% nonfat dry milk. Blocked membranes
will be incubated at 4
C for overnight in Tris-Buffered Saline TWEEN-20 (25 mM Tris-Buffered Saline,
0.1% TWEEN-20 ,
polyoxyethylene (20) sorbitan monolaureate) containing appropriate primary
antibodies as a probe.
Primary antibody probed blots will be washed three times for 15 minutes each
time in Tris-Buffered Saline
TWEEN-20 . Washed membranes will be incubated at room temperature for 2 hours
in Tris-Buffered
Saline TWEEN-20 containing an appropriate immunoglobulin G antibody
conjugated to horseradish
peroxidase as a secondary antibody. Secondary antibody-probed blots will be
washed three times for 15
minutes each time in Tris-Buffered Saline TWEEN-20 . Signal detection of the
labeled modified
Clostridial toxin will be visualized using the ECL PIusTM Western Blot
Detection System (Amersham
Biosciences, Piscataway, NJ) and will be imaged with a Typhoon 9410 Variable
Mode Imager (Amersham
Biosciences, Piscataway, NJ) for quantification of modified Clostridial toxin
expression levels.
[0452] Although aspects of the present invention have been described with
reference to the disclosed
embodiments, one skilled in the art will readily appreciate that the specific
examples disclosed are only
illustrative of these aspects and in no way limit the present invention.
Various modifications can be made
without departing from the spirit of the present invention.
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