Note: Descriptions are shown in the official language in which they were submitted.
CA 02592285 2015-03-16
COMPOSITIONS COMPRISING CONNEXIN POLYPEPTIDES AND METHODS
FOR PROMOTING WOUND HEALING AND TISSUE REGENERATION
USING CONNEXIN POLYPEPTIDES
ACKNOWLEDGEMENTS
This invention was made with government support under Grant RO-1 HL56728
awarded by the National Institutes of Health. The government of the United
States has
certain rights in the invention.
BACKGROUND OF THE INVENTION
Your average kid knows that if a skink lizard looses a tail it will eventually
grow
another one. Moreover, it is well understood among children and grown-ups who
make a
habit of studying such things that many lower animals are capable of
regenerating quite
complex structures, including whole limbs and organs following injury. For
example, fish
are able to grow back a heart after a significant part of the old heart of the
fish had been
sliced away (Poss et at., 2002). This is an astounding result when one
reflects on how
essential the heart is to the minute-to-minute survival of most animals.
However, regeneration of tissue, limbs and organs following injury in people
is not
as straightforward as it is in fish. While human tissues damaged by mechanical
wounding,
disease processes and other causes are capable of healing, complex tissue
structure and
function is rarely, if ever wholly restored. Instead, recovery of nearly all
tissues from injury
in humans and other higher vertebrates is dominated by the formation of scar
tissue. The
most familiar example of this is the discolored and fibrotic scars that linger
following the
healing of a skin cut or graze. Less well appreciated is that formation of
glial scar tissue
following injury to the brain or spinal chord is one of the main obstacles to
restoration of
neural function following damage to the central nervous system (Silver and
Miller RI,
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CA 02592285 2009-02-20
2004). There is currently no means of treating or preventing such scarring and
promoting
the rqgenpration of complex tissue structure and function following injury.
BRIEF SUMMARY OF THI INVENTION
An object of the present invention is to provide compositions and methods for
promoting wound healing and tissue regeneration.
In accordance with an aspect of the present invention there is provided,
a composition comprising an isolated polypeptide comprising the carboxy-
terminal
amino acid sequence of an alpha Connexin, or a conservative variant thereof,
wherein
the polypeptide does not comprise the full length alpha Connexin protein,
wherein the
composition is a formulation for topical administration.
Providedis an isolated polypeptide comprising a carboxy-terminal amino acid
sequence of an alpha Connexin (also referred to herein as an alpha Connexin
carboxy-
Terminal (ACT) polypeptide), or a conservative variant thereof.
Provided herein is a method of promoting wound healing following tissue injury
in a
subject, comprising administering to the subject one or more of the herein
provided
compositions (e.g., polypeptides, nucleic acids, or vectors) in a
pharmaceutically acceptable
carrier.
Additional advantages of the disclosed method and compositions will be set
forth in
part in the description which follows, and in part will be understood from the
description, or
may be learned by practice of the disclosed method and compositions. The
advantages of
the disclosed method and compositions will be realized and attained by means
of the
elements and combinations particularly pointed out in the appended claims. It
is to be
understood that both the foregoing general description and the following
detailed description
are exemplary and explanatory only and are not restrictive of the invention as
claimed.
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CA 02592285 2009-02-20
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and constitute a part of
this
specification, illustrate several embodiments of the disclosed method and
compositions and
together with the description, serve to explain the principles of the
disclosed method and
compositions.
Figure 1 shows that an alpha Connexin carboxy-Terminal (ACT) polypeptide
increases the extent of Cx43 gap junction formation in cultured neonatal
myocytes.
Myocytes from neonatal rat hearts were grown until forming a near-confluent
monolayer on
a tissue culture dish according to standard protocols. The cultures were
subsequently
allowed to culture for a further 5 days in culture medium comprising (a) 30 M
ACT 1
peptide (SEQ D NO:2), (b) 30 M non-active control peptide (SEQ lD NO:55), or
(c)
phosphate buffered saline (PBS) containing no ACT peptide or control. Culture
media with
added peptides or vehicle control was changed every 24 hours during the
experiment. (a)
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CA 02592285 2007-06-28
indicates that ACT peptide greatly increased the extent of Cx43 gap junction
formation (dots
and lines indicated by arrowheads) between myocytes relative to the control
conditions (b)
and (c). This increase in Cx43 gap junction formation in response to ACT
peptide is shared
by a number of cell types expressing Cx43.
Figure 2 shows that ACT peptide inhibits proliferation and migration of
transformed
fibroblasts (NILI-3T3 cells) injured by a scratch. An NIH-3T3 monolayer was
pre-treated
with ACT 1 peptide (SEQ ID NO:2) for 24 hrs, and "scratch-injured" with a p200
pipette
tip. The "scratch injury" was subsequently allowed to "heal" for 24 hours in
the presence of
(a, b) 30 M ACT 1 peptide (SEQ ID NO:2), (c, d) 30 M non-active control
peptide (SEQ
ID NO: 55), or (e, 0 vehicle control solution containing no ACT peptide or
control peptide.
The "scratch injury" of ACT peptide-treated cells remains relatively unhealed
after 24 hours
(a), with few cells (large arrow) repopulating the area within the initial
"scratch injury"
edges (i.e., within area marked by the small black arrowheads). By contrast,
in the control
conditions in (c) and (e), large numbers of cells (large arrows) have
repopulated the area
within the initial "scratch injury". The repopulation of the "scratch injury"
occurs in part via
migration of the transformed cells crawling into the "scratch injury" area.
Figures (b), (d)
and (f) show proliferating cell nuclear antigen (PCNA) immunolabeling of cells
in the
"scratch injury" or at the injury edge. ACT peptide treated cells (b) show
only low
luminosity consistent with background and non-proliferation. Only in the two
control
conditions shown in (d) and (f), are brightly labeled proliferating cells seen
(white arrows).
This indicates that the ACT peptide has also reduced proliferation of the
transformed cells.
Figure 3 shows quantification of the inhibition of migration by ACT peptides
following injury in an experimental cellular model. N1H-3T3 fibroblasts were
"scratch
injured" and subject to the continuous presence of 30 M ACT 1 peptide (SEQ ID
NO:2) for
24 hours or the control conditions as outlined in Figure 2. Figure (a) shows
the injury edge
of ACT peptide and non-active peptide-treated control cells at the end of the
24-hour period.
The cells have been labeled with fluorescent phalloidin to aid visualization.
ACT peptide-
treated cells show low levels of repopulation of the scratch injury area
(white double headed
arrows). Figure (b) shows a bar graph of the % area of cells repopulating the
scratch injury
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CA 02592285 2007-06-28
after 24 hours. The reduction of cells in the injury area in the presence of
ACT peptide is
dramatic, with a p <0.000001.
Figure 4 shows that expression of an ACT-peptide-encoding-polynucleotide
operably linked to a promoter in the epithelial cell WB-F344 inhibits
migration following
scratch injury in an experimental cellular model. WB-F344 cells are a
transformed rat
epithelial cell line derived by treatment of isolated rat liver cells with a
cancer-causing agent
(Tsao et al., 1984; Hayashi et al., 1997; Hayashi et al., 1998; Hayashi et
al., 2001). WB-
F344 cells were transfected with a cDNA expression plasmid construct and
selected under
antibiotic using standard protocols to generate cell lines that stably
expressed an ACT-
peptide-encoding-polynucleotide (SEQ ID NO:6) operably linked to a promoter
sequence or
a green fluorescent protein (GFP) polynucleotide operably linked to a promoter
sequence as
a control. The polynucleotide encoding the ACT peptide also encoded GFP. As
such,
expression of the ACT peptide could be assayed by standard GFP fluorescence
optics on a
light microscope. (a) and (b) show high magnification images of GFP
fluorescence in WB-
F344 cell lines expressing GFP plus the carboxy terminus ACT peptide sequence
(a) or GFP
alone (b). Near confluent monolayers of the WB-F344 cell lines were "scratch
injured" and
allowed to "heal" for 24 hours. Similar to the control cases of the NIH-3T3
cells treated with
vehicle or non-active control peptide, the control epithelial cell line
expressing GFP
repopulated the scratch injury (d). However, in the epithelial cell line that
stably expressed
the ACT-peptide-encoding-polynucleotide operably linked to a promoter
sequence, there
was inhibited repopulation of the scratch injury (c).
Figure 5 shows that ACT peptide reduces inflammation, improves healing and
reduces scarring following incisional skin injury in a neonatal mouse.
Neonatal mouse pups
were desensitized using hypothermia. A 4 mm long incisional skin injury was
made using a
scalpel through the entire thickness of the skin (down to the level of the
underlying muscle)
in the dorsal mid line between the shoulder blades. 30 I of a solution of 20
% pluronic (F-
127) gel containing either no (control) or dissolved ACT 1 peptide (SEQ ID NO:
2) at a
concentration of 60 M was then applied to the incisional injuries. Control or
ACT peptide
containing gel was applied subsequently 24 hours after the initial
application. No further
application of control and ACT peptide containing gel was made after the
second
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CA 02592285 2007-06-28
application. By 48 hours the ACT peptide treated injury (a) is significantly
more closed, less
inflamed, less swollen (note ridges at the wound edge), and generally more
healed in
appearance than the control injury that received no ACT peptide (b). These
differences in
inflammation, swelling and healing between the control and ACT peptide and
control treated
injury persisted at the 72 (c, d) and 96 (e, f) hour time points. At 7 days,
the ACT peptide
wound (g), had a smoother and less scarred appearance than the control peptide-
treated
injury (h). Note that images of the same injury on the same animal are shown
at the different
time points during the healing time course.
Figure 6 shows that ACT peptide reduces inflammation, improves healing and
reduces scarring following a large excisional skin injury in adult mice.
Anesthetized adult
mice had 8 mm wide circular excisional skin injuries made by fine surgical
scissors down to
the underlying muscle in the dorsal mid line between the shoulder blades
(i.e., as shown in
(a) an (b). The boundary of the injury was demarcated by an 8 mm wide circular
template
cut in a plastic sheet. 100 p.1 of a solution of 30% pluronic gel containing
either no (control)
or dissolved ACT 1 peptide (SEQ ID NO:2) at a concentration of 100 p.M was
then applied
to the excisional injuries. Control or ACT peptide containing gel was applied
subsequently
24 hours after the initial application. No further applications of control and
ACT peptide
containing gel were made after the second application. The ACT peptide-treated
large
excisional injury (a, c e, g, i) closed faster, was less inflamed in
appearance, healed faster
and scarred less than the control injury that received no ACT peptide (b, d,
f, h, j) over the
14 day time course. Indeed, the control injury at 14 days still shows a
partial scab indicating
that acute healing of the injury was incomplete (j). Note that images of the
same injury on
the same animal are shown at the different time points during the healing time
course.
Figure 7 shows that ACT peptide reduces the density of inflammatory cells
following excisional skin injury in adult mice. Skin biopsies of the entire
wound site were
taken from some of the mice 24 hours following the excisional injury in the
experiment
described in Figure 6. Figures (a) and (b) show low magnification survey views
of cross-
sections from near the center of the wound of control and ACT peptide treated
injuries
respectively. The wound edge (marked by the small arrows), bounded by skin of
normal
histological appearance, can be seen in both cases. A black rectangle is
placed over the
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CA 02592285 2007-06-28
images in (a) and (b) at the left hand wound edge. The histological structures
within these
two rectangles are shown at higher magnification in (c) and (d) for control
and ACT peptide
treated tissues, respectively. Of most interest is a "collar-like" tissue of
aligned fibrous
material (arrowed) projecting from basal parts of the injury to or toward the
wound edge and
exterior surface of injury. The aligned fibrous substrate has the appearance
of being much
more organized in the control injury (d) than in the ACT peptide treated
injury (c). Also,
there is a considerably lower density of inflammatory cells studding the
fibrous substrate in
the ACT peptide-treated tissue. This is confirmed in (e) and (f) where regions
of histological
section within the black rectangles shown in (c) and (d) are respectively
shown at higher
magnification. The inflammatory cells studding the aligned fibrous substrate
include mast
cells, neutrophils and macrophages. These inflammatory cells occur at much
higher density
in the control injury than in the ACT peptide treated injury.
Figure 8 shows that ACT peptide promotes healing, reduces scarring and
promotes
regeneration of complex tissue structure following excisional skin injury in
adult mice. At
the end of the 14 day period in the experiment described in Figure 6, skin
biopsies of the
entire excisional injury were taken and histological sections from these skin
samples were
H&E histochemically stained. Figures (a) and (b) show low magnification survey
views of
cross-sections from near the center of the injury of ACT peptide and control
respectively.
The wound edge (marked by the small arrows), bounded by skin of normal
histological
appearance, can be seen in both cases. A black rectangle is placed over the
images in (a) and
(b) near the center of each injury. The histological structures within these
two rectangles are
shown at higher magnification in (c) and (d) for the ACT peptide and control
tissues
respectively. It is evident that tissue within the ACT peptide treated injury
locus has
considerably more complexity. At the external surface of the ACT treated
wound, there is a
continuous layer of epithelial cells indicating that re-epithelization of the
injured surface is
complete, albeit that the epithelium is as yet relatively thin near the center
of the wound (c).
Regenerating hair follicles can already be seen differentiating de novo from
stem cells in the
new epithelium covering the healed injury (c, small arrows). By comparison, re-
epithelization of the injury surface is incomplete and there is no sign of
regenerating hair
follicles in the epithelium of the control injury (d). Beneath the reformed
epithelium of the
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CA 02592285 2007-06-28
,
ACT peptide treated injured skin, considerable restoration of normal
structural complexity is
seen, with glandular structures, fibrous and connective tissues, vascular
tissues, muscle and
fat cells all in evidence (a, c). As with, the hair follicles this tissue
complexity was
regenerated by differentiation of stem cells. By contrast, in the control
injury the wound
tissue is completely dominated by a uniform and large plug of fibrous scar
tissue (b, d), with
other complexity of tissue structure not particularly in evidence within this
scar tissue
Figure 9 shows that ACT peptides reduce inflammation, improve healing and
reduce
scarring following excisional skin injury in adult mice. Anesthetized adult
mice had 2 small
(5mm diameter) excisional skin wounds made by fine surgical scissors on the
neck and
(upper) back. The boundaries of the injuries were demarcated by a 5 mm wide
circular
template cut in a plastic sheet. 50-60 I of a solution of 20 % pluronic gel
containing either
no (control) or one of the ACT peptides (ACT 2-SEQ ID NO:1, ACT 1-SEQ ID NO:2,
ACT
3-SEQ ID NO:3, ACT 4-SEQ ID NO:4, ACT 5-SEQ lD NO:5) dissolved at
concentrations
of 100 M were then applied to the excisional injuries. Control or ACT peptide-
containing
gel was applied subsequently 24 hours after the initial application. No
further applications of
control and ACT peptide-containing gel were made after the second application.
It can be
noted in the case of ACT 1 (e-h), ACT 2 (i-1), ACT 3 (m-p), and ACT 5 (u-x)
peptides that
excisional injuries closed faster, were significantly less inflamed in
appearance, healed faster
and scarred less than the control injury that received no ACT peptide (a-d)
over the 240
hour time course (10 days). The ACT 4 peptide (q-t) also showed modest
improvement in
healing over the control during the time course, although less so than other
peptides. Note
that the same wound on the same animal is shown at the different time points
during the
healing time course.
Figure 10 shows that ACT peptide reduces the number and density of glial scar
forming astrocytes following penetration injury of brain in an adult rat. (b)
and (c) show low
magnification survey views of sections of brain tissue (cortex) surrounding
hollow fiber
membrane (HFM) implants filled with ACT peptide (100 M) plus vehicle gel (b)
or
collagen vehicle gel alone as control (c). In the control tissue (c), a high
density of
immunolabeled GFAP-positive astrocytes is observed near the site of injury
caused by the
HFM. The density of these cells appears to diminish slightly distal from the
injury. By
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CA 02592285 2007-06-28
contrast, a much lower density of GFAP-positive astrocytes is observed
adjacent the HFM
filled with ACT peptide (b). Indeed, the levels of GFAP positive cells are not
dissimilar to
those seen in normal uninjured brain tissue. The regions of tissue within the
white rectangles
in figures (b) and (c) are shown at higher magnification in (d) and (e)
respectively. In the
brain injury treated by ACT peptide (d), it can be seen that GFAP-positive
astrocytes are not
only less numerous, but are also smaller than those seen in the control injury
(e).
Figure 11 shows that ACT peptide promotes neuronal maintenance and neuronal
regeneration following penetration injury of brain in an adult rat. (a) and
(b) show low
magnification survey views of sections of brain tissue (cortex) surrounding
HFM implants
(implant or injury border is shown by arrows) filled with control collagen
vehicle gel or
ACT peptide plus vehicle gel at 1 week following brain penetration injury. In
the control
tissue (b), a high density of immunolabeled GFAP-positive astrocytes and a low
density of
NeuN immunolabeled neurons are observed near the site of injury caused by the
HFM. The
density of these cells appears to diminish and increase, respectively, distal
from the HFM.
By contrast, a much lower density of GFAP-positive astrocytes and higher
numbers NeuN
immunolabeled neurons are observed proximal (as well as distal) to the HFM
filled with
ACT peptide (a). The areas in (a) and (b) proximal to the HFMs are shown at
high
magnification views in (c) and (b), respectively. Again, in the control tissue
(d) a striking
increase in the density of GFAP-positive astrocytes and a reduced density of
NeuN-positive
neurons is observed compared to ACT peptide treated tissue (c). A
complementary pattern is
observed near the HFM containing ACT peptide, with NeuN positive neurons
predominating over astrocytes (c). Interestingly, the high magnification view
shown in (c)
reveals a high frequency of neurons in the process of fission relative to the
control (d).
DETAILED DESCRIPTION OF THE INVENTION
The disclosed method and compositions may be understood more readily by
reference to the following detailed description of particular embodiments and
the Examples
included therein and to the Figures and their previous and following
description.
Provided is an isolated polypeptide comprising a carboxy-terminal amino acid
sequence of an alpha Cormexin (also referred to herein as an alpha Connexin
carboxy-
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Terminal (ACT) polypeptide), or a conservative variant thereof. In one aspect,
following
tissue injury, the provided ACT polypeptide reduces inflammation, promotes
healing,
reduces scarring, increases tensile strength, and promotes complex tissue
regeneration. In
another aspect, the provided polypeptide increases the extent of gap
junctional channel
aggregates formed from Connexins.
It is to be understood that the disclosed compositions and methods are not
limited to
specific synthetic methods, specific analytical techniques, or to particular
reagents unless
otherwise specified, and, as such, may vary. It is also to be understood that
the terminology
used herein is for the purpose of describing particular embodiments only and
is not intended
to be limiting.
Disclosed are materials, compositions, and components that can be used for,
can be
used in conjunction with, can be used in preparation for, or are products of
the disclosed
method and compositions. These and other materials are disclosed herein, and
it is
understood that when combinations, subsets, interactions, groups, etc. of
these materials are
disclosed that while specific reference of each various individual and
collective
combinations and permutation of these compounds may not be explicitly
disclosed, each is
specifically contemplated and described herein. For example, if a vector is
disclosed and
discussed and a number of vector components including the promoters are
discussed, each
and every combination and permutation of promoters and other vector components
and the
modifications that are possible are specifically contemplated unless
specifically indicated to
the contrary. Thus, if a class of molecules A, B, and C are disclosed as well
as a class of
molecules D, E, and F and an example of a combination molecule, A-D is
disclosed, then
even if each is not individually recited, each is individually and
collectively contemplated.
Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D,
C-E, and C-
F are specifically contemplated and should be considered disclosed from
disclosure of A, B,
and C; D, E, and F; and the example combination A-D. Likewise, any subset or
combination of these is also specifically contemplated and disclosed. Thus,
for example, the
sub-group of A-E, B-F, and C-E are specifically contemplated and should be
considered
disclosed from disclosure of A, B, and C; D, E, and F; and the example
combination A-D.
This concept applies to all aspects of this application including, but not
limited to, steps in
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methods of making and using the disclosed compositions. Thus, if there are a
variety of
additional steps that can be performed it is understood that each of these
additional steps can
be performed with any specific embodiment or combination of embodiments of the
disclosed methods, and that each such combination is specifically contemplated
and should
be considered disclosed.
A variety of sequences are provided herein and these and others can be found
in
Genbank. Those of skill in the art understand how to
resolve sequence
discrepancies and differences and to adjust the compositions and methods
relating to a
particular sequence to other related sequences. Primers and/or probes can be
designed for
any sequence given the information disclosed herein and known in the art.
The herein provided polypeptide can be any polypeptide comprising the carboxy-
terminal most amino acids of an alpha Connexin, wherein the polypeptide does
not comprise
the flu-length alpha Connexin protein. Thus, in one aspect, the provided
polypeptide does
not comprise the cytoplasmic N-terminal domain of the alpha Connexin. In
another aspect,
the provided polypeptide does not comprise the two extracellular domains of
the alpha
Connexin. In another aspect, the provided polypeptide does not comprise the
four
transmembrane domains of the alpha Connexin. In another aspect, the provided
polypeptide
does not comprise the cytoplasmic loop domain of the alpha Connexin. In
another aspect,
the provided polypeptide does not comprise that part of the sequence of the
cytoplasmic
carboxyl terminal domain of the alpha Connexin proximal to the fourth
transmembrane
domain. There is a conserved proline or glycine residue in alpha Connexins
consistently
positioned some 17 to 30 amino acids from the carboxyl terminal-most amino
acid (Table
2). For example, for human Cx43 a proline residue at amino acid 363 is
positioned 19 amino
acids back from the carboxyl terminal most isoleucine. In another example, for
chick Cx43 a
proline residue at amino acid 362 is positioned 18 amino acids back from the
carboxyl
terminal-most isoleucine. In another example, for human Cx45 a glycine residue
at amino
acid 377 is positioned 19 amino acids back from the carboxyl terminal most
isoleucine. In
another example for rat Cx33, a proline residue at amino acid 258 is
positioned 28 amino
acids back from the carboxyl terminal most methionine. Thus, in another
aspect, the
provided polypeptide does not comprise amino acids proximal to said conserved
proline or
CA 02592285 2007-06-28
glycine residue of the alpha Connexin. Thus, the provided polypeptide can
comprise the c-
terminal-most 4 to 30 amino acids of the alpha Connexin, including the c-
terminal most 4, 5,
6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30
amino acids of the alpha Connexin.
The carboxy-terminal most amino acids of an alpha Connexin in the provided
peptides can be flanked by non-alpha Connexin or non-ACT peptide Connexin
amino acids.
Examples of the flanking non-alpha Connexin and non-ACT Connexin amino acids
are
provided herein. An example of non-ACT Connexin amino acids are the carboxy-
terminal
20 to 120 amino acids of human Cx43 (SEQ ID NO: 72). Another example would be
the
.. carboxy-terminal 20 to 120 amino acids of chick Cx43 (SEQ ID NO: 73).
Another example
would be the carboxy-terminal 20 to 120 amino acids of human Cx45 (SEQ ID NO:
74).
Another example would be the carboxy-terminal 20 to 120 amino acids of chick
Cx45 (SEQ
ID NO: 75). Another example would be the carboxy-terminal 20 to 120 amino of
human
Cx37 (SEQ ID NO: 76). Another example would be the carboxy-terminal 20 to 120
amino
acids of rat Cx33 (SEQ ID NO: 77).
An example of a non-alpha Connexin is the 239 amino acid sequence of enhanced
green fluorescent protein (ACT1 is shown functionally fused to GFP in Figure
4; SEQ ID
NO: 78). In another aspect, given that ACT1 is shown to be functional when
fused to the
carboxy terminus of the 239 amino acid sequence of GFP (e.g., Figure 4), ACT
peptides are
expected to retain function when flanked with non-Connexin polypeptides of up
to at least
239 amino acids. Indeed, as long as the ACT sequence is maintained as the free
carboxy
terminus of a given polypeptide, and the ACT peptide is able to access its
targets. Thus,
polypeptides exceeding 239 amino acids in addition to the ACT peptide can
function in
reducing inflammation, promoting healing, increasing tensile strength,
reducing scarring and
promoting tissue regeneration following injury.
Connexins are the sub-unit protein of the gap junction channel which is
responsible
for intercellular communication (Goodenough and Paul, 2003). Based on patterns
of
conservation of nucleotide sequence, the genes encoding Connexin proteins are
divided into
two families termed the alpha and beta Connexin genes. The carboxy-terminal-
most amino
acid sequences of alpha Connexins are characterized by multiple distinctive
and conserved
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CA 02592285 2007-06-28
features (see Table 2). This conservation of organization is consistent with
the ability of
ACT peptides to form distinctive 3D structures, interact with multiple
partnering proteins,
mediate interactions with lipids and membranes, interact with nucleic acids
including DNA,
transit and/or block membrane channels and provide consensus motifs for
proteolytic
cleavage, protein cross-linking, ADP-ribosylation, glycosylation and
phosphorylation. Thus,
the provided polypeptide interacts with a domain of a protein that normally
mediates the
binding of said protein to the carboxy-terminus of an alpha Connexin. For
example,
nephroblastoma overexpressed protein (NOV) interacts with a Cx43 c-terminal
domain (Fu
et al., J Biol Chem. 2004 279(35):36943-50). It is considered that this and
other proteins
interact with the carboxy-terminus of alpha Connexins and further interact
with other
proteins forming a macromolecular complex. Thus, the provided polypeptide can
inhibit the
operation of a molecular machine, such as, for example, one involved in
regulating the
aggregation of Cx43 gap junction channels.
As used herein, "inhibit," "inhibiting," and "inhibition" mean to decrease an
activity,
response, condition, disease, or other biological parameter. This can include,
but is not
limited to, the complete loss of activity, response, condition, or disease.
This can also
include, for example, a 10% reduction in the activity, response, condition, or
disease as
compared to the native or control level. Thus, the reduction can be a 10, 20,
30, 40, 50, 60,
70, 80, 90, 100%, or any amount of reduction in between as compared to native
or control
levels.
The ACT sequence of the provided polypeptide can be from any alpha Connexin.
Thus, the alpha Connexin component of the provided polypeptide can be from a
human,
murine, bovine, monotrene, marsupial, primate, rodent, cetacean, mammalian,
avian,
reptilian, amphibian, piscine, chordate, protochordate or other alpha
Connexin.
Thus, the provided polypeptide can comprise an ACT of a Connexin selected from
the group consisting of mouse Connexin 47, human Connexin 47, Human Connexin
46.6,
Cow Connexin 46.6, Mouse Connexin 30.2, Rat Connexin 30.2, Human Connexin
31.9,
Dog Connexin 31.9, Sheep Connexin 44, Cow Connexin 44, Rat Connexin 33, Mouse
Connexin 33, Human Connexin 36, mouse Connexin 36, rat Connexin 36, dog
Connexin 36,
chick Connexin 36, zebrafish Connexin 36, morone Connexin 35, morone Connexin
35,
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,
,
Cynops Connexin 35, Tetraodon Connexin 36, human Connexin 37, chimp Connexin
37,
dog Connexin 37, Cricetulus Connexin 37, Mouse Connexin 37, Mesocricetus
Connexin 37,
Rat Connexin 37, mouse Connexin 39, rat Connexin 39, human Connexin 40.1,
Xenopus
Connexin 38, Zebrafish Connexin 39.9, Human Connexin 40, Chimp Connexin 40,
dog
Connexin 40, cow Connexin 40, mouse Connexin 40, rat Connexin 40, Cricetulus
Connexin
40, Chick Connexin 40, human Connexin 43, Cercopithecus Connexin 43,
Oryctolagus
Connexin 43, Spermophilus Connexin 43, Cricetulus Connexin 43, Phodopus
Connexin 43,
Rat Connexin 43, Sus Connexin 43, Mesocricetus Connexin 43, Mouse Connexin 43,
Cavia
Connexin 43, Cow Connexin 43, Erinaceus Connexin 43, Chick Connexin 43,
Xenopus
Connexin 43, Oryctolagus Connexin 43, Cyprinus Connexin 43, Zebrafish Connexin
43,
Danio aequipinnatus Connexin 43, Zebrafish Connexin 43.4, Zebrafish Connexin
44.2,
Zebrafish Connexin 44.1, human Connexin45, chimp Connexin 45, dog Connexin 45,
mouse Connexin 45, cow Connexin 45, rat Connexin 45, chick Connexin 45,
Tetraodon
Connexin 45, chick Connexin 45, human Connexin 46, chimp Connexin 46, mouse
Connexin 46, dog Connexin 46, rat Connexin 46, Mesocricetus Connexin 46,
Cricetulus
Connexin 46, Chick Connexin 56, Zebrafish Connexin 39.9 , cow Connexin 49,
human
Connexin 50, chimp Connexin 50, rat Connexin 50, mouse Connexin 50, dog
Connexin 50,
sheep Connexin 49, Mesocricetus Connexin 50, Cricetulus Connexin 50, Chick
Connexin
50, human Connexin 59, or other alpha Connexin. Amino acid sequences for alpha
connexins are known in the art and include those identified in Table 1 by
accession number.
Table 1: Alpha Connexins
Protein Accession No. Protein Accession
No.
mouse Connexin 47 NP 536702 Phodopus Connexin 43
AAR33085
human Connexin 47 AAH89439 Rat Connexin 43
AAH81842
Human Connexin46.6 AAB94511 Sus Connexin 43
AAR33087
Cow Connexin 46.6 XP 582393 Mesocricetus Connexin 43
AA061857
Mouse Connexin 30.2 NP 848711 Mouse Connexin 43
AAH55375
Rat Connexin 30.2 XP 343966 Cavia Connexin 43
AAU06305
Human Connexin 31.9 AAM18801 Cow Connexin 43
NP 776493
Dog Connexin 31.9 XP_548134 Erinaceus Connexin 43
AAR33083
Sheep Connexin 44 AAD56220 Chick Connexin 43
AAA53027
Cow Connexin 44 146053 Xenopus Connexin 43
NP 988856
Rat Connexin 33 P28233 Oryctolagus Connexin 43
AA589649
Mouse Connexin 33 AAR28037 Cyprinus Connexin 43
AAG17938
Human Connexin 36 Q9UKL4 Zebrafish Connexin 43
CAH69066
mouse Connexin 36 NP 034420 Danio aequipinnatus Connexin 43
AAC19098
rat Connexin 36 NP_062154 Zebrafish Connexin 43.4
NP 571144
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CA 02592285 2007-06-28
dog Connexin 36 XP 544602 Zebrafish Connexin 44.2
AA1145279
chick Connexin 36 NP 989913 Zebrafish Connexin 44.1 NP
571884
zebrafish Connexin 36 NP 919401 human Connexin45 138430
morone Connexin 35 AA¨C31884 chimp Connexin45 XP
511557
morone Connexin 35 AAC31885 dog Connexin 45 XP
548059
Cynops Connexin 35 BAC22077 mouse Connexin 45
AA1171230
Tetraodon Connexin 36 CAG06428 cow Connexin 45 XP
588395
human Connexin 37 155593 rat Connexin 45
AAN17802
chimp Connexin 37 XP 524658 chick Connexin45 NP
990834
dog Connexin 37 XP 539602 Tetraodon Connexin 45
CA93782
.A Cricetulus Connexin 37 A7k98615 chick Connexin 45.6 150219
Mouse Connexin 37 AAH56613 human Connexin 46 NP
068773
Mesocricetus Connexin37 AAS83433 chimp Connexin 46 XP
522616
Rat Connexin37 AAH86576 mouse Connexin 46 NP
058671
mouse Connexin 39 NP 694726 dog Connexin 46 XP
543178
rat Connexin 39 AAT\117801 rat Connexin 46 NP
077352
human Connexin 40.1 NP 699199 Mesocricetus Connexin 46
AAS83437
Xenopus Connexin38 AAH73347 Cricetulus Connexin 46
AAS77618
Zebrafish Connexin 39.9 NP_997991 Chick Connexin 56 A45338
Human Connexin 40 NP 859054 Zebrafish Connexin 39.9 NP
997991
Chimp Connexin 40 XP-513754 cow Connexin 49 XP
602360
dog Connexin 40 XP 540273 human Connexin 50 P4fi65
cow Connexin 40 XP 587676 chimp Connexin 50 XP
524857
mouse Connexin 40 AA1153054 rat Connexin 50 NP
703195
rat Connexin 40 AAH70935 mouse Connexin 50
AAG59880
Cricetulus Connexin 40 AAP37454 dog Connexin 50 XP
540274
Chick Connexin 40 NP 990835 sheep Connexin 49
AAT01367
human Connexin 43 P1T302 Mesocricetus Connexin 50
AAS83438
Cercopithecus Connexin 43 AAR33082 Cricetulus Connexin 50
AAR98618
Oryctolagus Connexin 43 AAR33084 Chick Connexin 50
BAA05381
Spermophilus Connexin 43 AAR33086 human Connexin 59
AAG09406
Cricetulus Connexin 43 AA061858
Thus, the provided polypeptide can comprise the amino acid sequence SEQ ID
NO:1, 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:43, SEQ ID NO:90 or JD NO:91 or
conservative variants or fragments thereof.
The 20-30 carboxy-terminal-most amino acid sequence of alpha Connexins are
characterized by a distinctive and conserved organization. This distinctive
and conserved
organization would include a type II PDZ binding motif (1.-x-4; wherein x =
any amino
acid and (I) = a Hydrophobic amino acid; e.g., Table 2, BOLD) and proximal to
this motif,
Proline (P) and/or Glycine (G) hinge residues; a high frequency phospho-Serine
(S) and/or
14
CA 02592285 2007-06-28
phospho-Threonine (T) residues; and a high frequency of positively charged
Arginine (R),
Lysine (K) and negatively charged Aspartic acid (D) or Glutamic acid (E) amino
acids. For
many alpha Connexins, the P and G residues occur in clustered motifs (e.g.,
Table 2,
italicized) proximal to the carboxy-terminal type II PDZ binding motif. The S
and T
phosphor-amino acids of most alpha Connexins also are typically organized in
clustered,
repeat-like motifs (e.g., Table 2, underlined). This organization is
particularly the case for
Cx43, where 90% of 20 carboxyl terminal-most amino acids are comprised of the
latter
seven amino acids. In a further example of the high conservation of the
sequence, ACT
peptide organization of Cx43 is highly conserved from humans to fish (e.g.,
compare Cx43
ACT sequences for humans and zebrafish in Table 2). In another example, the
ACT peptide
organization of Cx45 is highly conserved from humans to birds (e.g., compare
Cx45 ACT
sequences for humans and chick in Table 2). ). In another example, the ACT
peptide
organization of Cx36 is highly conserved from primates to fish (e.g., compare
Cx36 ACT
sequences for chimp and zebrafish in Table 2).
Table 2. Alpha Connexin Carboxy-Terminal (ACT) Amino Acid Sequences
Gene Sequence
SEQ ID NO
Human alpha Cx43 P SSRA SSRA SSR PRP D DLEI
(SEQ ID NO:1)
Chick alpha Cx43 P S RA SSRA SSR PRP D DLEI
(SEQ ID NO:29)
Zebrafish alpha
P CSRA SSRM SSRA R P D DLDV
(SEQ ID NO:90)
Cx43
Human alpha Cx45 G SNKS TA SSKS GDG KN SVIVI
(SEQ ID NO:30)
Chick alpha Cx45 G SNKSS A SSKS GDGKN WWI
(SEQ ID NO:31)
Human alpha Cx46 G RA SKAS RASS GRARP E DLAI
SEQ ID NO: 32)
Human alpha Cx46.6 G SASS RD G K TVWI
(SEQ ID NO:33)
Chimp alpha Cx36 P RVSV PNFG R TQ SSD SAYV
(SEQ ID NO:34)
Chick alpha Cx36 P RMSM PNFG R TQ SSD S AYV
(SEQ lD NO:35)
Zebrafish alpha
P RMSM PNFG R TQ SSD S AYV
(SEQ ID NO: 91)
Cx36
Human alpha Cx47 P RAGSEK G SASS R DG KT TVWI
(SEQ ID NO:36)
Human alpha Cx40 G HRL PHG YHSDKRRL SKASS KARSD DLSV (SEQ ID NO:37)
Human alpha Cx50 P ELTTDDAR P LSRL SKASS RARSD DLTV
(SEQ ID NO:38)
Human alpha Cx59 P NHVV SLTN NLI GRRVP T DLQI
(SEQ ID NO:39)
Rat alpha Cx33
P S CV SSS A VLTTIC SS DQVV PVG L SS FYM (SEQ ID NO:40)
CA 02592285 2007-06-28
Sheep alpha Cx44 G R SSKA SKSS GG RARAA DLAI
(SEQ ID NO:41)
Human beta Cx26 LC YLLIR YCSGK SKKPV
(SEQ ID NO:42)
Thus, in one aspect, the provided polypeptide comprises one, two, three or all
of the
amino acid motifs selected from the group consisting of 1) a type II PDZ
binding motif, 2)
Proline (P) and/or Glycine (G) hinge residues; 3) clusters of phospho-Serine
(S) and/or
phospho-Threonine (T) residues; and 4) a high frequency of positively charged
Arginine (R)
and Lysine (K) and negatively charged Aspartic acid (D) and/or Glutamic acid
(E) amino
acids). In another aspect, the provided polypeptide comprises a type II PDZ
binding motif at
the carboxy-terminus, Proline (P) and/or Glycine (G) hinge residues proximal
to the PDZ
binding motif, and positively charged residues (K, R, D, E) proximal to the
hinge residues.
PDZ domains were originally identified as conserved sequence elements within
the
postsynaptic density protein PSD95/SAP90, the Drosophila tumor suppressor dig-
A, and the
tight junction protein ZO-1. Although originally referred to as GLGF or DHR
motifs, they
are now known by an acronym representing these first three PDZ-containing
proteins
(PSD95/DLG/Z0-1). These 80-90 amino acid sequences have now been identified in
well
.. over 75 proteins and are characteristically expressed in multiple copies
within a single
protein. Thus, in one aspect, the provided polypeptide can inhibit the binding
of an alpha
Connexin to a protein comprising a PDZ domain. The PDZ domain is a specific
type of
protein-interaction module that has a structurally well-defined interaction
'pocket' that can
be filled by a PDZ-binding motif, referred to herein as a "PDZ motif'. PDZ
motifs are
consensus sequences that are normally, but not always, located at the extreme
intracellular
carboxyl terminus. Four types of PDZ motifs have been classified: type I (SIT-
x-(13), type II
(43-x-CF), type III (1F-x-4.) and type IV (D-x-V), where x is any amino acid,
(I) is a
hydrophobic residue (V, I, L, A, G,W, C, M, F) and NY is a basic, hydrophilic
residue (H, R,
K). (Songyang, Z., et al. 1997. Science 275,73-77). Thus, in one aspect, the
provided
polypeptide comprises a type II PDZ binding motif.
It is noted that the 18 carboxy-terminal-most amino acid sequence of alpha
Cx37
represents an exceptional variation on the ACT peptide theme. The Cx37 ACT-
like
sequence is GQKPPSRPSSSASICKQ*YV (SEQ ID NO: 43). Thus the carboxy terminal 4
amino acids of Cx37 conform only in part to a type II PDZ binding domain.
Instead of a
16
CA 02592285 2007-06-28
classical type II PDZ binding domain, Cx37 has a neutral Q* at position 2
where a
hydrophobic amino acid would be expected. As such Cx37 comprises what might be
termed
a type II PDZ binding domain ¨like sequence. Nonetheless, Cx37 strictly
maintains all other
aspects of ACT peptide organization including clustered serine residues,
frequent R and K
residues and a P-rich sequence proximal to the PDZ binding domain-like
sequence. Given
this overall level of conservation of ACT-like organization in common with the
other >70
alpha Connexins listed above, it is understood that the Cx37 ACT-like carboxy
terminus
functions in the provided capacity.
For comparison, the beta Connexin Cx26 is shown in Table 2. Cx26 has no
carboxyl
terminal type II PDZ binding motif; less than 30% of the carboxyl terminal
most amino
acids comprise S, T, R, D or E residues; it has no evidence of motifs proximal
to a type II
PDZ binding motif or PDZ binding like motif containing clusters of P and G
hinge residues;
and no evidence of clustered, repeat-like motifs of serine and threonine
phospho-amino
acids. Cx26 does have three Lysine (K) residues, clustered one after the other
near the
carboxy terminus of the sequence. However, no alpha Connexin surveyed in the
>70 alpha
Connexins listed above was found to display this feature of three repeated K
residues
domain at carboxy terminus (Cx26 is a beta connexin, thus by definition does
not have an
ACT domain).
As provided herein, the unique functional characteristics of this relatively
short
stretch of amino acids encompass unexpected roles in reducing inflammation,
promoting
healing, reducing scarring, increasing tensile strength, and promoting
regeneration of
complex tissue structure and function following injury in tissues as diverse
as skin and brain.
Thus, in one aspect, the provided polypeptide comprises a type II PDZ binding
motif (40-x-
4); wherein x any amino acid and 4) = a Hydrophobic amino acid). In another
aspect,
greater than 50%, 60%, 70%, 80%, 90% of the amino acids of the provided ACT
polypeptide is comprised one or more of Proline (P), Glycine (G), phospho-
Serine (S),
phospho-Threonine (T), Arginine (R), Lysine (K), Aspartic acid (D), or
Glutamic acid (E)
amino acid residues.
The amino acids Proline (P), Glycine (G), Arginine (R), Lysine (K), Aspartic
acid
(D), and Glutamic acid (E) are necessary determinants of protein structure and
function.
17
CA 02592285 2007-06-28
,
Proline and Glycine residues provide for tight turns in the 3D structure of
proteins, enabling
the generation of folded conformations of the polypeptide required for
function. Charged
amino acid sequences are often located at the surface of folded proteins and
are necessary
for chemical interactions mediated by the polypeptide including protein-
protein interactions,
protein-lipid interactions, enzyme-substrate interactions and protein-nucleic
acid
interactions. Thus, in another aspect Proline (P) and Glycine (G) Lysine (K),
Aspartic acid
(D), and Glutamic acid (E) rich regions proximal to the type II PDZ binding
motif provide
for properties necessary to the provided actions of ACT peptides. In another
aspect, the
provided polypeptide comprises Proline (P) and Glycine (G) Lysine (K),
Aspartic acid (D),
and/or Glutamic acid (E) rich regions proximal to the type II PDZ binding
motif.
Phosphorylation is the most common post-translational modification of proteins
and
is crucial for modulating or modifying protein structure and function. Aspects
of protein
structure and function modified by phosphorylation include protein
conformation, protein-
protein interactions, protein-lipid interactions, protein-nucleic acid
interactions, channel
gating, protein trafficking and protein turnover. Thus, in one aspect the
phospho-Serine (S)
and/or phospho-Threonine (T) rich sequences are necessary for modifying the
function of
ACT peptides, increasing or decreasing efficacy of the polypeptides in their
provided
actions. In another aspect, the provided polypeptide comprise Serine (S)
and/or phospho-
Threonine (T) rich sequences or motifs.
In another example, respecting definition of an ACT peptide, it is highly
auspicious,
in light of the high degree of tissue/organ regeneration potential in lower
animals such as
fish, that a methionine occurs near the amino terminus of the ACT sequence of
zebrafish
Cx43 (Table 2). In addition to encoding methionine, the methionine base pair
triplet is an
alternate translation start site. If translation initiated from this
methionine, the sequence
SSRARPDDLDV (SEQ ID NO:90), would be produced. This translation product
maintains
all the conserved and distinctive features of a canonical ACT peptide.
Specifically this
peptide comprises a carboxy terminal type II PDZ binding domain and has a
domain
enriched in P, R and D residues proximal to the PDZ binding domain. In
addition, the
sequence comprises a clustered S motif, with potential to modulate ACT peptide
function at
its amino terminal. This raises the interesting prospect that animals with
high tissue/organ
regeneration potential such as fish may translate ACT peptides sequences
directly.
18
CA 02592285 2007-06-28
,
,
,
Thus, the provided polypeptide can comprise the c-terminal sequence of human
Cx43. Thus, the provided polypeptide can comprise the amino acid sequence SEQ
ID NO:1
or SEQ ID NO:2. The polypeptide can comprise 9 amino acids of the carboxy
terminus of
human Cx40. Thus, the polypeptide can comprise the amino acid sequence SEQ ID
NO:5.
When specific proteins are referred to herein, variants, derivatives, and
fragments are
contemplated. Protein variants and derivatives are well understood to those of
skill in the art
and in can involve amino acid sequence modifications. For example, amino acid
sequence
modifications typically fall into one or more of three classes:
substitutional, insertional or
deletional variants. Insertions include amino and/or carboxyl terminal fusions
as well as
intrasequence insertions of single or multiple amino acid residues. Insertions
ordinarily will
be smaller insertions than those of amino or carboxyl terminal fusions, for
example, on the
order of one to four residues. Deletions are characterized by the removal of
one or more
amino acid residues from the protein sequence. These variants ordinarily are
prepared by
site specific mutagenesis of nucleotides in the DNA encoding the protein,
thereby producing
DNA encoding the variant, and thereafter expressing the DNA in recombinant
cell culture.
Techniques for making substitution mutations at predetermined sites in DNA
having a
known sequence are well known and include, for example, M13 primer mutagenesis
and
PCR mutagenesis. Amino acid substitutions are typically of single residues,
but can occur at
a number of different locations at once; insertions usually will be on the
order of about from
1 to 10 amino acid residues. Deletions or insertions preferably are made in
adjacent pairs,
i.e., a deletion of 2 residues or insertion of 2 residues. Substitutions,
deletions, insertions or
any combination thereof may be combined to arrive at a final construct. The
mutations must
not place the sequence out of reading frame and preferably will not create
complementary
regions that could produce secondary mRNA structure unless such a change in
secondary
structure of the mRNA is desired. Substitutional variants are those in which
at least one
residue has been removed and a different residue inserted in its place. Such
substitutions
generally are made in accordance with the following Table 3 and are referred
to as
conservative substitutions.
TABLE 3: Amino Acid Substitutions
Original Residue Exemplary Substitutions
Ala Ser
19
CA 02592285 2007-06-28
Arg Lys
Asn Gin
Asp Glu
Cys Ser
Gin Asn
Glu Asp
Gly Pro
His Gin
Ile Leu; Val
Leu Ile; Val
Lys Arg; Gin
Met Leu; Ile
Phe Met; Leu; Tyr
Pro Gly
Ser Thr
Thr Ser
Trp Tyr
Tyr Tip; Phe
Val Ile; Leu
For example, the replacement of one amino acid residue with another that is
biologically and/or chemically similar is known to those skilled in the art as
a conservative
substitution. For example, a conservative substitution would be replacing one
hydrophobic
residue for another, or one polar residue for another. The substitutions
include combinations
shown in Table 3. Conservatively substituted variations of each explicitly
disclosed
sequence are included within the polypeptides provided herein.
Typically, conservative substitutions have little to no impact on the
biological
activity of a resulting polypeptide. In a particular example, a conservative
substitution is an
amino acid substitution in a peptide that does not substantially affect the
biological function
of the peptide. A peptide can include one or more amino acid substitutions,
for example 2-
10 conservative substitutions, 2-5 conservative substitutions, 4-9
conservative substitutions,
such as 2, 5 or 10 conservative substitutions.
A polypeptide can be produced to contain one or more conservative
substitutions by
manipulating the nucleotide sequence that encodes that polypeptide using, for
example,
standard procedures such as site-directed mutagenesis or PCR. Alternatively, a
polypeptide
can be produced to contain one or more conservative substitutions by using
standard peptide
synthesis methods. An alanine scan can be used to identify which amino acid
residues in a
CA 02592285 2007-06-28
protein can tolerate an amino acid substitution. In one example, the
biological activity of the
protein is not decreased by more than 25%, for example not more than 20%, for
example not
more than 10%, when an alanine, or other conservative amino acid (such as
those listed
below), is substituted for one or more native amino acids.
Further information about conservative substitutions can be found in, among
other
locations, in Ben-Bassat et al., (I Bacteriol. 169:751-7, 1987), O'Regan et
al., (Gene
77:237-51, 1989), Sahin-Toth etal., (Protein Sci. 3:240-7, 1994), Hochuli et
al.,
(Rio/Technology 6:1321-5, 1988) and in standard textbooks of genetics and
molecular
biology.
Substitutional or deletional mutagenesis can be employed to insert sites for N-
glycosylation (Asn-X-Thr/Ser) or 0-glycosylation (Ser or Thr). Deletions of
cysteine or
other labile residues also may be desirable. Deletions or substitutions of
potential
proteolysis sites, e.g. Arg, is accomplished for example by deleting one of
the basic residues
or substituting one by glutaminyl or histidyl residues.
Certain post-translational derivatizations are the result of the action of
recombinant
host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues
are frequently
post-translationally deamidated to the corresponding glutamyl and asparyl
residues.
Alternatively, these residues are deamidated under mildly acidic conditions.
Other post-
translational modifications include hydroxylation of proline and lysine,
phosphorylation of
.. hydroxyl groups of seryl or threonyl residues, methylation of the o-amino
groups of lysine,
arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and
Molecular
Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation
of the N-
terminal amine and, in some instances, amidation of the C-terminal carboxyl.
It is understood that there are numerous amino acid and peptide analogs which
can
be incorporated into the disclosed compositions. For example, there are
numerous D amino
acids or amino acids which have a different functional substituent than the
amino acids
shown in Table 3. The opposite stereoisomers of naturally occurring peptides
are disclosed,
as well as the stereoisomers of peptide analogs. These amino acids can readily
be
incorporated into polypeptide chains by charging tRNA molecules with the amino
acid of
choice and engineering genetic constructs that utilize, for example, amber
codons, to insert
21
CA 02592285 2013-11-15
the analog amino acid into a peptide chain in a site specific way (Thorson et
al., Methods in
Malec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348-
354 (1992);
Ibba, Biotechnology & Genetic Enginerring Reviews 13:197-216 (1995), Cahill et
al., T1BS,
14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba and Hennecke,
Bio/technology, 12:678-682 (1994)). =
Molecules can be produced that resemble polypeptides, but which are not
connected
via a natural peptide linkage. For example, linkages for amino acids or amino
acid analogs
can include CII2NH--, --CH2S--, --CH2--CH2 --CII=CH-- (cis and trans), --COCH2
-CII(OH)CH2--, and --CHH2S0¨(These and others can be found in Spatola, A. F.
in
Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.
Weinstein, eds.,
Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March
1983), Vol. 1,
Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm
Sci
(1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (-
-CH2NH--,
CH2CH2--); Spatola et al. Life Sci 38:1243-1249 (1986) (--CH H2--S); Hann J.
Chem. Soc
Perkin Trans. 1307-314 (1982) (--CH--CH--, cis and trans); Ahnquist etal. Med.
Chem.
23:1392-1398 (1980) (--COCH2--); Jennings-White etal. Tetrahedron Lett 23:2533
(1982)
(--COCH2 --); Szelke etal. European Appin, EP 45665 CA (1982): 97:39405 (1982)
(--
CH(OH)CH2--); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--
C(OH)CH2--);
and Hruby Life Sci 31:189-199 (1982) (--CH2--S--). It is understood that
peptide analogs
can have more than one atom between the bond atoms, such as b-alanine, g-
aminobutyric
acid, and the like.
Amino acid analogs and peptide analogs often have enhanced or desirable
properties,
such as, more economical production, greater chemical stability, enhanced
pharmacological
properties (half-life, absorption, potency, efficacy, etc.), altered
specificity (e.g., abroad-
spectrum of biological activities), reduced antigenicity, greater ability to
cross biological
barriers (e.g., gut, blood vessels, blood-brain-barrier), and others.
D-amino acids can be used to generate more stable peptides, because D amino
acids
are not recognized by peptidases and such. Systematic substitution of one or
more amino
22
CA 02592285 2007-06-28
acids of a consensus sequence with a D-amino acid of the same type (e.g., D-
lysine in place
of L-lysine) can be used to generate more stable peptides. Cysteine residues
can be used to
cyclize or attach two or more peptides together. This can be beneficial to
constrain peptides
into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61:387
(1992)).
Thus, the provided polypeptide can comprise a conservative variant of the c-
terminus
of an alpha Connexin (ACT). As shown in Table 4, an example of a single
conservative
substitution within the sequence SEQ ID NO:2 is given in the sequence SEQ ID
NO:3. An
example of three conservative substitutions within the sequence SEQ ID NO:2 is
given in
the sequence SEQ ID NO:4. Thus, the provided polypeptide can comprise the
amino acid
SEQ ID NO:3 or SEQ ID NO:4.
Table 4. ACT Polypeptide Variants
Sequence SEQ ID NO
RPRPDDLEI SEQ ID NO:2
RPRPDDLEV SEQ ID NO:3
RPRPDDVPV SEQ ID NO:4
SSRASSRASSRPRPDDLEV SEQ ID NO: 44
RPKPDDLEI SEQ ID NO: 45
SSRASSRASSRPKPDDLEI SEQ ID NO: 46
RPKPDDLDI SEQ ID NO: 47
SSRASSRASSRPRPDDLDI SEQ ID NO: 48
SSRASTRASSRPRPDDLEI SEQ ID NO: 49
RPRPEDLEI SEQ ID NO: 50
SSRASSRASSRPRPEDLEI SEQ ID NO: 51
GDGKNSVWV SEQ ID NO: 52
SKAGSNKSTASSKSGDGKNSVWV SEQ ID NO: 53
GQKPPSRPSSSASKKLYV SEQ ID NO: 54
It is understood that one way to define any variants, modifications, or
derivatives of
the disclosed genes and proteins herein is through defining the variants,
modification, and
derivatives in terms of sequence identity (also referred to herein as
homology) to specific
23
CA 02592285 2013-11-15
known sequences. Specifically disclosed are variants of the nucleic acids and
polypeptides
herein disclosed which have at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
percent sequence
identity to the stated or known sequence. Those of skill in the art readily
understand how to
determine the sequence identity of two proteins or nucleic acids. For example,
the sequence
identity can be calculated after aligning the two sequences so that the
sequence identity is at
its highest level.
Another way of calculating sequence identity can be performed by published
algorithms. Optimal alignment of sequences for comparison may be conducted by
the local
sequence identity algorithm of Smith and Waterman Adv. App!. Math. 2: 482
(1981), by the
sequence identity alignment algorithm of Needleman and Wunsch, I. MoL Biol.
48: 443
(1970), by the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci.
U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms
(GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics
Computer Group, 575 Science Dr., Madison, WI), or by inspection. These
references teach
the methods of calculating sequence identity.
The same types of sequence identity can be obtained for nucleic acids by, for
example, the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger
et al. Proc.
Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et at. Methods Enzymol.
183:281-306,
1989.
Thus, the provided polypeptide can comprise an amino acid sequence with at
least
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent sequence identity to the c-
terminus of an
alpha ConnexM (ACT). Thus, in one aspect, the provided polypeptide comprises
an amino
acid sequence with at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent
sequence identity
to SEQ ID NO:1, 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 LD NO:39, SEQ NO:40, SEQ ID NO:41, SEQ ID NO: 90 or SEQ DI) NO:91. As an
24
CA 02592285 2007-06-28
example, provided is a polypeptide (SEQ ID NO:4) having 66% sequence identity
to the
same stretch of 9 amino acids occurring on the carboxy-terminus of human Cx43
(SEQ ID
NO:2).
The herein provided polypeptides can be added directly to a tissue injury in a
subject.
However, efficiency of cytoplasmic localization of the provided polypeptide is
enhanced by
cellular internalization transporter chemically linked in cis or trans with
the polypeptide.
Efficiency of cell internalization transporters are enhanced further by light
or co-
transduction of cells with Tat-HA peptide.
Thus, the provided polypeptide can comprise a cellular internalization
transporter or
sequence. The cellular internalization sequence can be any internalization
sequence known
or newly discovered in the art, or conservative variants thereof. Non-limiting
examples of
cellular internalization transporters and sequences include Antennapedia
sequences, TAT,
HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP
(model
amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynBl, Pep-7, TIN-1,
BGSC
(Bis-Guanidinium-Spermidine-Cholesterol, and BGTC (Bis-Guanidinium-Tren-
Cholesterol)
(see Table 5).
Table 5: Cell Internalization Transporters
Name Sequence SEQ ID NO
Antp RQPKIWFPNRRKPWKK (SEQ ID NO:7)
HIV-Tat GRKKRRQRPPQ (SEQ ID NO:14)
Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO:15)
Antp-3A RQIAIWFQNRRMKWAA (SEQ ID NO:16)
Tat RKKRRQRRR (SEQ ID NO:17)
Buforin II TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO:18)
Transportan GWTLNSAGYLLGKINKALAALA (SEQ ID NO:19)
KKIL
model amphipathic KLALKLALKALKAALKLA (SEQ ID NO:20)
peptide (MAP)
K-FGF AAVALLPAVLLALLAP (SEQ ID NO:21)
Ku70 VPMLK- PMLKE (SEQ ID NO:22)
Prion MANLGYWLLALFVTMWTDVGL (SEQ ID NO:23)
CKKRPKP
pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO:24)
Pep-1 KETWWETWWTEWSQPKKKRKV (SEQ ID NO:25)
CA 02592285 2007-06-28
SynB1 RGGRLSYSRRRFSTSTGR (SEQ ID NO:26)
Pep-7 SDLWEMMMVSLACQY (SEQ ID NO:27)
HN-1 TSPLNIFINGQKL (SEQ ID NO:28)
BGSC (Bis-
Guanidinium- H2N,
Ho+ip-NH-(CH3
Spermidine-
Cholesterol) 74))C-NH-(CH12:
BGSC
BGTC (Bis-
Guanidinium-Tren-
Cholesterol)
fr11-C-
3C¨NH
H2N
BGTC
Thus, the provided polypeptide can further comprise the amino acid sequence
SEQ
ID NO:7, SEQ ID NO:14 (Bucci, M. etal. 2000. Nat. Med. 6, 1362-1367), SEQ ID
NO:15
(Derossi, D., et al. 1994. BioLChem. 269, 10444-10450), SEQ ID NO:16 (Fischer,
P.M. et
al. 2000.1 Pept. Res. 55, 163-172), SEQ ID NO:17 (Frankel, A. D. & Pabo, C. 0.
1988.
Cell 55,1189-1193; Green, M. & Loewenstein, P. M. 1988. Cell 55, 1179-1188),
SEQ lD
NO:18 (Park, C. B., et al. 2000. Proc. Natl Acad. Sci. USA 97, 8245-8250), SEQ
ID NO:19
(Pooga, M., et al. 1998. FASEB J. 12, 67-77), SEQ ID NO:20 (Oehlke, J. etal.
1998.
Biochim. Biophys. Acta. 1414, 127-139), SEQ ID NO:21 (Lin, Y. Z., et al. 1995.
1 Biol.
Chem. 270, 14255-14258), SEQ ID NO:22 (Sawada, M., et al. 2003. Nature Cell
Biol. 5,
352-357), SEQ lD NO:23 (Lundberg, P. et al. 2002. Biochem. Biophys. Res.
Commun. 299,
85-90), SEQ ID NO:24 (Elmquist, A., et al. 2001. Exp. Cell Res. 269, 237-244),
SEQ ID
NO:25 (Morris, M. C., et al. 2001. Nature BiotechnoL 19, 1173-1176), SEQ ID
NO:26
(Rousselle, C. et al. 2000. Mot PharmacoL 57,679-686), SEQ ID NO:27 (Gao, C.
et al.
2002. Bioorg. Med. Chem. 10, 4057-4065), or SEQ ID NO:28 (Hong, F. D. &
Clayman, G.
L. 2000. Cancer Res. 60, 6551-6556). The provided polypeptide can further
comprise
BGSC (Bis-Guanidinium-Spermidine-Cholesterol) or BGTC (Bis-Guanidinium-Tren-
Cholesterol) (Vigneron, J.P. et al. 1998. Proc. Natl. Acad. Sci. USA. 93, 9682-
9686). The
preceding references teach cellular internalization vectors and sequences. Any
other
26
CA 02592285 2007-06-28
internalization sequences now known or later identified can be combined with a
peptide of
the invention.
The provided polypeptide can comprise any ACT sequence (e.g, any of the ACT
peptides disclosed herein) in combination with any of the herein provided cell
internalization sequences. Examples of said combinations are given in Table 6.
Thus, the
provided polypeptide can comprise an Antennapedia sequence comprising amino
acid
sequence SEQ ID NO:7. Thus, the provided polypeptide can comprise the amino
acid
sequence SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID
NO:12.
Table 6: ACT Polypeptides with Cell Internalization Sequences (CIS)
CIS/
Sequence SEQ ID
NO
ACT
Antp/
RQPKIWFPNRRKPWKK P S S RAS S RAS SRPRPDD LE I SEQ ID NO:8
ACT 2
Antp/ RQPKIWFPNRRKPWKK RPRPDDLEI SEQ ID
NO:9
ACT 1
Antp/ RQPKIWFPNRRKPWKK RPRPDDLEV SEQ ID NO:10
ACT 3
Antp/ RQPKIWFPNRRKPWKK RPRPDDVPV SEQ ID NO:11
ACT 4
Antp/ RQPKIWFPNRRKPWKK KARSDDLSV SEQ ID NO:12
ACT 5
HIV-Tat/ GRKKRRQRPPQ RPRPDDLEI SEQ ID NO:56
ACT 1
Penetratin/ RQIKIWFQNRRMKWKK RPRPDDLEI SEQ ID N057
ACT 1
Antp-3A/ RQIAIWFQNRRMKWAA RPRPDDLEI SEQ ID NO:58
ACT 1
Tat/ RKKRRQRRR RPRPDDLEI SEQ ID NO:59
ACT 1
Buforin II/
TRSSRAGLQFPVGRVHRLLRK RPRPDDLEI SEQ ID NO:60
ACT 1
Transportan/
GWTLNSAGYLLGKINKALAALAKKIL RPRPDDLEI SEQ ID NO:61
ACT 1
MAP/
KLALKLALICALICAALICLA RPRPDDLEI SEQ ID NO:62
ACT 1
K-FGF/
AAVALLPAVLLALLAP RPRPDDLEI SEQ ID NO:63
ACT 1
Ku70/
VPMLKPMLKE RPRPDDLEI SEQ ID NO:64
ACT 1
Prion/ MANLGYWLLALFVTMWTDVGLCKKRPKP
ACT 1 RPRPDDLEI SEQ ID NO:65
pVEC/
LLIILRRRIRKQAHAHSK RPRPDDLEI SEQ ID NO:66
ACT 1
Pep-1/ KETWWETWWTEWSQPKKKRKV RPRPDDLEI SEQ ID NO:67
27
CA 02592285 2007-06-28
ACT 1
Syn131/
RGGRLSYSRRRFSTSTGR RPRPDDLEI SEQ ID NO:68
ACT 1
Pep-7/
SDLWEMMMVSLACQY RPRPDDLEI SEQ ID NO:69
ACT 1
HN-1/
TSPLNIHNGQICL RPRPDDLEI SEQ ID NO:70
ACT 1
Also provided are isolated nucleic acids encoding the polypeptides provided
herein.
The disclosed nucleic acids are made up of for example, nucleotides,
nucleotide analogs, or
nucleotide substitutes. Non-limiting examples of these and other molecules are
discussed
herein. It is understood that for example, when a vector is expressed in a
cell, the expressed
mRNA will typically be made up of A, C, G, and U.
By "isolated nucleic acid" or "purified nucleic acid" is meant DNA that is
free of the
genes that, in the naturally-occurring genome of the organism from which the
DNA of the
invention is derived, flank the gene. The term therefore includes, for
example, a
recombinant DNA which is incorporated into a vector, such as an autonomously
replicating
plasmid or virus; or incorporated into the genomic DNA of a prokaryote or
eukaryote (e.g., a
transgene); or which exists as a separate molecule (e.g., a cDNA or a genomic
or cDNA
fragment produced by PCR, restriction endonuclease digestion, or chemical or
in vitro
synthesis). It also includes a recombinant DNA which is part of a hybrid gene
encoding
additional polypeptide sequence. The term "isolated nucleic acid" also refers
to RNA, e.g.,
an mRNA molecule that is encoded by an isolated DNA molecule, or that is
chemically
synthesized, or that is separated or substantially free from at least some
cellular components,
e.g., other types of RNA molecules or polypeptide molecules.
Thus, provided is an isolated nucleic acid encoding a polypeptide comprising
the
amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or
SEQ ID NO:12.
Thus, the provided nucleic acid can comprise the nucleic acid sequence SEQ ID
NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84,
SEQ ID NO:85, SEQ ID NO:86, SEQ NO:87SEQ ID NO:88, or SEQ ID NO:89.
28
CA 02592285 2007-06-28
The herein provided nucleic acid can be operably linked to an expression
control
sequence. Also provided is a vector comprising one or more of the herein
provided nucleic
acids, wherein the nucleic acid is operably linked to an expression control
sequence. There
are a number of compositions and methods which can be used to deliver nucleic
acids to
cells, either in vitro or iin vivo. These methods and compositions can largely
be broken
down into two classes: viral based delivery systems and non-viral based
delivery systems.
For example, the nucleic acids can be delivered through a number of direct
delivery systems
such as, electroporation, lipofection, calcium phosphate precipitation,
plasmids, viral
vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via
transfer of genetic
material in cells or carriers such as cationic liposomes. Appropriate means
for transfection,
including viral vectors, chemical transfectants, or physico-mechanical methods
such as
electroporation and direct diffusion of DNA, are described by, for example,
Wolff, J. A., et
al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,
(1991). Such
methods are well known in the art and readily adaptable for use with the
compositions and
methods described herein. In certain cases, the methods will be modifed to
specifically
function with large DNA molecules. Further, these methods can be used to
target certain
diseases and cell populations by using the targeting characteristics of the
carrier.
Transfer vectors can be any nucleotide construction used to deliver genes into
cells
(e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as
part of recombinant
retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).
As used herein, plasmid or viral vectors are agents that transport the
disclosed
nucleic acids, such as SEQ ID NO:6, into the cell without degradation and
include a
promoter yielding expression of the gene in the cells into which it is
delivered. In some
embodiments the promoters are derived from either a virus or a retrovirus.
Viral vectors are,
for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus,
Polio virus,
AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including
these viruses
with the HIV backbone. Also disclosed are any viral families which share the
properties of
these viruses which make them suitable for use as vectors. Retroviruses
include Murine
Maloney Leukemia virus, MMLV, and retroviruses that express the desirable
properties of
.. MMLV as a vector. Retroviral vectors are able to carry a larger genetic
payload, i.e., a
29
CA 02592285 2013-11-15
transgene or marker gene, than other viral vectors, and for this reason are a
commonly used
vector. However, they are not as useful in non-proliferating cells. Adenovirus
vectors are
relatively stable and easy to work with, have high titers, and can be
delivered in aerosol
formulation, and can transfect non-dividing cells. Pox viral vectors are large
and have
several sites for inserting genes, they are thermostable and can be stored at
room
temperature. Also disclosed is a viral vector which has been engineered so as
to suppress
the immune response of the host organism, elicited by the viral antigens.
Vectors of this
type can carry coding regions for Interleulcin 8 or 10.
Viral vectors can have higher transaction (ability to introduce genes)
abilities than
chemical or physical methods to introduce genes into cells. Typically, viral
vectors contain,
nonstructural early genes, structural late genes, an RNA polyrnerase III
transcript, inverted
terminal repeats necessary for replication and encapsidation, and promoters to
control the
transcription and replication of the viral genome. When engineered as vectors,
viruses
typically have one or more of the early genes removed and a gene or
gene/promotor cassette
is inserted into the viral genome in place of the removed viral DNA.
Constructs of this type
can carry up to about 8 kb of foreign genetic material. The necessary
functions of the
removed early genes are typically supplied by cell lines which have been
engineered to
express the gene products of the early genes in trans.
A retrovirus is an animal virus belonging to the virus family of Retrovitidae,
including any types, subfamilies, genus, or tropisms. Retroviral vectors, in
general, are
described by Verma, I.M., Retroviral vectors for gene transfer. In
Microbiology-1985,
American Society for Microbiology, pp. 229-232, Washington, (1985).
Examples of methods for using retroviral vectors for gene
therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT
applications WO
90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)).
A retrovirus is essentially a package which has packed into it nucleic acid
cargo.
The nucleic acid cargo carries with it a packaging signal, which ensures that
the replicated
daughter molecules will be efficiently packaged within the package coat. In
addition to the
package signal, there are a number of molecules which are needed in cis, for
the replication,
and packaging of the replicated virus. Typically a retroviral genome, contains
the gag, poi,
CA 02592285 2007-06-28
and env genes which are involved in the making of the protein coat. It is the
gag, poi, and
env genes which are typically replaced by the foreign DNA that it is to be
transferred to the
target cell. Retrovirus vectors typically contain a packaging signal for
incorporation into
the package coat, a sequence which signals the start of the gag transcription
unit, elements
necessary for reverse transcription, including a primer binding site to bind
the tRNA primer
of reverse transcription, terminal repeat sequences that guide the switch of
RNA strands
during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as
the priming site
for the synthesis of the second strand of DNA synthesis, and specific
sequences near the
ends of the LTRs that enable the insertion of the DNA state of the retrovirus
to insert into
the host genome. The removal of the gag, pol, and env genes allows for about 8
kb of
foreign sequence to be inserted into the viral genome, become reverse
transcribed, and upon
replication be packaged into a new retroviral particle. This amount of nucleic
acid is
sufficient for the delivery of a one to many genes depending on the size of
each transcript.
Since the replication machinery and packaging proteins in most retroviral
vectors
.. have been removed (gag, pot, and env), the vectors are typically generated
by placing them
into a packaging cell line. A packaging cell line is a cell line which has
been transfected or
transformed with a retrovirus that contains the replication and packaging
machinery, but
lacks any packaging signal. When the vector carrying the DNA of choice is
transfected into
these cell lines, the vector containing the gene of interest is replicated and
packaged into
new retroviral particles, by the machinery provided in cis by the helper cell.
The genomes
for the machinery are not packaged because they lack the necessary signals.
The construction of replication-defective adenoviruses has been described
(Berkner
etal., J. Virology 61:1213-1220 (1987); Massie et at., Mol. Cell. Biol. 6:2872-
2883 (1986);
Haj-Alunad et al., J. Virology 57:267-274 (1986); Davidson et at., J. Virology
61:1226-
1239 (1987); Zhang "Generation and identification of recombinant adenovirus by
liposome-
mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The
benefit
of the use of these viruses as vectors is that they are limited in the extent
to which they can
spread to other cell types, since they can replicate within an initial
infected cell, but are
unable to form new infectious viral particles. Recombinant adenoviruses have
been shown
to achieve high efficiency gene transfer after direct, iin vivo delivery to
airway epithelium,
31
CA 02592285 2007-06-28
hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue
sites
(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest.
92:381-387
(1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature
Genetics 4:154-
159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem.
267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner,
Nature
Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993);
Bout,
Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud,
Eur. J.
Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507
(1993)).
Recombinant adenoviruses achieve gene transduction by binding to specific cell
surface
receptors, after which the virus is internalized by receptor-mediated
endocytosis, in the same
manner as wild type or replication-defective adenovirus (Chardormet and Dales,
Virology
40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973);
Svensson and
Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655
(1984); Seth, et
al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-
6070 (1991);
Wickham et al., Cell 73:309-319 (1993)).
A viral vector can be one based on an adenovirus which has had the El gene
removed, and these virons are generated in a cell line such as the human 293
cell line. In one
aspect, both the El and E3 genes are removed from the adenovirus genome.
Another type of viral vector is based on an adeno-associated virus (AAV). This
defective parvovirus can infect many cell types and is nonpathogenic to
humans. AAV type
vectors can transport about 4 to 5 kb and wild type AAV is known to stably
insert into
chromosome 19. As an example, this vector can be the P4.1 C vector produced by
Avigen,
San Francisco, CA, which can contain the herpes simplex virus thymidine kinase
gene,
HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent
protein,
GFP.
In another type of AAV virus, the AAV contains a pair of inverted terminal
repeats
(ITRs) which flank at least one cassette containing a promoter which directs
cell-specific
expression operably linked to a heterologous gene. Heterologous in this
context refers to
any nucleotide sequence or gene which is not native to the AAV or B19
parvovirus.
32
CA 02592285 2013-11-15
Typically the AAV and B19 coding regions have been deleted, resulting in a
safe,
noncytotoxic vector. The AAV flits, or modifications thereof, confer
infectivity and site-
specific integration, but not cytotoxicity, and the promoter directs cell-
specific expression.
The disclosed vectors thus provide DNA molecules which are capable of
integration
into a mammalian chromosome without substantial toxicity.
The inserted genes in viral and retroviral usually contain promoters, and/or
enhancers to help control the expression of the desired gene product. A
promoter is
generally a sequence or sequences of DNA that function when in a relatively
fixed location
in regard to the transcription start site. A promoter contains core elements
required for basic
interaction of RNA polymerase and transcription factors, and may contain
upstream
elements and response elements.
Molecular genetic experiments with large human herpes viruses have provided a
means whereby large heterologous DNA fragments can be cloned, propagated and
established in cells permissive for infection with herpes viruses (Sun et al.,
Nature genetics
8: 33-41, 1994; Cotter and Robertson,. Curr Opin Mol Ther 5: 633-644, 1999).
These large
DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the
potential
to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV
recombinants can maintain large pieces of DNA in the infected B-cells as
episomal DNA.
Individual clones carried human genomie inserts up to 330 kb appeared
genetically stable.
The maintenance of these episomes requires a specific EBV nuclear protein,
EBNA1,
constitutively expressed during infection with EBV. Additionally, these
vectors can be used
for transfection, where large amounts of protein can be generated transiently
in vitro.
Herpesvirus amplicon systems are also being used to package pieces of DNA >
220 kb and
to infect cells that can stably maintain DNA as episomes.
Other useful systems include, for example, replicating and host-restricted non-
replicating vaccinia virus vectors.
The disclosed compositions can be delivered to the target cells in a variety
of ways.
For example, the compositions can be delivered through electroporation, or
through
33
CA 02592285 2007-06-28
lipofection, or through calcium phosphate precipitation. The delivery
mechanism chosen
will depend in part on the type of cell targeted and whether the delivery is
occurring for
example iin vivo or in vitro.
Thus, the compositions can comprise, in addition to the disclosed
polypeptides,
nucleic acids or vectors, for example, lipids such as liposomes, such as
cationic liposomes
(e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can
further
comprise proteins to facilitate targeting a particular cell, if desired.
Administration of a
composition comprising a compound and a cationic liposome can be administered
to the
blood afferent to a target organ or inhaled into the respiratory tract to
target cells of the
respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. I Resp.
Cell. MoL Biol.
1:95-100 (1989); Felgner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987);
U.S. Pat.
No.4,897,355. Furthermore, the compound can be administered as a component of
a
microcapsule that can be targeted to specific cell types, such as macrophages,
or where the
diffusion of the compound or delivery of the compound from the microcapsule is
designed
for a specific rate or dosage.
In the methods described above which include the administration and uptake of
exogenous DNA into the cells of a subject (i.e., gene transduction or
transfection), delivery
of the compositions to cells can be via a variety of mechanisms. As one
example, delivery
can be via a liposome, using commercially available liposome preparations such
as
LLPOFECTIN, LIPOFECTAMINE (GLBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT
(Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,
WI), as well as other liposomes developed according to procedures standard in
the art. In
addition, the disclosed nucleic acid or vector can be delivered iin vivo by
electroporation, the
technology for which is available from Genetronics, Inc. (San Diego, CA) as
well as by
means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).
Nucleic acids that are delivered to cells which are to be integrated into the
host cell
genome, typically contain integration sequences. These sequences are often
viral related
sequences, particularly when viral based systems are used. These viral
integration systems
can also be incorporated into nucleic acids which are to be delivered using a
non-nucleic
34
CA 02592285 2007-06-28
acid based system of deliver, such as a liposome, so that the nucleic acid
contained in the
delivery system can be come integrated into the host genome.
Other general techniques for integration into the host genome include, for
example,
systems designed to promote homologous recombination with the host genome.
These
systems typically rely on sequence flanking the nucleic acid to be expressed
that has enough
homology with a target sequence within the host cell genome that recombination
between
the vector nucleic acid and the target nucleic acid takes place, causing the
delivered nucleic
acid to be integrated into the host genome. These systems and the methods
necessary to
promote homologous recombination are known to those of skill in the art.
The compositions can be delivered to the subjectOs cells iin vivo and/or ex
vivo by a
variety of mechanisms well known in the art (e.g., uptake of naked DNA,
liposome fusion,
intramuscular injection of DNA via a gene gun, endocytosis and the like).
If ex vivo methods are employed, cells or tissues can be removed and
maintained
outside the body according to standard protocols well known in the art. The
compositions
can be introduced into the cells via any gene transfer mechanism, such as, for
example,
calcium phosphate mediated gene delivery, electroporation, microinjection or
proteoliposomes. The transduced cells can then be infused (e.g., in a
pharmaceutically
acceptable carrier) or homotopically transplanted back into the subject per
standard methods
for the cell or tissue type. Standard methods are known for transplantation or
infusion of
various cells into a subject.
The nucleic acids that are delivered to cells typically contain expression
controlling
systems. For example, the inserted genes in viral and retroviral systems
usually contain
promoters, and/or enhancers to help control the expression of the desired gene
product. A
promoter is generally a sequence or sequences of DNA that function when in a
relatively
fixed location in regard to the transcription start site. A promoter contains
core elements
required for basic interaction of RNA polymerase and transcription factors,
and may contain
upstream elements and response elements.
Promoters controlling transcription from vectors in mammalian host cells may
be
obtained from various sources, for example, the genomes of viruses such as:
polyoma,
.. Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus,
cytomegalovirus, or
CA 02592285 2007-06-28
from heterologous mammalian promoters, e.g. beta actin promoter. The early and
late
promoters of the SV40 virus are conveniently obtained as an SV40 restriction
fragment
which also contains the SV40 viral origin of replication (Fiers et al.,
Nature, 273: 113
(1978)). The immediate early promoter of the human cytomegalovirus is
conveniently
obtained as a HindIII E restriction fragment (Greenway, P.J. et al., Gene 18:
355-360
(1982)). Of course, promoters from the host cell or related species also are
useful herein.
Enhancer generally refers to a sequence of DNA that functions at no fixed
distance
from the transcription start site and can be either 5' (Laimins, L. et al.,
Proc. Natl. Acad. Sci.
78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3: 1108 (1983)) to
the
transcription unit. Furthermore, enhancers can be within an intron (Banerji,
J.L. et al., Cell
33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F.,
et al., Mol. Cell
Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and
they function
in cis. Enhancers function to increase transcription from nearby promoters.
Enhancers also
often contain response elements that mediate the regulation of transcription.
Promoters can
also contain response elements that mediate the regulation of transcription.
Enhancers often
determine the regulation of expression of a gene. While many enhancer
sequences are now
known from manunalian genes (globin, elastase, albumin, a-fetoprotein and
insulin),
typically one will use an enhancer from a eukaryotic cell virus for general
expression.
Examples are the SV40 enhancer on the late side of the replication origin (bp
100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side
of the
replication origin, and adenovirus enhancers.
The promotor and/or enhancer may be specifically activated either by light or
specific chemical events which trigger their function. Systems can be
regulated by reagents
such as tetracycline and dexamethasone. There are also ways to enhance viral
vector gene
expression by exposure to irradiation, such as gamma irradiation, or
alkylating
chemotherapy drugs.
In certain embodiments the promoter and/or enhancer region can act as a
constitutive
promoter and/or enhancer to maximize expression of the region of the
transcription unit to
be transcribed. In certain constructs the promoter and/or enhancer region be
active in all
eukaryotic cell types, even if it is only expressed in a particular type of
cell at a particular
36
CA 02592285 2007-06-28
time. A promoter of this type is the CMV promoter (650 bases). Other such
promoters are
SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector
LTR.
It has been shown that all specific regulatory elements can be cloned and used
to
construct expression vectors that are selectively expressed in specific cell
types such as
melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been
used to
selectively express genes in cells of glial origin.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal,
human or nucleated cells) may also contain sequences necessary for the
termination of
transcription which may affect mRNA expression. These regions are transcribed
as
polyadenylated segments in the untranslated portion of the mRNA encoding
tissue factor
protein. The 3' untranslated regions also include transcription termination
sites. The
transcription unit can also contain a polyadenylation region. One benefit of
this region is
that it increases the likelihood that the transcribed unit will be processed
and transported like
mRNA. The identification and use of polyadenylation signals in expression
constructs is
well established. Homologous polyadenylation signals can be used in the
transgene
constructs. In certain transcription units, the polyadenylation region is
derived from the
SV40 early polyadenylation signal and consists of about 400 bases. Transcribed
units an
contain other standard sequences alone or in combination with the above
sequences improve
expression from, or stability of, the construct.
The viral vectors can include nucleic acid sequence encoding a marker product.
This
marker product is used to determine if the gene has been delivered to the cell
and once
delivered is being expressed. Example marker genes are the E. Coli lacZ gene,
which
encodes 13-galactosidase, and green fluorescent protein.
In some embodiments the marker may be a selectable marker. Examples of
suitable
selectable markers for mammalian cells are dihydrofolate reductase (DHFR),
thymidine
kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such
selectable markers are successfully transferred into a mammalian host cell,
the transformed
mammalian host cell can survive if placed under selective pressure. There are
two widely
used distinct categories of selective regimes. The first category is based on
a cell's
metabolism and the use of a mutant cell line which lacks the ability to grow
independent of a
37
CA 02592285 2007-06-28
supplemented media. Two examples are: Chinese hamster ovary (CHO) DHFR- cells
and
mouse LTK- cells. These cells lack the ability to grow without the addition of
such nutrients
as thymidine or hypoxanthine. Because these cells lack certain genes necessary
for a
complete nucleotide synthesis pathway, they cannot survive unless the missing
nucleotides
are provided in a supplemented media. An alternative to supplementing the
media is to
introduce an intact DHFR or TK gene into cells lacking the respective genes,
thus altering
their growth requirements. Individual cells which were not transformed with
the DHFR or
TK gene will not be capable of survival in non-supplemented media.
The second category is dominant selection which refers to a selection scheme
used in
any cell type and does not require the use of a mutant cell line. These
schemes typically use
a drug to arrest growth of a host cell. Those cells which have a novel gene
would express a
protein conveying drug resistance and would survive the selection. Examples of
such
dominant selection use the drugs neomycin, (Southern P. and Berg, P., J.
Molec. Appl.
Genet. 1:327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science
209: 1422
(1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413
(1985)). The three
examples employ bacterial genes under eukaryotic control to convey resistance
to the
appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or
hygromycin,
respectively. Others include the neomycin analog G418 and puramycin.
Also provided is a cell comprising one or more of the herein provided vectors.
As
used herein, "cell", "cell line", and "cell culture" may be used
interchangeably and all such
designations include progeny. The disclosed cell can be any cell used to clone
or propagate
the vectors provided herein. Thus, the cell can be from any primary cell
culture or
established cell line. The method may be applied to any cell, including
prokaryotic or
eukaryotic, such as bacterial, plant, animal, and the like. The cell type can
be selected by one
skilled in the art based on the choice of vector and desired use.
Disclosed are animals produced by the process of transfecting a cell within
the
animal with any of the nucleic acid molecules or vectors disclosed herein.
Disclosed are
animals produced by the process of transfecting a cell within the animal any
of the nucleic
acid molecules or vectors disclosed herein, wherein the animal is a mammal.
Also disclosed
are animals produced by the process of transfecting a cell within the animal
any of the
38
CA 02592285 2007-06-28
=
nucleic acid molecules or vectors disclosed herein, wherein the mammal is
mouse, rat,
rabbit, cow, sheep, pig, or primate.
Provided is a composition comprising one or more of the herein provided
polypeptides, nucleic acids, or vectors in a pharmaceutically acceptable
carrier. Thus,
provided is a composition comprising a combination of two or more of any of
the herein
provided ACT polypeptides in a pharmaceutically acceptable carrier. For
example, provided
is a composition comprising SEQ ID NO:1 and SEQ ED NO:5 in a pharmaceutically
acceptable carrier.
By "pharmaceutically acceptable" is meant a material that is not biologically
or
otherwise undesirable, i.e., the material may be administered to a subject,
along with the
nucleic acid or vector, without causing any undesirable biological effects or
interacting in a
deleterious manner with any of the other components of the pharmaceutical
composition in
which it is contained. The carrier would naturally be selected to minimize any
degradation
of the active ingredient and to minimize any adverse side effects in the
subject, as would be
well known to one of skill in the art.
The herein provide composition can further comprise any known or newly
discovered substance that can be administered to a wound, tissue injury, site
of inflammation
or cancer. For example, the provided composition can further comprise one or
more of
classes of antibiotics (e.g. Aminoglycosides, Cephalosporins, Chloramphenicol,
.. Clindamycin, Erythromycins, Fluoroquinolones, Macrolides, Azolides,
Metronidazole,
Penicillin's, Tetracycline's, Trimethoprim-sulfamethoxazole, Vancomycin),
steroids (e.g.
Andranes (e.g. Testosterone), Cholestanes (e.g. Cholesterol), Cholic acids
(e.g. Cholic acid),
Corticosteroids (e.g. Dexamethasone), Estraenes (e.g. Estradiol), Pregnanes
(e.g.
Progesterone), narcotic and non-narcotic analgesics (e.g. Morphine, Codeine,
Heroin,
Hydromorphone, Levorphanol, Meperidine, Methadone, Oxydone, Propoxyphene,
Fentanyl,
Methadone, Naloxone, Buprenorphine, Butoiphanol, Nalbuphine, Pentazocine),
chemotherapy (e.g. anti-cancer drugs such as but not limited to Altretamine,
Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil,
Cisplatin,
Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Diethylstilbesterol,
Ethinyl
estradiol, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide,
Goserelin,
39
CA 02592285 2007-06-28
Hydroxyurea, Idarubicin, Ifosfamide, Leuprolide, Levamisole, Lomustine,
Mechlorethamine, Medroxyprogesterone, Megestrol, Melphalan, Mercaptopurine,
Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Paclitaxel, pentastatin,
Pipobroman,
Plicamycin, Prednisone, Procarbazine, Streptozocin, Tamoxifen, Teniposide,
Vinblastine,
Vincristine), anti-inflammatory agents (e.g. Alclofenac; Alclometasone
Dipropionate;
Algestone Acetonide; alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium;
Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;
Balsalazide
Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;
Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;
Clobetasol
Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate;
Cormethasone
Acetate; Cortodoxone; Decanoate; Deflazacort; Delatestryl; Depo-Testosterone;
Desonide;
Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac
Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate;
Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam
Sodium;
Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac;
Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic
Acid;
Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin
Butyl;
Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone
Propionate;
Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone
Acetate;
Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap;
Indomethacin;
Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;
Isoxepac;
Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol
Etabonate;
Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic
Acid;
Mesalamine; Meseclazone; Mesterolone; Methandrostenolone; Methenolone;
Methenolone
Acetate; Methylprednisolone Suleptanate; Momiflumate; Nabumetone; Nandrolone;
Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;
Orpanoxin; Oxandrolane; Oxaprozin; Oxyphenbutazone; Oxymetholone ; Paranyline
Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate;
Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;
Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole
Citrate;
CA 02592285 2013-11-15
Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride;
Seclazone; Sermetacin; Stanozolol; Sudoxicam; Sulindac; Suprofen; Talmetacin;
Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam;
Tesicam;
Tesimide; Testosterone; Testosterone Blends; Tetrydamine; Tiopinac; Tixocortol
Pivalate;
Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidornetacin; Zomepirac
Sodium),
or anti-histaminic agents (e.g. Ethanolamines (like diphenhydrmine
carbinoxamine),
Ethylenediamine (like tripelennamine pyrilatnine), Alkylamine (like
chlorpheniramine,
dexchlorpheniramine, brompheniramine, triprolidine), other anti-histamines
like astemizole,
loratadine, fexofenadine, Bropheniramine, Clemastine, Acetaminophen,
Pseudoephedrine,
Triprolidine).
The compositions may be administered topically, orally, or parenterally. For
example, the compositions can be administered extracorporeally,
intracranially,
intravaginally, intraanally, subcutaneously, intrademially, intracardiac,
intragashic,
intravenously, intramuscularly, by intraperitoneal injection, transdermally,
intranasally, or
by inhalant. As used herein, "intracranial administration" means the direct
delivery of
substances to the brain including, for example, intrathecal, intracisternal,
intraventricular or
trans-sphenoidal delivery via catheter or needle.
Parenteral administration of the composition, if used, is generally
characterized by
injection. Injectables can be prepared in conventional forms, either as liquid
solutions or
suspensions, solid forms suitable for solution of suspension in liquid prior
to injection, or as
emulsions. A more recently revised approach for parenteral administration
involves use of a
slow release or sustained release system such that a constant dosage is
maintained. See, e.g.,
U.S. Patent No. 3,610,795.
As used herein, "topical intranasal administration" means delivery of the
.. compositions into the nose and nasal passages through one or both of the
nares and can
comprise delivery by a spraying mechanism or droplet mechanism, or through
aerosolization
of the nucleic acid or vector. Administration of the compositions by inhalant
can be through
the nose or mouth via delivery by a spraying or droplet mechanism. Delivery
can also be
directly to any area of the respiratory system (e.g., lungs) via intubation.
41
CA 02592285 2007-06-28
=
The exact amount of the compositions required will vary from subject to
subject,
depending on the species, age, weight and general condition of the subject,
the severity of
the allergic disorder being treated, the particular nucleic acid or vector
used, its mode of
administration and the like. Thus, it is not possible to specify an exact
amount for every
composition. However, an appropriate amount can be determined by one of
ordinary skill in
the art using only routine experimentation given the teachings herein.
The materials may be in solution or suspension (for example, incorporated into
microparticles, liposomes, or cells). These may be targeted to a particular
cell type via
antibodies, receptors, or receptor ligands. The following references are
examples of the use
of this technology to target specific proteins to tumor tissue (Senter, et
al., Bioconjugate
Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989);
Bagshawe,
et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.,
4:3-9, (1993);
Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz
and McKenzie,
Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.
Pharmacol, 42:2062-
2065, (1991)). Vehicles such as "stealth" and other antibody conjugated
liposomes
(including lipid mediated drug targeting to colonic carcinoma), receptor
mediated targeting
of DNA through cell specific ligands, lymphocyte directed tumor targeting, and
highly
specific therapeutic retroviral targeting of murine glioma cells in vivo. The
following
references are examples of the use of this technology to target specific
proteins to tumor
tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger
and Huang,
Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors
are involved
in pathways of endocytosis, either constitutive or ligand induced. These
receptors cluster in
clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass
through an acidified
endosome in which the receptors are sorted, and then either recycle to the
cell surface,
become stored intracellularly, or are degraded in lysosomes. The
internalization pathways
serve a variety of functions, such as nutrient uptake, removal of activated
proteins, clearance
of macromolecules, opportunistic entry of viruses and toxins, dissociation and
degradation
of ligand, and receptor-level regulation. Many receptors follow more than one
intracellular
pathway, depending on the cell type, receptor concentration, type of ligand,
ligand valency,
and ligand concentration. Molecular and cellular mechanisms of receptor-
mediated
42
CA 02592285 2007-06-28
endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6,
399-409
(1991)).
Suitable carriers and their formulations are described in Remington: The
Science and
Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company,
Easton, PA
1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt
is used in the
formulation to render the formulation isotonic. Examples of the
pharmaceutically-
acceptable carrier include, but are not limited to, saline, Ringer's solution
and dextrose
solution. The pH of the solution can be from about 5 to about 8, from about 7
to about 7.5.
Further carriers include sustained release preparations such as semipermeable
matrices of
solid hydrophobic polymers containing the antibody, which matrices are in the
form of
shaped articles, e.g., films, liposomes or microparticles. It will be apparent
to those persons
skilled in the art that certain carriers may be more preferable depending
upon, for instance,
the route of administration and concentration of composition being
administered.
Pharmaceutical carriers are known to those skilled in the art. These most
typically
would be standard carriers for administration of drugs to humans, including
solutions such
as sterile water, saline, and buffered solutions at physiological pH. The
compositions can be
administered intramuscularly or subcutaneously. Other compounds will be
administered
according to standard procedures used by those skilled in the art.
Pharmaceutical compositions may include carriers, thickeners, diluents,
buffers,
preservatives, surface active agents and the like in addition to the molecule
of choice.
Pharmaceutical compositions may also include one or more active ingredients
such as
antimicrobial agents, antiinfiamrnatory agents, anesthetics, and the like.
The pharmaceutical composition may be administered in a number of ways
depending on whether local or systemic treatment is desired, and on the area
to be treated.
Administration may be topically (including ophthahnically, vaginally,
rectally, intranasally),
orally, by inhalation, or parenterally, for example by intravenous drip,
subcutaneous,
intraperitoneal or intramuscular injection.
Preparations for parenteral administration include sterile aqueous or non-
aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous solvents are
propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable
organic esters
43
CA 02592285 2007-06-28
such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions
or suspensions, including saline and buffered media. Parenteral vehicles
include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's, or
fixed oils. Intravenous vehicles include fluid and nutrient replenishers,
electrolyte
replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and
other additives may also be present such as, for example, antimicrobials, anti-
oxidants,
chelating agents, and inert gases and the like.
Formulations for topical administration may include ointments, lotions,
creams, gels
(e.g., poloxamer gel), drops, suppositories, sprays, liquids and powders.
Conventional
.. pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the
like may be
necessary or desirable. The disclosed compositions can be administered, for
example, in a
microfiber, polymer (e.g., collagen), nanosphere, aerosol, lotion, cream,
fabric, plastic,
tissue engineered scaffold, matrix material, tablet, implanted container,
powder, oil, resin,
wound dressing, bead, microbead, slow release bead, capsule, injectables,
intravenous drips,
pump device, silicone implants, or any bio-engineered materials.
In one aspect the provided pharmaceutically acceptable carrier is a poloxamer.
Poloxamers, referred to by the trade name Pluronics , are nonionic surfactants
that form
clear thermoreversible gels in water. Poloxamers are polyethylene oxide-
polypropylene
oxide-polyethylene oxide (PEO-PPO-PEO) tri-block copolymers. The two
polyethylene
oxide chains are hydrophilic but the polypropylene chain is hydrophobic. These
hydrophobic and hydrophilic characteristics take charge when placed in aqueous
solutions.
The PEO-PPO-PEO chains take the form of small strands where the hydrophobic
centers
would come together to form micelles. The micelle, sequentially, tend to have
gelling
characteristics because they come together in groups to form solids (gels)
where water is just
slightly present near the hydrophilic ends. When it is chilled, it becomes
liquid, but it
hardens when warmed. This characteristic makes it useful in pharmaceutical
compounding
because it can be drawn into a syringe for accurate dose measurement when it
is cold. When
it warms to body temperature (when applied to skin) it thickens to a perfect
consistency
(especially when combined with soy lecithin/isopropyl palmitate) to facilitate
proper
inunction and adhesion. Pluronic F127 (F127) is widely used because it is
obtained easily
44
CA 02592285 2007-06-28
,
,
and thus it is used in such pharmaceutical applications. F127 has a EO:PO:E0
ratio of
100:65:100, which by weight has a PEO:PPO ratio of 2:1. Pluronic gel is an
aqueous
solution and typically contains 20-30% F-127. Thus, the provided compositions
can be
administered in F127.
Compositions for oral administration include powders or granules, suspensions
or
solutions in water or non-aqueous media, capsules, sachets, or tablets.
Thickeners,
flavorings, diluents, emulsifiers, dispersing aids or binders may be
desirable..
Some of the compositions may potentially be administered as a pharmaceutically
acceptable acid- or base- addition salt, formed by reaction with inorganic
acids such as
hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic
acid, sulfuric
acid, and phosphoric acid, and organic acids such as formic acid, acetic acid,
propionic acid,
glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic
acid, maleic acid,
and fumaric acid, or by reaction with an inorganic base such as sodium
hydroxide,
ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-,
trialkyl
and aryl amines and substituted ethanolamines.
Effective dosages and schedules for administering the compositions may be
determined empirically, and making such determinations is within the skill in
the art. The
dosage ranges for the administration of the compositions are those large
enough to produce
the desired effect in which the symptoms disorder are effected. The dosage
should not be so
large as to cause adverse side effects, such as unwanted cross-reactions,
anaphylactic
reactions, and the like. Generally, the dosage will vary with the age,
condition, sex and
extent of the disease in the patient, route of administration, or whether
other drugs are
included in the regimen, and can be determined by one of skill in the art. The
dosage can be
adjusted by the individual doctor in the event of any counterindications.
Dosage can vary,
and can be administered in one or more dose administrations daily, for one or
several days.
Guidance can be found in the literature for appropriate dosages for given
classes of
pharmaceutical products. The range of dosage largely depends on the
application of the
compositions herein, severity of condition, and its route of administration.
For example, in applications as a laboratory tool for research, the ACT
peptide
compositions can be used in doses as low as 0.01% w/v. The dosage can be as
low as 0.02%
CA 02592285 2007-06-28
w/v and possibly as high as 2% w/v in topical skin wound treatments.
Significantly higher
concentrations of the compositions by themselves or in combination with other
compounds
may be used in applications like cancer/tumor therapy or as an early
concentrated bolus
immediately following an acute tissue injury. Thus, upper limits of the
provided
polypeptides may be up to 2-5 % w/v or v/v if given as an initial bolus
delivered for
example directly into a tumor mass. Recommended upper limits of dosage for
parenteral
routes of administration for example intramuscular, intracerebral,
intracardicardiac and
intraspinal could be up to 1 % w/v or v/v depending on the severity of the
injury. This upper
dosage limit may vary by formulation, depending for example on how the
polypeptide(s) is
combined with other agents promoting its action or acting in concert with the
polypeptide(s).
For continuous delivery of the provided polypeptides, for example, in
combination
with an intravenous drip, upper limits of 0.01g /Kg body weight over time
courses
determined by the doctor based on improvement in the condition can be used. In
another
example, upper limits of concentration of the provided nucleic acids delivered
topically, for
example, in skin wounds would be 5-10 ptg/cm2 of wound depending for example
on how
the nucleic acid is combined with other agents promoting its action or acting
in concert with
the nucleic acids. This would be repeated at a frequency determined by the
Doctor based on
improvement. In another example, upper limits of concentration of the provided
nucleic
acids delivered internally for example, intramuscular, intracerebral,
intracardicardiac and
intraspinal would be 50-1001.1g/m1 of solution. Again, the frequency would be
determined
by the Doctor based on improvement.
Also disclosed is the pre-conditioning of an area with the provided
polypeptides
prior to surgery. The concentration of the polypeptides can be 10200 AM mixed
in with 10-
% pluronic gel or any such carrier that enables penetration of the peptide(s)
within the
25 site of interest for a period of at least 3-6 hours prior to surgery.
This pre-procedural
conditioning can improve the subsequent healing response to surgery, including
reduced
inflammatory response.
Viral vectors remain highly experimental tools that nonetheless show
considerable
potential in clinical applications. As such, caution is warranted in
calculation of expected
30 dosage regimes for viral vectors and will depend considerably on the
type of vector used.
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CA 02592285 2007-06-28
For example, retroviral vectors infect dividing cells such as cancer cells
efficiently,
intercalating into the host cell genome and continuing expression of encoded
proteins
indefinitely. Typical dosages of retroviruses in an animal model setting are
in the range of
107 to 109 infectious units per ml. By contrast, adenoviruses most efficiently
target post-
mitotic cells, but cells are quickly eliminated by the host immune system or
virus is
eventually lost if infected cells resume proliferation and subsequently dilute
the viral
episomal DNA. Indeed, this transient time course of infection may be useful
for short-term
delivery of the composition described herein in certain clinical situations,
for example in
amelioration of a small injury. In animal models, concentrations of 108-1011
infectious units
per ml of adenovirus are typical for uses in research. Dose ranges of vectors
based on data
derived from animal models would be envisaged to be used eventually in
clinical setting(s),
pending the development of pharmaceutically acceptable formulation(s).
Two topical applications of ACT compositions at 0.02 % w/v; one applied
acutely
and the second applied 24 hours later are sufficient to reduce inflammation,
promote
healing, reduce scarring, increase tensile strength, and promote tissue
regeneration.
However, in a clinical setting an increased frequency of up to 3 applications
per day
topically at a concentration of up to 5 % is recommended until significant
improvement is
achieved as determined by a Doctor. For internal administration, for example,
intravenously,
intramuscularly, intracerebral, intracardicardiac and intraspinally and
increased frequency of
up to 3 dosages of 1 % w/v or v/v per day is recommended until significant
improvement is
determined by the Doctor.
Following administration of a disclosed composition, such as a polypeptide,
for
promoting wound healing, the efficacy of the therapeutic composition can be
assessed in
various ways well known to the skilled practitioner. For instance, one of
ordinary skill in
.. the art will understand that a composition, such as a polypeptide,
disclosed herein is
efficacious in promoting wound healing in a subject by observing that the
composition can
reduce scar tissue formation, reduce fibrotic tissue formation, improve tissue
regeneration,
or reduce inflammation in the subject following tissue injury. Methods for
measuring these
criteria are known in the art and discussed herein.
47
CA 02592285 2007-06-28
Also provided are materials comprising the herein provided compositions (e.g.,
polypeptides, nucleic acids, or vectors). For example, provided are materials
used to treat
wounds, wherein the materials are coated with an ACT polypeptide. Non-limiting
examples
of materials used to treat wounds include bandages, steri-strip, sutures,
staples, or grafts
(e.g., skin grafts).
For example, the material (e.g., bandage, steri-strip, suture, staple, graft)
can be
soaked in the provided polypeptide at a concentration ranging from 10-200 M.
The
material can then be dried and sealed in a sterile container. The material can
also be
immersed in liquid 10-30% pluronic gel at 4 C containing polypeptide at 10-
200 ttM
concentration. The material can then be brought to approximate room
temperature so that
the gel polymerizes, leaving a coat of polypeptide-impregnated gel surrounding
the material,
which can be sealed in a sterile container. The polypeptide can also be
incorporated into a
cross-linkable hydrogel system, such as the poly(lactic-co-glycolic acid)
(PLGA) or
polyurethane, which can then be fashioned into materials for treating wounds
(e.g., bandage,
steri-strip, suture, staple, graft). Thus, provided are composite hydrogel-
peptide materials.
Also disclosed are medical implants coated with the provided polypeptide
before
implantation in a subject. For example, a common problem in such implant
surgeries is the
formation of a contraction capsule around the implant from scar tissue
formation that leads
to undue hardening, contraction and ultimately mis shaping of the tissue of
interest. The use
of the present polypeptides in or on the implant can reduce or prevent this
misshaping. Non-
limiting examples of medical implants include: limb prostheses, breast
implants, penile
implants, testicular implants, artificial eyes, facial implants, artificial
joints, heart valve
prostheses, vascular prostheses, dental prostheses, facial prosthesis, tilted
disc valve, caged
ball valve, ear prosthesis, nose prosthesis, pacemakers, cochlear implants,
and skin
substitutes (e.g., porcine heterograft/ pigskin, BIOBRANE, cultured
keratinocytes).
A. Methods
Provided herein is a method of promoting wound healing following tissue injury
in a
subject, comprising administering to the subject one or more of the herein
provided
compositions (e.g., polypeptides, nucleic acids, or vectors) in a
pharmaceutically acceptable
carrier. Further provided is a method of treating a subject with tissue
injury, comprising
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CA 02592285 2007-06-28
administering to the subject one or more of the herein provided compositions
(e.g.,
polypeptides, nucleic acids, or vectors) in a pharmaceutically acceptable
carrier.
"Promote," "promotion," and "promoting" refer to an increase in an activity,
response, condition, disease, or other biological parameter. This can include
but is not
limited to the initiation of the activity, response, condition, or disease.
This may also
include, for example, a 10% increase in the activity, response, condition, or
disease as
compared to the native or control level. Thus, the increase can be a 10, 20,
30, 40, 50, 60,
70, 80, 90, 100%, or any amount of increase in between as compared to native
or control
levels.
By "treat" or "treatment" is meant a method of reducing the effects of a
disease or
condition. Treatment can also refer to a method of reducing the underlying
cause of the
disease or condition itself rather than just the symptoms. The treatment can
be any
reduction from native levels and can be but is not limited to the complete
ablation of the
disease, condition, or the symptoms of the disease or condition. For example,
a disclosed
method for promoting wound healing is considered to be a treatment if there is
a 10%
reduction in one or more symptoms of the disease in a subject with the disease
when
compared to native levels in the same subject or control subjects. Thus, the
reduction can be
a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in
between as
compared to native or control levels.
As used herein, "subject" includes, but is not limited to, animals, plants,
bacteria,
viruses, parasites and any other organism or entity that has nucleic acid. The
subject may be
a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit,
dog, sheep, goat,
non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a
reptile or an
amphibian. The subject can be an invertebrate, more specifically an arthropod
(e.g., insects
and crustaceans). The term does not denote a particular age or sex. Thus,
adult and newborn
subjects, as well as fetuses, whether male or female, are intended to be
covered. A patient
refers to a subject afflicted with a disease or disorder. The term "patient"
includes human
and veterinary subjects.
The provided method can reduce scar tissue formation in a subject following
tissue
injury. By "scar tissue" is meant the fibrous (fibrotic) connective tissue
that forms at the site
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CA 02592285 2007-06-28
of injury or disease in any tissue of the body, caused by the overproduction
of disorganized
collagen and other connective tissue proteins, which acts to patch the break
in the tissue.
Scar tissue may replace injured skin and underlying muscle, damaged heart
muscle, or
diseased areas of internal organs such as the liver. Dense and thick, it is
usually paler than
the surrounding tissue because it is poorly supplied with blood, and although
it structurally
replaces destroyed tissue, it cannot perform the functions of the missing
tissue. It is
composed of collagenous fibers, which will often restrict normal elasticity in
the tissue
involved. Scar tissue may therefore limit the range of muscle movement or
prevent proper
circulation of fluids when affecting the lymphatic or circulatory system.
Glial scar tissue
following injury to the brain or spinal chord is one of the main obstacles to
restoration of
neural function following damage to the central nervous system. A reduction in
scar tissue
can be assessed by the population of cell types within the injured site. For
example, a
reduction in glial scar tissue can be estimated by an increased ratio of
neuronal to astrocytic
cells. A reduction in scar tissue formation can be measured by a simple
measurement of scar
width or area of scar tissue (Wilgus et al., 2003). In addition histological
assessments can be
made about the restoration of structural complexity within healed tissue in
comparison to
normal tissue.
In addition to reducing fibrotic tissue formation in a subject in following
tissue
injury, the provided compositions and methods can also be used to treat
disorders associated
with pathological increases in fibrotic tissue formation in a subject, such as
for example,
psoriasis, cutaneous and systemic mastocytosis, asthma, eczema, sinusitis,
atherosclerosis,
rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis,
pulmonary fibrosis and
cystic fibrosis. A reduction in fibrotic tissue formation in a subject can be
measured by
clinical judgment of a doctor assessing whether a regain in normal structure
and function of
a given tissue and/or organ in a subject has resulted following a treatment.
As an example,
for psoriasis a doctor would assess the subject's skin to determine whether
there has been a
reduction in patches of raised red skin covered by flaky white buildup.
Certain kinds of
psoriasis, are characterized by a pimple-ish (pustular psoriasis) or burned
(erythrodermic)
appearance. In such cases, the doctor would determine whether treatment has
resulted in the
reduction of these symptoms. In the case of an tissue or organ in which a
subject where a
doctor judges that a biopsy is clinically available and/or necessary or in an
animal model of
CA 02592285 2007-06-28
the human disease, tissue fragments of bioposies would be prepared and tissue
histological
structure would be assessed by a clinical pathologist and/or trained
histopathologist to
determine if reduction in fibrosis and restoration of normal tissue structure
and function has
occurred. The area of fibrosis to normal tissue could also be quantitatively
assessed on such
histological preparations.
The provided method can restore normal tissue mechanical properties such as
tensile
strength following tissue injury in a subject. "Tensile strength" refers to
the amount of stress
or strain required to break the tissue or wound.
The tensile strength of treated wounds can be 60, 65, 70, 75, 80, 85, 90, 95,
100%
.. that of uninjured tissue within 3 months after treatment. Thus, provided is
a method of
restoring tissue mechanical properties, including increasing tensile strength
of a healed
injury to approach or reach that of normal uninjured tissue, in a subject
comprising
administering to the subject one or more of the herein provided compositions
(e.g.,
polypeptides, nucleic acids, or vectors) in a pharmaceutically acceptable
carrier.
The type of wounds that would be important with respect to tensile strength/
extensibility would include injuries to musculoskeletal structures/ tissues,
and the skin
covering these structures. For example, the provided methods can improve
tensile strength
of articulating joints, bone, cartilage, tendons, or ligaments. The provided
methods can also
improve tensile strength of skin under higher degrees of stress /strain, such
as the skin
covering the elbow, knee, or foot. The most common problems associated with
healing of
joint injuries is that excessive scarring in these areas leads to contraction,
and non-
extensibility of the healed joint area. This has serious cosmetic and
psychological
consequences. The properties of the peptides will help modulate and lessen the
formation of
such scar tissue leading to greater mobility of the joint.
The provided method can improve tissue regeneration following tissue injury in
a
subject. By "regeneration" is meant the renewal, re-growth, or restoration of
a body or a
bodily part, tissue, or substance after injury or as a normal bodily process.
In contrast to
scarring, tissue regeneration involves the restoration of the tissue to its
original structural,
functional, and physiological condition. This is also referred to herein as
tissue
"complexity". The restoration can be partial or complete, meaning 10, 20, 30,
40, 50, 60,
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CA 02592285 2007-06-28
,
70, 80, 90, 100% restoration, or any amount of restoration in between as
compared to native
or control levels. As an example, in the case of a skin injury, tissue
regeneration can involve
the restoration of hair follicles, glandular structures, blood vessels,
muscle, or fat. In the case
of a brain injury, tissue regeneration can involve maintenance or restoration
of neurons. As
an example in the case of skin an improvement in tissue regeneration can be
assessed by
measurements of the volume of fibrous scar tissue to normal regenerated skin
as a ratio. As
another example, counts can be made of discrete regenerating structures such
as
regenerating skin glands normalized to the volume of the wound area.
In one aspect, tissue regeneration involves the recruitment and
differentiation of stem
cells to replace the damaged cells. As used herein, a "stem cell" is an
undifferentiated cell
found among differentiated cells in a tissue or organ, or introduced from an
external source
for e.g., Embryonic stem cells, Adult Bone Marrow stem cells, that can renew
itself and
differentiate to yield the major specialized cell types of the tissue or
organ. The primary
roles of stem cells in a living organism are to maintain and repair the tissue
in which they
are found. By stem cell differentiation is meant the process whereby an
unspecialized cell
(e.g., stem cell) acquires the features of a specialized cell such as a skin,
neural, heart, liver,
or muscle cell. As an example, in the case of a skin injury, tissue
regeneration can involve
the differentiation of stem cells present in the epithelium into hair
follicles (Alonso and
Fuchs, 2003). In the case of a brain injury, tissue regeneration can involve
the differentiation
of stem cells into neurons. The provided method can enhance stem cell
differentiation
following tissue injury in a subject. Enhanced stem cell differentiation can
be measured by
providing a clinically acceptable genetic or other means of marking endogenous
or engrafted
stem cells and determining the frequency of differentiation and incorporation
of marked
stem cells into normal tissue structures. As another example, certain
structures such as hair
follicles are known to be regenerated from endogenous stem cells following
tissue injury. As
such, counts of hair follicles normalized to tissue injury area would serve as
a quantitative
assessment of enhanced stem cell differentiation.
The provided method can reduce inflammation in a subject. By "inflammation",
"inflammatory response" or "immune response" is meant the reaction of living
tissues to
injury, infection or irritation characterized by redness, warmth, swelling,
pain, and loss of
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function, produced as the result of increased blood flow and an influx of
immune cells and
secretions. Inflammation is the body's reaction to invading infectious
microorganisms and
results in an increase in blood flow to the affected area, the release of
chemicals that draw
white blood cells, an increased flow of plasma, and the arrival of monocytes
(or astrocytes in
the case of the brain) to clean up the debris. Anything that stimulates the
inflammatory
response is said to be inflammatory. Thus, in addition to reducing
inflammation in a subject
in response to tissue injury, the provided compositions and methods can also
be used to treat
disorders associated with pathological increases in levels of inflammatory
cells, including,
for example, asthma, eczema, sinusitis, atherosclerosis, rheumatoid arthritis,
inflammatory
bowel disease, cutaneous and systemic mastocytosis, psoriasis, and multiple
sclerosis.
Treatment with the provided polypeptide can also reduce itching, for example
of healing
wounds. Generally, itching results from histamine release by mast cells. The
provided
polypeptide can reduce mast cell de-granulation and histamine release. Thus,
the provided
polypeptide can be used to treat conditions involving histamine release,
including, but not
limited to, itching, scratching, sinus irritation, allergic cough, red eyes,
asthma, and eczema.
A reduction in inflammation can be measured by a reduction in the density of
inflammatory cell types such as, for example, monocytes or astrocytes. A
reduction in
inflammation can be measured by a reduction in the density of inflammatory
cell types such
as, for example, neutrophils, mast cells, basophils, and monocytes. A
reduction in
inflammation can be calculated by an in vivo measurement of neutrophil
activity (Jones et
al., 1994). In addition factors like frequency of mast cell degranulation or
measurement of
histamine levels or levels of reactive oxygen species can be used as
measurements of
reduction in inflammation. The level of inflammation can also be indirectly
measured by
checking for transcription levels of certain genes by qRT-PCR for e.g. genes
like,
Interferon-alpha, -beta and ¨gamma, Tumor Necrosis Factor-alpha, Interleukine
lbeta, -2, -
4, -5, -6, -8, -12, -18, -23, -27, CD4, CD28, CD80, CD86, MHCII, and iNOS.
Measurement
of pro-inflammatory cytokine levels in the tissues and or bodily fluids of the
subject
including plasma can measure a reduction in inflammation. It is noteworthy
that a
mechanism of ACT peptide action may be by inhibition of inflammatory cell
migration
and/or inhibition of pro-inflammatory chemicals (histamine, reactive oxygen
species) and
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CA 02592285 2007-06-28
pro-inflammatory cytokines such as Interleukin (IL)-1, IL-6, IL-8 and tumor
necrosis factor
(TNF).
The provided method can inhibit proliferation of a transformed cell in a
subject (see
Figure 2). By transformed cell is meant a neoplasm, cancer, or tumor cell that
divides and
reproduces abnormally with uncontrolled growth. Thus, inhibition of
proliferation (i.e.,
hyperplasia) of said transformed cell results in a reduction in the growth and
thus
malignancy of the cancer. A representative but non-limiting list of cancers
that the disclosed
compositions and methods can be used to treat is the following: glioma,
lymphoma, B cell
lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid
leukemia,
bladder cancer, brain cancer, nervous system cancer, head and neck cancer,
squamous cell
carcinoma of head and neck, kidney cancer, lung cancers such as small cell
lung cancer and
non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer,
pancreatic cancer,
prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas
of the
mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical
carcinoma, breast
cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary
cancer,
esophageal carcinoma, head and neck carcinoma, large bowel cancer,
hematopoietic
cancers, testicular cancer, colon and rectal cancers, prostatic cancer, or
pancreatic cancer.
Thus, the provided method can be used to treat cancer in a subject. For
example, the
provided method can be used to treat glioma in a subject.
An inhibition in transformed cell proliferation can be measured by a variety
of cell
proliferation markers and kits for e.g. Ki67/MIB-1 immunostaining, tritiated
thymidine or
bromodeoxyuridine labeling indices, DNA S-phase fraction, proliferating cell
nuclear
antigen expression, potential doubling time and analysis of the nucleolar
organizer region
associated proteins (AgNORs). Since the proliferative activity of the tumor
depends both on
the proportion of cells committed to the cycle (growth fraction) and the speed
of the cell
cycle, the actual proliferative activity of a tumor could well be measured by
the equation [PA
= Ki67 or MIB-1 scores x AgNORs] (Pich et al., 2004). In another example,
histopathologists are skilled in assessing biopsy tissue sections using simple
qualitative and
quantitative indices of mitosis to determine proliferation in transformed cell
populations
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CA 02592285 2007-06-28
Various mouse models have been developed for cancer research. There are
specific
mouse models for specific types of cancers. For example, Bladder cancer,
Cervical cancer,
Endometrial cancer, Gastrointestinal cancer, Genitourinary cancer, Head and
Neck cancer,
Hematopoietic cancer, Kidney cancer, Lung cancer, Mammary Gland cancer,
Melanoma,
Myeloma, Nervous System cancer, Oral cancer, Ovarian cancer, Pancreatic
cancer, Prostate
cancer, Sarcoma, Skin cancer. These models are well described and used. The
favorable
effects of the polypeptides, nucleic acids or vectors provided herein can be
studied in any of
these models. For example the skin cancer mouse model can be easily used for
demonstration. Cancers can be cultivated applying the xenograft model of
growing human
cancerous tissues using the specific pathogen free, homo inbred mouse (a nude
mouse)
(Yoo, 2004). The polypeptides, nucleic acids or vectors provided herein can be
locally
administered for e.g. bioengineered materials such as a hollow fiber membranes
(Orlandini
and Margaria. 1983; Ming Chu et al., 1998) and microfibers, slow release
beads,
hypodermic needles, indwelling catheters, which can be inserted locally into
the cancerous
growth, or systemically administered to reach its target for e.g. intravenous
infusions,
intramuscularly, intraperitoneal injection. This treatment can be administered
by itself or in
combination with other therapeutic compounds for e.g. Chemotherapeutic agents.
The provided method can inhibit metastasis of a transformed cell in a subject.
By
"metastasis" is meant the transmission of cancer cells from an original site
to one or more
sites elsewhere in the body, usually by way of the blood vessels or
lymphatics. Metastatis
can be broken down into a series of events. First, cancer cell migration
begins the process
by which tumor cells leave the primary site of growth, often penetrating the
basement
membrane and moving towards the local vasculature. Intravasation describes the
process of
cancer cell entry into the vasculature, and distribution to distant sites.
Extravasation refers
to the process of cancer cell egression from the vasculature. Finally,
proliferation of cancer
cells at the distant site is profoundly influenced by localized growth factor
availability,
influences of stromal cells, and the surrounding extracellular matrix milieu
(the so-called
"soil") as well as the availability of nutrients and factors provided by the
resultant
vascularization of the growing tumor. Thus, the provided compositions and
methods can
inhibit metastasis of a transformed cell in a subject by inhibiting migration
(i.e., metastatic
CA 02592285 2007-06-28
migration) of said cell. Tumourigenesis is the result of cell cycle
disorganization, leading to
an uncontrolled cellular proliferation. Specific cellular processes-mechanisms
that control
cell cycle progression and checkpoint traversation through the intermitotic
phases are
deregulated. Normally, these events are highly conserved due to the existence
of
conservatory mechanisms and molecules such as cell cycle genes and their
products. An
inhibition in metastatic migration can be measured by the levels of such cell
cycle genes and
products for e.g. cyclins, cyclin dependent kinases (Cdks), Cdk inhibitors
(CKI) and extra
cellular factors (i.e. growth factors). Revolutionary techniques using laser
cytometry and
commercial software are available to quantify and evaluate cell cycle
processes and cellular
growth. S-phase fraction measurements, including ploidy values, using
histograms and
estimation of indices such as the mitotic index and tumour-doubling time
indices, provide
adequate information to the clinician to evaluate tumour aggressiveness.
As used herein, tissue injury can result from, for example, a scrape, cut,
laceration
wound, crush wound, compression wound, stretch injury, bite wound, graze,
bullet wound,
.. explosion injury, body piercing, stab wound, burn wound, wind burn, sun
burn, chemical
burn, surgical wound, surgical intervention, medical intervention, host
rejection following
cell, tissue or organ grafting, pharmaceutical effect, pharmaceutical side-
effect, bed sore,
radiation injury, cosmetic skin wound, internal organ injury, disease process
(e.g., asthma,
cancer), infection, infectious agent, developmental process, maturational
process (e.g.,
acne), genetic abnormality, developmental abnormality, environmental toxin,
allergen, scalp
injury, facial injury, jaw injury, foot injury, toe injury, finger injury,
bone injury, sex organ
injury, joint injury, excretory organ injury, eye injury, corneal injury,
muscle injury, adipose
tissue injury, lung injury, airway injury, hernia, anus injury, piles, ear
injury, retinal injury,
skin injury, abdominal injury, arm injury, leg injury, athletic injury, back
injury, birth injury,
premature birth injury, toxic bite, sting, tendon injury, ligament injury,
heart injury, heart
valve injury, vascular system injury, cartilage injury, lymphatic system
injury,
craniocerebral trauma, dislocation, esophageal perforation, fistula, nail
injury, foreign body,
fracture, frostbite, hand injury, heat stress disorder, laceration, neck
injury, self mutilation,
shock, traumatic soft tissue injury, spinal cord injury, spinal injury,
sprain, strain, tendon
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CA 02592285 2007-06-28
injury, ligament injury, cartilage injury, thoracic injury, tooth injury,
trauma, nervous system
injury, aging, aneurism, stroke, digestive tract injury, infarct, or ischemic
injury.
The peptides and/or other formulations embodying the invention will modulate
cell
migration and proliferation, thereby reducing inflammation, accelerating wound
healing,
reduce scarring and ultimately promote repair, regeneration and restoration of
structure and
function in all tissues. Healing of wounds, post-peptide application will
involve
significantly reduced fibrosis, consequently reduced scarring in skin wounds
and fibrous
patches in internal tissue injuries, promoting tissue regeneration and
restoring tissue and
organ structure and function.
An additional embodiment of the invention comprises an in vitro scratch wound
assay of cell migration that the peptide alters and modulates migration and
proliferation of
various cultured cell types, including but not limited to,
fibroblasts/mesenchymal cells,
tumor cells and epithelial cells.
Further, said peptides and/or formulations embodying the invention can be used
to
treat external wounds caused by, but not limited to scrapes, cuts, lacerated
wounds, bite
wounds, bullet wounds, stab wounds, burn wounds, sun burns, chemical burns,
surgical
wounds, bed sores, radiation injuries, all kinds of acute and chronic wounds,
wounds or
lesions created by cosmetic skin procedures and also ameliorate the effects of
skin aging.
The actions of said peptides and/or other formulations will accelerate wound
healing in all
kinds of external wounds and improve the cosmetic appearance of wounded areas,
and skin
subject to aging and disease. Said peptides and/or other formulations can be
used to treat
internal injury caused by, but not limited to, disease, surgery, gunshots,
stabbing, accidents,
infarcts, ischemic injuries, to organs and tissues including but not limited
to heart, bone,
brain, spinal cord, retina, peripheral nerves and other tissues and organs
commonly subject
to acute and chronic injury, disease, congenital and developmental
malformation and aging
processes. Injury to internal organs causes a fibrotic response, which leads
to loss of
structure and function in organ systems. In central nervous system (CNS) this
response to
injury is mediated by astrocytes (fibroblast-like cells in the CNS) and thus
will subsequently
be referred to as an astrocytic response. Embodiments of our invention will
alleviate this
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CA 02592285 2007-06-28
=
fibrotic/astrocytic response hence helping in repair and regeneration of
injured tissues and
restoration of tissue and organ structure and function.
Further embodiments of the inventions include the use of said peptides and/or
other
formulations to improve angiogenesis by stimulating angiogenic factors like,
but not limited
to VEGF, and improve differentiation of vascular tissues thereby improving
blood flow to
the site of tissue injury.
Increased blood supply to the wound site stimulated by our treatments will
result in
reduced scarring in external and internal wounds and promote improved repair
and
regeneration of tissues and organs.
Additional embodiments of the invention comprises the use of said peptides
and/or
other formulations for tissue and organ regeneration, when administered in
association with
stem cells and/or drugs and/or other endogenous and/or clinical regimens
promoting stem
cell mobilization and/or tissue regeneration. Stem cells will help in tissue
regeneration and
our treatment will promote differentiation directly and/or indirectly by
processes that
.. include, but are not limited to reduced fibrotic/astrocytic scar formation,
thereby restoring
normal tissue structure and function. Our treatment will promote the
generation of a
permissive environment in vivo for regeneration and restoration of structure
and function of
tissues and organs. Regenerative processes aided by our peptide include, but
are not limited
to internal and external injury, regeneration of tissues, organs, or other
body parts, healing
and restoration of function following vascular occlusion and ischemia, brain
stroke,
myocardial infarction, spinal cord damage, brain damage, peripheral nerve
damage, retinal
damage, bone damage and other insults to tissues causing destruction, damage
or otherwise
resulting from, but not limited to, injury, surgery, cancer, congenital and
developmental
malformation, and diseases causing progressive loss of tissue structure and
function,
.. including but not limited to diabetes, bacterial, viral and prion-
associated diseases,
Alzheimer's disease, Parkinson's disease, AlDs and other genetically
determined,
environmentally determined or idiopathic disease processes causing loss of
tissue/organ/body part structure and function. In addition, we claim that our
peptide can be
administered with drugs or other compounds promoting tissue and cellular
regeneration
.. including, but not limited to, trophic factors in processes including, but
not limited to, brain,
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CA 02592285 2007-06-28
retina, spinal cord and peripheral nervous system regeneration (e.g., NGFs,
FGFs,
Neurtrophins, Neuregulins, Endothelins, GDNFs, BDNF. BMPs, TGFs, Wnts).
Said peptides and/or other formulations can be used in bioengineering
approaches to
tissue and organ repair, regeneration and restoration of structure and
function, including but
not limited to, application with bioengineered delivery vehicles. These
include but are not
limited to, nanoparticles, fibers, gels, polymers, polyethylene glycol and
other bioengineered
materials designed for the purpose of promoting tissue repair and/or targeted
and/or
sustained release of our peptide and tissue scaffolds, polymer matrices and
other
bioengineered surfaces or structures coated or otherwise treated to release,
maintain or
.. localize the effects of said peptides and/or other formulations in
association with the other
beneficial effects or otherwise of these bioenigineered materials.
Additional embodiments of the invention comprise the use of said peptides
and/or
other formulations in vitro and/or in animal models humanized or otherwise to
promote
and/or assist in the regeneration of tissues, organs and body parts for use,
but not limited to
organ/tissue or body part transplantation.
A further embodiment of the invention comprises the use of said peptides
and/or
other formulations to alleviate the symptoms of Multiple Sclerosis (MS). MS is
a chronic
disease of the central nervous system. Pathologically, MS is characterized by
the presence of
areas of demyelination and T-cell predominant perivascular inflammation in the
brain white
matter. The anti-inflammatory and regenerative properties of our treatment
will help in the
treatment of MS and other conditions similar to it. Said peptide will help
with conditions
like, but not limited to psoriasis, scleroderma, acne, eczema and other
diseases of skin and
connective tissues. Psoriasis, a chronic, inflammatory skin disease
characterized by an
uncontrolled shedding of the skin and afflicts millions of people throughout
the world. The
effects of our treatment on fibroblasts and inflammatory response of the
treatments, as stated
in the claims above, will help alleviate Psoriasis. Eczema is characterized by
painful
swelling, oozing of the skin, bleeding cracks, severe scaling, itching and
burning. As stated
above, the effects of our treatment on fibroblasts and inflammatory response,
combined with
accelerated healing properties will relieve symptoms of eczema.
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CA 02592285 2007-06-28
Said peptides and/or other formulations will help with repair after cosmetic
and/or
clinical procedures involving, but not limited to, controlled damage - e.g.,
corneal laser
surgery, laser and dermabrasion/ dermaplaning, skin resurfacing, and punch
excision.
Application of our treatment immediately after surgery or any cosmetic
procedure will
reduce or eliminate scarring. Uses of said peptides and/or other formulations
will reduce
keloid scar formation. Keloid scars are common in dark skin people of Asian,
African, or
Middle Eastern descent. Keloid scar is a thick, puckered, itchy cluster of
scar tissue that
grows beyond the edges of a wound or incision. Keloid scars are sometimes very
nodular in
nature, and they are often darker in color than surrounding skin. They occur
when the body
continues to produce tough, fibrous protein (known as collagen) after a wound
has healed.
Application of our treatment will ameliorate formation of these Keloid scars.
Additional uses of said peptides and/or other formulations will help correct
other
diseases and other conditions (e.g., congenital and developmental defects,
aging) associated
with inflammatory response, fibrosis and connective tissue disorders. Fibrosis
is a common
condition noted after trauma to any bodily organ or tissue. Excessive fibrosis
results in loss
of structure and function and scarring at the trauma site. Our treatment will
reduce fibrosis
and promote regeneration, and restoration of structure and function.
Said peptides and/or other formulations will modulate cell proliferation and
can be
used alone or in association with drugs used in the treatment of uncontrolled
proliferation
(e.g., anti-cancer drugs) and procedures (e.g., radiation therapy). Diseases
of uncontrolled
cell proliferation, or hyperplasias, are common health problems. Examples of
diseases of
cell over-proliferation include but are not limited to psoriasis, seborrhea,
scleroderma,
eczema, benign prostate hyperplasia, congenital adrenal hyperplasia,
endometrial
hyperplasia, squamous cell (vulvular) hyperplasia, sebaceous hyperplasia,
Crohn's Disease,
leukemia, carcinoma, sarcoma, glioma, and lymphoma. Our peptides limits
undesirable
cellular proliferation and will thus improve prognosis of conditions
associated with
excessive cell proliferation.
Said peptides and/or other formulations will have effects on cell migration,
proliferation and differentiation and thus will assist in preventing
metastasis. Said peptides
and/or other formulations can be administered alone or in association with
drugs or
CA 02592285 2007-06-28
procedures used in the treatment of metastasis like but not limited to,
Altretamine,
Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil,
Cisplatin,
Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Diethylstilbesterol,
Ethinyl
estradiol, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide,
Goserelin,
Hydroxyurea, Idarubicin, Ifosfamide, Leuprolide, Levamisole, Lomustine,
Mechlorethamine, Medroxyprogesterone, Megestrol, Melphalan, Mercaptopurine,
Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Paclitaxel, pentastatin,
Pipobroman,
Plicamycin, Prednisone, Procarbazine, Streptozocin, Tamoxifen, Teniposide,
Vinblastine,
Vincristine. Metastasis is the spread of cancer from its primary site to other
places in the
body. Cell migration is the movement of cells from one part of the body to
another. Our
treatments effects on cell migration demonstrates its ability to inhibit
spread of tumors.
Additional embodiments of the invention comprise the delivery of said peptides
and/or other formulations using techniques such as, but not limited to, all
Antennapedia
sequences, and related cell internalization vectors (e.g., TAT protein
transduction domain,
all TAT peptides, all TAT fusion proteins), viral gene delivery vectors, DNA
expression
vectors, and any other delivery method that can help get our peptide to the
tissue and/or
cellular site of action by itself or in association with other agents
including but not limited to
co-factors assisting this delivery (e.g., including but limited to TAT-HA2)
and/or stem cells,
drugs and other formulations which help in repair, regeneration and
restoration of organ and
tissue structure and function.
B. Methods of making the compositions
The compositions disclosed herein and the compositions necessary to perform
the
disclosed methods can be made using any method known to those of skill in the
art for that
particular reagent or compound unless otherwise specifically noted.
For example, the provided nucleic acids can be made using standard chemical
synthesis methods or can be produced using enzymatic methods or any other
known method.
Such methods can range from standard enzymatic digestion followed by
nucleotide fragment
isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd
Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)
Chapters 5,
6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite
method
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CA 02592285 2013-11-15
using a Milligen or Beckman System 1Plus DNA synthesizer (for example, Model
8700
automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model
380B).
Synthetic methods useful for making oligonucleotides are also described by
Ilcuta et al.,
Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester
methods),
and Narang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriester
method). Protein
nucleic acid molecules can be made using known methods such as those described
by
Nielsen etal., Bioconjug. Chem. 5:3-7 (1994).
One method of producing the disclosed polypeptides, such as SEQ ID NO:2, is to
link two or more peptides or polypeptides together by protein chemistry
techniques. For
example, peptides or polypeptides can be chemically synthesized using
currently available
laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc
(tert
-butyloxycarbonoyl) chemistry. (Applied Biosysterns, Inc., Foster City, CA).
One skilled in
the art can readily appreciate that a peptide or polypeptide corresponding to
the disclosed
proteins, for example, can be synthesized by standard chemical reactions, For
example, a
peptide or polypeptide can be synthesized and not cleaved from its synthesis
resin whereas
the other fragment of a peptide or protein can be synthesized and subsequently
cleaved from
the resin, thereby exposing a terminal group which is functionally blocked on
the other
fragment. By peptide condensation reactions, these two fragments can be
covalently joined
via a peptide bond at their carboxyl and amino termini, respectively, to form
a protein, or
fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H.
Freeman and
Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide
Synthesis.
Springer-Verlag Inc., NY).
Alternatively, the peptide or polypeptide is independently
synthesized iin vivo as described herein. Once isolated, these independent
peptides or
polypeptides may be linked to form a peptide or fragment thereof via similar
peptide
= condensation reactions.
For example, enzymatic ligation of cloned or synthetic peptide segments allow
relatively short peptide fragments to be joined to produce larger peptide
fragments,
polypeptides or whole protein domains (Abrahmsen Let al., Biochemistry,
30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can be utilized
to synthetically
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CA 02592285 2007-06-28
construct large peptides or polypeptides from shorter peptide fragments. This
method
consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins
by Native
Chemical Ligation. Science, 266:776-779 (1994)). The first step is the
chemoselective
reaction of an unprotected synthetic peptide¨thioester with another
unprotected peptide
segment containing an amino-terminal Cys residue to give a thioester-linked
intermediate as
the initial covalent product. Without a change in the reaction conditions,
this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a native peptide
bond at the
ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I
et al.,
J.Biol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128
(1991);
Raj arathnam K et al., Biochemistry 33:6623-30 (1994)).
Alternatively, unprotected peptide segments are chemically linked where the
bond
formed between the peptide segments as a result of the chemical ligation is an
unnatural
(non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)). This
technique has been
used to synthesize analogs of protein domains as well as large amounts of
relatively pure
proteins with full biological activity (deLisle Milton RC et al., Techniques
in Protein
Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).
Disclosed are processes for making the compositions as well as the
intermediates
leading to the compositions. There are a variety of methods that can be used
for making
these compositions, such as synthetic chemical methods and standard molecular
biology
methods. It is understood that the methods of making these and the other
disclosed
compositions are specifically disclosed. Disclosed are nucleic acid molecules
produced by
the process comprising linking in an operative way a nucleic acid encoding a
polypeptide
disclosed herein and a sequence controlling the expression of the nucleic
acid. Disclosed are
cells produced by the process of transforming the cell with any of the herein
disclosed
nucleic acids. Disclosed are any of the disclosed peptides produced by the
process of
expressing any of the herein disclosed nucleic acids. Disclosed are animals
produced by the
process of transfecting a cell within the animal with any of the nucleic acid
molecules
disclosed herein. Disclosed are animals produced by the process of
transfecting a cell within
the animal any of the nucleic acid molecules disclosed herein, wherein the
animal is a
mammal. Also disclosed are animals produced by the process of transfecting a
cell within
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CA 02592285 2007-06-28
,
the animal any of the nucleic acid molecules disclosed herein, wherein the
mammal is
mouse, rat, rabbit, cow, sheep, pig, or primate. Also disclose are animals
produced by the
process of adding to the animal any of the cells disclosed herein.
C. Kits
The materials described above as well as other materials can be packaged
together in
any suitable combination as a kit useful for performing, or aiding in the
performance of, the
disclosed method. It is useful if the kit components in a given kit are
designed and adapted
for use together in the disclosed method. For example disclosed are kits for
promoting
wound healing, the kit comprising one or more of the polypeptides, nucleic
acids or vectors
provided herein in a pharmaceutically acceptable carrier. Such kits can also
include gels,
bandages, Millipore tapes, Medicated Q-tips, Sprays, Drops, Syrups, Liquids,
Disposable
tubes or pouches. The kits also can contain instructions for proper use and
safety
information of the product or formulation. The kits may contain dosage
information based
on the application and method of administration as determined by a doctor.
D. Uses
The disclosed methods and compositions are applicable to numerous areas
including,
but not limited to, laboratory research tools. These formulations play
regulatory roles in
several cellular processes for e.g. Cell Proliferation, Cell Migration. These
formulations can
be used in the laboratory in both in vitro and iin vivo model systems for
studying various
cellular processes, cell cycle regulations, cell behavior, responses of cells,
organs or tissues
to test compounds etc. The formulations can be supplied by themselves or in
combination
with other compounds or as part of a kit, such as a kit for cell proliferation
assay. The kit
may contain the formulations mentioned herein by themselves or in combination
with other
compounds. Such a kit would include instructions designed to facilitate the
experiment.
Other uses are disclosed, apparent from the disclosure, and/or will be
understood by those in
the art.
E. Specific Embodiments
Provided herein is a method of promoting healing following tissue injury in a
subject, comprising administering to the subject an isolated polypeptide
comprising the
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CA 02592285 2007-06-28
=
carboxy-terminal amino acid sequence of an alpha Connexin, or a conservative
variant
thereof, wherein the polypeptide does not comprise the full length alpha
Connexin protein.
In some aspects, the disclosed method promotes wound closure. In some aspects,
the
disclosed method reduces scar formation. In some aspects, the disclosed method
promotes
tissue regeneration. In some aspects, the disclosed method increases the
amount of stress or
strain required to break the tissue.In some aspects, the disclosed method
enhances stem cell
differentiation in the tissue.
Also provided is a method of reducing inflammation in a tissue of a subject,
comprising administering to the subject an isolated polypeptide comprising the
carboxy-
terminal amino acid sequence of an alpha Connexin, or a conservative variant
thereof,
wherein the polypeptide does not comprise the full length alpha Connexin
protein.
Also provided is a method of reducing fibrotic tissue formation in a subject,
comprising administering to the subject an isolated polypeptide comprising the
carboxy-
terminal amino acid sequence of an alpha Connexin, or a conservative variant
thereof,
wherein the polypeptide does not comprise the full length alpha Connexin
protein.
In some aspects of the herein disclosed methods, the subject is undergoing
cosmetic
surgery. In some aspects of the herein disclosed methods, the tissue injury
results from a
scrape, cut, incision, laceration, burn, bed sore, body piercing, bite wound,
stab wound,
gunshot wound, surgical wound, stretch injury, crush wound, compression wound,
fracture,
sprain, strain, stroke, infarction, aneurism, herniation, ischemia, fistula,
dislocation,
radiation, cell, tissue or organ grafting, or cancer. The tissue injury can be
a skin injury,
muscle injury, brain injury, eye injury, or spinal cord injury, tissue injury
can be a joint
injury, back injury, heart injury, vascular system injury, soft tissue injury,
cartilage injury,
lymphatic system injury, tendon injury, ligament injury, or abdominal injury.
In some aspects of the disclosed method, the polypeptide comprises from 4 to
30
contiguous amino acids of the carboxy-terminus of the alpha Connexin. In some
aspects,
the polypeptide comprises from 5 to 19 contiguous amino acids of the carboxy-
terminus of
the alpha Connexin.
In some aspects of the disclosed method, the conservative variant comprises a
deletion of one amino acid from the carboxy-terminal amino acid sequence.
CA 02592285 2007-06-28
In some aspects of the disclosed method, the alpha Connexin is selected from a
group consisting of Connexin 30.2, Connexin 31.9, Connexin 33, Connexin 35,
Connexin
36, Connexin 37, Connexin 38, Connexin 39, Connexin 39.9, Connexin 40,
Connexin 40.1,
Connexin 43, Connexin 43.4, Connexin 44, Connexin 44.2, Connexin 44.1,
Connexin 45,
Connexin 46, Connexin 46.6, Connexin 47, Connexin 49, Connexin 50, Connexin
56, or
Connexin 59. Thus, the alpha Connexin can be Connexin 37, Connexin 40,
Connexin 43, or
Connexin 45. Thus, the polypeptide can comprise the amino acid sequence
selected from the
group consisting of SEQ JD NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ 1D NO: 4,
and
SEQ ID NO: 5. Thus, the polypeptide can comprise the amino sequence of SEQ ID
NO: 2.
.. Thus, the polypeptide can comprise an amino acid sequence with at least 65%
sequence
identity to the c-terminal most 9 amino acids of SEQ ID NO: 1. Thus, the
polypeptide can
comprise an amino acid sequence with at least 75% sequence identity to the c-
terminal most
9 amino acids of SEQ ID NO: 1. Thus, the polypeptide can comprise an amino
acid sequence
with at least 85% sequence identity to the c-terminal most 9 amino acids of
SEQ ID NO: 1.
In some aspects of the disclosed method, the polypeptide further comprises a
cellular
internalization sequence. For example, the cellular internalization sequence
can comprise an
amino acid sequence of a protein selected from a group consisting of
Antennapedia, TAT,
HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP
(model
amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynBl, Pep-7, HN-1,
BGSC
(Bis-Guanidinium-Spermidine-Cholesterol and BGTC (Bis-Guanidinium-Tren-
Cholesterol.
Thus, for example, the the amino acid sequence can be from Antermapedia and
comprise the
amino acid sequence SEQ ID NO:7. Thus, the disclosed polypeptide can comprise
the amino
acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9,
SEQ ID
NO:10, SEQ lD NO:11, and SEQ ID NO:12.
Also provided is a method of pre-conditioning a cell or tissue prior to
injury,
comprising administering to the cells or tissue an isolated polypeptide
comprising the
carboxy-terminal amino acid sequence of an alpha Connexin, or a conservative
variant
thereof, wherein the polypeptide does not comprise the full length alpha
Connexin protein.
Provided herein is a composition comprising an isolated polypeptide comprising
the
carboxy-terminal amino acid sequence of an alpha Connexin, or a conservative
variant
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CA 02592285 2007-06-28
=
..
thereof, wherein the polypeptide does not comprise the full length alpha
Connexin protein,
wherein the composition is a formulation for topical administration.
For example, the composition can comprise a collagen polymer. The formulation
can
be an ointment, lotion, cream, or gel. For example, the formulation can be a
pluronic gel.
The formulation can be a poloxamer.
In some aspects of the disclosed composition, the polypeptide comprises from 4
to
30 contiguous amino acids of the carboxy-terminus of the alpha Connexin. In
some aspects
of the disclosed composition, the polypeptide comprises from 5 to 19
contiguous amino
acids of the carboxy-terminus of the alpha Connexin.
In some aspects of the disclosed composition, the alpha Connexin is selected
from a
group consisting of Connexin 30.2, Connexin 31.9, Connexin 33, Connexin 35,
Connexin
36, Connexin 37, Connexin 38, Connexin 39, Connexin 39.9, Connexin 40,
Connexin 40.1,
Connexin 43, Connexin 43.4, Connexin 44, Connexin 44.2, Connexin 44.1,
Connexin 45,
Connexin 46, Connexin 46.6, Connexin 47, Connexin 49, Connexin 50, Connexin
56, or
Connexin 59. Thus, the alpha Connexin can be Connexin 37, Connexin 40,
Connexin 43, or
Connexin 45. Thus, the polypeptide can comprise the amino acid sequence
selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ lD NO: 3, SEQ ID NO: 4,
and
SEQ ID NO: 5. Thus, the polypeptide can comprise the amino sequence of SEQ ID
NO: 2.
Thus, the polypeptide can comprise an amino acid sequence with at least 65%
sequence
identity to the c-terminal most 9 amino acids of SEQ ID NO:1 (SEQ ID NO:2).
Thus, the
polypeptide can comprise an amino acid sequence with at least 75% sequence
identity to the
c-terminal most 9 amino acids of SEQ ID NO:1 (SEQ ID NO:2). Thus, the
polypeptide can
comprise an amino acid sequence with at least 85% sequence identity to the c-
terminal most
9 amino acids of SEQ ID NO:1 (SEQ ID NO:2).
In some aspects of the disclosed composition, the polypeptide further
comprises a
cellular internalization sequence. For example, the cellular internalization
sequence can
comprise an amino acid sequence of a protein selected from a group consisting
of
Antennapedia, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II,
Transportan,
MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynBl, Pep-
7, HN-
1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol and BGTC (Bis-Guanidinium-Tren-
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CA 02592285 2007-06-28
Cholesterol. Thus, for example, the the amino acid sequence can be from
Antennapedia and
comprise the amino acid sequence SEQ ID NO:7. Thus, the disclosed polypeptide
can
comprise the amino acid sequence selected from the group consisting of SEQ ID
NO:8, SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
The disclosed composition can further comprise an antibiotic, steroid,
analgesic,
anti-inflammatory agent, anti-histaminic agent, chemotherapy agent, or other
therapeutic
peptide, or combination thereof.
Also provided is an isolated nucleic acid selected from the group consisting
of the
nucleic acid set forth as SEQ ID NOS: 6, 13, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, and 89.
In some aspects, the disclosed nucleic acid is operably linked to an
expression
control sequence. Also disclosed is a vector comprising a nucleic acid
disclosed herein
operably linked to an expression control sequence. IN some aspects, the vector
is a virus.
Also disclosed is a cell comprising a nucleic acid disclosed herein. Also
disclosed is
a cell comprising a vector disclosed herein. Also disclosed is an organism
comprising a
nucleic acid disclosed herein. Also disclosed is an organism comprising a
vector disclosed
herein.
Also disclosed is a kit comprising a polypeptide comprising the carboxy-
terminal
amino acid sequence of an alpha Connexin, or a conservative variant thereof,
wherein the
polypeptide does not comprise the full length alpha Connexin protein;and a
pharmaceutically acceptable carrier, further comprising gels, bandages,
millipore tapes,
medicated Q-tips, sprays, drops, syrups, liquids, disposable tubes, or
pouches.
Also disclosed is an isolated polypeptide comprising the carboxy-terminal
amino
acid sequence of Connexin 37, or a conservative variant thereof, wherein the
polypeptide
does not comprise the full length Connexin 37 protein. In some aspects, the
polypeptide
comprises from 4 to 30 contiguous amino acids of the carboxy-terminus of
Connexin 37. In
some aspects, the polypeptide comprises from 5 to 19 contiguous amino acids of
the
carboxy-terminus of Connexin 37. In some aspects, the polypeptide comprises an
amino
acid sequence with at least 65% sequence identity to SEQ ID NO:43. In some
aspects, the
polypeptide comprises an amino acid sequence with at least 75% sequence
identity to SEQ
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CA 02592285 2007-06-28
ID NO:43. In some aspects, the polypeptide comprises an amino acid sequence
with at least
85% sequence identity to SEQ ID NO:43.'
Also provided is an isolated polypeptide comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 3, 4, 5,10, 11, and 12.
Also provided is a material coated with a polypeptide comprising the carboxy-
terminal amino acid sequence of an alpha Connexin, or a conservative variant
thereof,
wherein the polypeptide does not comprise the full length alpha Connexin
protein. The
material can be a bio-engineered material. The material can be a tissue
engineered scaffold.
The material can be a medical implant selected from the group consisting of
limb prostheses,
breast implants, penile implants, testicular implants, artificial eyes, facial
implants, artificial
joints, heart valve prosthesis, vascular prostheses, dental prostheses, facial
prostheses, titled
disc valve, caged ball valve, ear prostheses, nose prostheses, pacemakers,
cochlear implants,
and skin substitutes. The material can be selected from the group consisting
of bandages,
steri-strips, sutures, staples, and grafts.
Also provided is a method of inhibiting fibroblast migration at a wound site
comprising administering to the wound site an isolated polypeptide comprising
the carboxy-
terminal amino acid sequence of an alpha Connexin, or a conservative variant
thereof,
wherein the polypeptide does not comprise the full length alpha Connexin
protein.
Also provided is a method of reducing inflammatory cell migration following an
event that generates an inflammatory response comprising administering an
isolated
polypeptide comprising the carboxy-terminal amino acid sequence of an alpha
Connexin, or
a conservative variant thereof, wherein the polypeptide does not comprise the
full length
alpha Connexin protein. In some aspects, the inflammatory cells are
neutrophils.
Also provided is a method of reducing the density of inflammatory cells
following an
event that generates an inflammatory response comprising administering an
isolated
polypeptide comprising the carboxy-terminal amino acid sequence of an alpha
Connexin, or
a conservative variant thereof, wherein the polypeptide does not comprise the
full length
alpha Connexin protein.
Also provided is a method of re-epithelization of a wound site comprising
administering to the wound site an isolated polypeptide comprising the carboxy-
terminal
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CA 02592285 2007-06-28
amino acid sequence of an alpha Connexin, or a conservative variant thereof,
wherein the
polypeptide does not comprise the full length alpha Connexin protein.
Also provided is a method of regenerating hair follicles following injury
comprising
administering an isolated polypeptide to the site of injury comprising the
carboxy-terminal
amino acid sequence of an alpha Connexin, or a conservative variant thereof,
wherein the
polypeptide does not comprise the full length alpha Connexin protein.
Also provided is a method of reducing the density of astrocytes following an
event
that generates astrocytes comprising administering an isolated polypeptide
comprising the
carboxy-terminal amino acid sequence of an alpha Connexin, or a conservative
variant
thereof, wherein the polypeptide does not comprise the full length alpha
Connexin protein.
Also provided is a method of increasing the density of neurons following brain
injury
comprising administering an isolated polypeptide to the site of injury
comprising the
carboxy-terminal amino acid sequence of an alpha Connexin, or a conservative
variant
thereof, wherein the polypeptide does not comprise the full length alpha
Connexin protein.
Also provided is a method of reducing proliferation of transformed cells
comprising
administering an isolated polypeptide comprising the carboxy-terminal amino
acid sequence
of an alpha Connexin, or a conservative variant thereof, wherein the
polypeptide does not
comprise the full length alpha Connexin protein.
Examples
Example 1: In vitro Scratch Injury
Myocytes from neonatal rat hearts were grown until forming a near-confluent
monolayer on a tissue culture dish according to standard protocols. The
cultures were
subsequently allowed to culture for a further 5 days culture medium comprising
30 I.LM ACT
1 peptide (SEQ ID NO:2), 30 pi.M non-active control peptide (SEQ ID NO:55), or
phosphate
buffered saline (PBS) containing no ACT peptide or control peptide. The non-
active control
peptide comprises a polypeptide with a carboxy terminus in which the ACT
peptide
sequence has been reversed. The amino terminus of ACT and control peptides are
both
biotinylated, enabling detection (i.e., assay) of the peptides in the cell
cytoplasm using
standard microscopic or biochemical methods based on high affinity
streptavidin binding to
biotin.
CA 02592285 2007-06-28
Culture media with added peptides or vehicle control was changed every 24
hours
during the experiment. Figure la indicates that ACT peptide greatly increased
the extent of
Cx43 gap junction formation between myocytes relative to the control
conditions (Fig. lb
and 1c). As shown in Figure 4, this increase in Cx43 gap junction formation in
response to
ACT peptide is shared by a number of cell types expressing CX43.
NIH-3T3 cells were grown over 2-3 days until forming a near-confluent
monolayer
on a tissue culture dish according to standard protocols and the monolayer was
then pre-
treated with ACT 1 peptide (SEQ ID NO:2) for 24 hrs, and "scratch-injured"
with a p200
pipette tip. The "scratch injury" was subsequently allowed to repopulate for
24 hours in the
presence of 30 11M ACT 1 peptide (SEQ ID NO:2) dissolved in the culture media
(Fig.2a, b)
or in presence of two control conditions (Fig.2c-0. In the first control
condition, the
"scratch-injured" cells were allowed to repopulate for 24 hours in the
presence of a non-
active control peptide (as in Figure 1) dissolved in the culture media at a
concentration of 30
jiM (Fig.2c, d). In the second control condition, phosphate buffered saline
(PBS) was added
to the culture media and the "scratch-injured" cells were allowed to
repopulate in the
presence of this vehicle control solution containing no ACT peptide or control
peptide
(Fig.2e, 0. The "scratch injury" of ACT peptide-treated cells remains
relatively repopulated
after 24 hours (Fig.2a), with few cells (large arrow) repopulating the area
within the initial
"scratch injury" edges (i.e., within area marked by the small black
arrowheads). By contrast,
in the control conditions in (Fig.2c, e), large numbers of cells (large
arrows) have
repopulated the area within the initial "scratch injury". The repopulation of
the "scratch
injury" occurs in part via migration of the transformed cells crawling into
the "scratch
injury" area. Figures (Fig.2b, d, and 0 show proliferating cell nuclear
antigen (PCNA)
immunolabeling of cells in the "scatch injury" or at the injury edge. ACT
peptide treated
cells (Fig.2b) show only low luminosity consistent with background and non-
proliferation.
Only in the two control conditions shown in figures (Fig.2d, 0, are brightly
labeled
proliferating cells seen (white arrows). This indicates that the ACT peptide
has also reduced
proliferation of the transformed cells in this experimental cellular model.
Figure3a shows the injury edge of ACT peptide and non-active peptide-treated
control cells at the end of the 24-hour period. The cells were labeled with
fluorescent
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CA 02592285 2007-06-28
phalloidin to aid visualization. ACT peptide-treated cells show low levels of
repopulation of
the scratch injury area (white double headed arrows). Figure 3b shows a bar
graph of the %
area of cells repopulating the scratch injury after 24 hours. The reduction of
cells in the
injury area in the presence of ACT peptide is dramatic, with a p value of less
than 0.000001.
WB-F344 cells are a transformed rat epithelial cell line derived by treatment
of
isolated rat liver cells with a cancer-causing agent (Tsao et al., 1984;
Hayashi et al., 1997;
Hayashi et al., 1998; Hayashi et al., 2001). WB-F344 cells were transfected
with a cDNA
expression plasmid construct and selected under antibiotic using standard
protocols to
generate cell lines that stably expressed an ACT-peptide-encoding-
polynucleotide (SEQ ED
NO:6) operably linked to a promoter sequence or a green fluorescent protein
(GFP)
polynucleotide operably linked to a promoter sequence as a control. The
polynucleotide
encoding the ACT peptide also encoded GFP. As such, expression of the ACT
peptide could
be assayed by standard GFP fluorescence optics on a light microscope. Figure
4a, b show
high magnification images of GFP fluorescence in WB-F344 cell lines expressing
GFP
alone (Fig.4a) or GFP plus the carboxy terminus ACT peptide sequence (Fig.4a)
or GFP
alone (Fig.4b). Near confluent monolayers of the WB-F344 cell lines were
"scratch injured"
and allowed to repopulate for 24 hours. Similar to the control cases of the
NIH-3T3 cells
treated with vehicle or non-active control peptide, the control epithelial
cell line expressing
GFP repopulated the scratch injury (Fig.4c). However, in the epithelial cell
line that stably
expressed the ACT-peptide-encoding-polynucleotide operably linked to a
promoter
sequence, there was inhibited repopulation of the scratch injury (Fig.4d). In
addition to WB-
F344 cells lines, NIH-3T3 cell lines have been made that stably express an ACT-
peptide-
encoding-polynucleotide operably linked to a promoter
Example 2: In vivo Wound Healing
Neonatal mouse pups were desensitized using hypothermia. A 4 mm long
incisional
skin injury was made using a scalpel through the entire thickness of the skin
(down to the
level of the underlying muscle) in the dorsal mid line between the shoulder
blades. 30 1 of a
solution of 20 % pluronic (F-127) gel containing either no (control) or
dissolved ACT 1
peptide (SEQ ID NO:2) at a concentration of 60 NI was then applied to the
incisional
.. injuries. Pluronic gel has mild surfactant properties that may aid in the
uniform dispersion of
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CA 02592285 2007-06-28
the ACT peptide in micelles. More importantly, 20% pluronic gel stays liquid
at
temperatures below 15 C, but polymerizes at body temperature (37 C). This
property of
pluronic gel probably aided in the controlled release of peptide into the
tissue at the site of
incisional injury, protecting the peptide from break-down in the protease-rich
environment
of the wound and also enabling active concentrations of the peptide to
maintained over
prolonged periods. Control or ACT peptide containing gel was applied
subsequently 24
hours after the initial application. No further application of control and ACT
peptide
containing gel was made after the second application. By 48 hours it can be
noted that the
ACT peptide treated injury (Fig.5a) is significantly more closed, less
inflamed, less swollen
(note ridges at the wound edge), and generally more healed in appearance than
the control
injury that received no ACT peptide (Fig.5b). These differences in
inflammation, swelling
and healing between the control and ACT peptide and control treated injury
persisted at the
72 (Fig.5c, d) and 96 (Fig.5e, 0 hour time points. At 7 days, the ACT peptide
wound
(Fig.5g), had a smoother and less scarred appearance than the control peptide-
treated injury
(Fig.5h). Note that images of the same injury on the same animal are shown at
the different
time points during the healing time course.
Anesthetized adult mice had 8 mm wide circular excisional skin injuries made
by
fine surgical scissors down to the underlying muscle in the dorsal mid line
between the
shoulder blades (Fig.6a, b). The boundary of the injury was demarcated by an 8
mm wide
circular template cut in a plastic sheet. 100 IA of a solution of 30% pluronic
gel containing
either no (control) or dissolved ACT 1 peptide (SEQ 1D NO:2) at a
concentration of 100 M
was then applied to the excisional injuries. Control or ACT peptide containing
gel was
applied subsequently 24 hours after the initial application. No further
applications of control
and ACT peptide containing gel were made after the second application. The ACT
peptide-
treated large excisional injury (Fig.6a, c, e, g, i) closed faster, was less
inflamed in
appearance, healed faster and scarred less than the control injury that
received no ACT
peptide (Fig.6b, d, f, h, j) over the 14 day time course. Indeed, the control
injury at 14 days
still shows a partial scab indicating that acute healing of the injury was
incomplete (Fig.6j).
Skin biopsies of the entire wound site were taken from some of the 24 hours
following the excisional injury. These skin samples were fixed in 2%
paraformaldehyde,
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CA 02592285 2007-06-28
paraffin¨embedded, sectioned and Hemotoxylin and Eosin (H&E) histochemically
stained
using standard protocols. Figures 7a and 7b show low magnification survey
views of cross-
sections from near the center of the wound of ACT peptide and control treated
injuries,
respectively. The wound edge (marked by the small arrows), bounded by skin of
normal
histological appearance, can be seen in both cases. A black rectangle is
placed over the
images in Figure 7a and 7b at the left hand wound edge. The histological
structures within
the black rectangle placed over the left hand wound edges in Figures 7a and 7b
are shown at
higher magnification in Figure 7c and 7d for ACT peptide and control treated
tissues,
respectively. Of interest is a "collar-like" tissue of aligned fibrous
material (arrowed)
projecting from basal parts of the injury to or toward the wound edge and
exterior surface of
injury. Fibrous material serves as a substrate for migration of inflammatory
cells moving to
the injury surface (Elder et al., 1997). Interestingly, the aligned fibrous
substrate has the
appearance of being much more organized in the control injury (Fig.7d) than in
the ACT
peptide treated injury (Fig.c). Also, there is a considerably lower density of
inflammatory
.. cells studding the fibrous substrate in the ACT peptide-treated tissue.
This is confirmed in
(Fig.7f) and (Fig.7e) where regions of histological section within the black
rectangles shown
in (Fig.7d) and (Fig.7c) are respectively shown at higher magnification. The
inflammatory
cells studding the aligned fibrous substrate include mast cells, neutrophils
and macrophages.
These inflammatory cells occur at much higher density in the control injury
than in the ACT
peptide treated injury.
At the end of the 14 day period, skin biopsies of the entire excisional injury
were
taken and histological sections from these skin samples were H&E
histochemically stained.
Figures 8a and 8b show low magnification survey views of cross-sections from
near the
center of the injury of ACT peptide and control, respectively. The wound edge
(marked by
the small arrows), bounded by skin of normal histological appearance, can be
seen in both
cases. A black rectangle is placed over the images in Figures 8a and 8b near
the center of
each injury. The histological structures within these two rectangles are shown
at higher
magnification in Figures 8c and 8d for the ACT peptide and control tissues,
respectively. It
is evident that tissue within the ACT peptide treated injury locus has
considerably more
.. complexity. At the external surface of the ACT treated wound, there is a
continuous layer of
74
CA 02592285 2007-06-28
=
epithelial cells indicating that re-epithelization of the injured surface is
complete, albeit that
the epithelium is as yet relatively thin near the center of the wound
(Fig.8c). Unusually,
regenerating hair follicles can already be seen differentiating de novo from
stem cells in the
new epithelium covering the healed injury (Fig.8c, small arrows). By
comparison, re-
epithelization of the injury surface is incomplete and there is no sign of
regenerating hair
follicles in the epithelium of the control injury. Beneath the reformed
epithelium of the ACT
peptide treated injured skin, considerable restoration of normal structural
complexity is seen,
with glandular structures, fibrous and connective tissues, vascular tissues,
muscle and fat
cells all in evidence (Fig.8a, c). As with the hair follicles, this tissue
complexity was
regenerated by differentiation of stem cells. By contrast, in the control
injury the wound
tissue is completely dominated by a uniform and large plug of fibrous scar
tissue (Fig.8b, d),
with other complexity of tissue structure not particularly in evidence within
this scar tissue.
Anesthetized adult mice had 2 small (5mm diameter) excisional skin wounds made
by fine surgical scissors on the neck and (upper) back. The boundaries of the
injuries were
demarcated by a 5 mm wide circular template cut in a plastic sheet. 50-60 1
of a solution of
30 % pluronic gel containing either no (control) or one of the ACT peptides
(ACT 2 - SEQ
ID NO:1, ACT 1 - SEQ ID NO:2, ACT 3 - SEQ ID NO:3, ACT 4 - SEQ ID NO:4, ACTS-
SEQ ID NO:5) dissolved at concentrations of 100 0,4 were then applied to the
excisional
injuries. Control or ACT peptide-containing gels were applied subsequently 24
hours after
the initial application. No further applications of control and ACT peptide-
containing gel
were made after the second application. It can be noted in the case of ACT 1
(Fig.9e-h),
ACT 2 (Fig.9i-l), ACT 3 (Fig.9m-p), and ACT 5 (Fig.9u-x) peptides that
excisional injuries
closed faster, were less inflamed in appearance, healed faster and scarred
less than the
control injury that received no ACT peptide (Fig.9a-d) over the 240 hour time
course (10
days). The ACT 4 peptide (Fig.9q-t) also appeared to show modest improvement
in healing
over the control during the time course. Note that the same wound on the same
animal is
shown at the different time points during the healing time course.
The area of open wound was measured during the time course using NIH image
according to standard protocols on multiple (-5 mice per control or treatment
condition)
adult mice. These individual area measurements were then normalized to (i.e.,
divided by)
CA 02592285 2007-06-28
the average area measured for the control injuries for a given time point,
multiplied by 100
to give a % of unclosed wound relative to the control and then plotted against
time. A Mann-
Whitney U-test was used to statistically assess the effects of ACT peptides
over the time
course. ACT 1, ACT 2, ACT 3, and ACT 5 peptides significantly improved wound
closure
rates following excisional injury. These treatments provided results with
significant p
values. The ACT 1 and ACT 3 quantifiably gave the most pronounced improvements
over
the control. A more modest, although consistent, improvement was also observed
for the
ACT 4 peptide over the control.
Anesthetized adult rats were positioned in a stereota.xic apparatus. A midline
incision
was made on the scalp to expose the skull. A stereotaxic drill was sighted 2
mm posterior to
the bregma and 2 holes were drilled with a 1 mm spherical bit, each at 2.5 mm
to the right
and left of the bregma, and 3.5 mm below the dura. A cerebral lesion was made
by inserting
an 18-gauge needle. The coordinates were determined from the atlas by Paxinos
and Watson
(1986). The hollow fiber membrane (HFM) was inserted in the hole and external
skin
sutures were placed to cover the stab. The ACT peptide was dissolved at 100
tEM
concentration in a 2 % collagen vehicle solution contained within the HFM.
Studies of
isolated HFMs indicated that these bioengineered constructs were capable of
slow release of
detectable levels of ACT peptide (as assayed by biotin-streptavidin reaction)
in aqueous
solutions for periods of at least 7 days. Reactive astrocytosis associated
with inflammation
and subsequently with glial scar formation follows a well characterized time
course after
brain injury in rodent models (Norenberg, 1994; Fawcett and Asher, 1999).
Typically, the
astrocytic response in rat brain peaks after a week, together with loss of
neurons and other
aspects of brain tissue complexity. Subsequently with the emergence of glial
scar tissue, the
density of GFAP-positive astrocytes decreases. Figures 10b and 10c show low
magnification
survey views of sections of brain tissue (cortex) surrounding HFM implants
filled with ACT
peptide plus vehicle gel or control collagen vehicle gel or ACT peptide plus
vehicle gel a
week following brain penetration injury. In the control tissue (Fig.10c), a
high density of
immunolabeled GFAP-positive astrocytes is observed near the site of injury
caused by the
HFM. The density of these cells appears to diminish slightly distal from the
injury. By
contrast, a much lower density of GFAP-positive astrocytes is observed
adjacent the HFM
76
CA 02592285 2007-06-28
filled with ACT peptide (Fig.10b). Indeed, the levels of GFAP positive cells
are not
dissimilar to those seen in normal uninjured brain tissue. The regions of
tissue within the
white rectangles in Figures 10b and 10c are shown at higher magnification in
Figures 10d
and 10e, respectively. In the brain injury treated by ACT peptide (Fig. 10d),
it can be seen
that GFAP-positive astrocytes are not only less numerous, but are also smaller
than those
seen in the control injury (Fig. 10e).
Figures 1 1 a and 1 lb show low magnification survey views of sections of
brain tissue
(cortex) surrounding HFM implants (implant or injury border is shown by
arrows) filled
with control collagen vehicle gel (Fig.11b) or ACT peptide plus vehicle gel
(Fig.11a) at 1
week following brain penetration injury. In the control tissue (Fig.11b), a
high density of
immunolabeled GFAP-positive astrocytes and low density of NeuN immunolabeled
neurons
are observed near the site of injury caused by the HFM. The density of these
cells appears to
diminish and increase distal from the HFM, respectively. By contrast, a much
lower density
of GFAP-positive astrocytes and higher numbers NeuN immunolabeled neurons are
observed proximal (as well as distal) to the HFM filled with ACT peptide
((Fig.11a). The
areas in Figures lla and llb proximal to the HFMs are shown at high
magnification views
of in Figures 11c and 11d, respectively. Again, in the control tissue (Fig.
lid) a striking
increase in the density of GFAP-positive astrocytes and a reduced density of
NeuN-positive
neurons is observed compared to ACT peptide treated tissueseen (Fig.11c). A
complementary pattern is observed near the HFM containing ACT peptide, with
NeuN
positive neurons predominating over astrocytes (Fig.11c). Interestingly, the
high
magnification view shown in Figure lid reveals a high frequency of neurons in
the process
of fission relative to the control (Fig.11c). This suggests that the high
density of neurons
associated with ACT peptide treatment may be from generation of new neurons.
ACT
peptide can also increase neuronal density in part by sparing neurons from
cell death
following brain injury.
Example 3: Treatment of Acute Spinal Cord Injury
Subjects with acute spinal cord injuries represent a seriously problematic
group for
whom even a small neurological recovery of function can have a major influence
on their
subsequent independence. In one example, a subject with acute spinal cord
injury receives a
77
CA 02592285 2007-06-28
bolus infusion of a 0.02% to 0.1 % solution of ACT peptide (e.g., SEQ ID NO:1)
over 15
min within 8 h directly into the site of acute spinal cord injury, followed 45
min later by an
infusion of 0.01 % solution of ACT peptide for a subsequent 23 to 48 hours. In
another
example, ACT peptide is used to coat slow release nanoparticles loaded within
8 h directly
into the site of acute spinal cord injury or tissue engineered bioscaffolds
designed to
promote neural reconnection across the zone of acute spinal cord lesion.
Improvement in
function are assessed by a doctor at intervals (e.g., 6, 12, 26 and 52 weeks)
following
treatment by neurological outcome tests including assessments designed to
measure motor
activity, pinprick skin sensitivity and recovery of sensation.
Example 4: Quantitative assessment of wound closure, tissue regeneration,
and tensile strength of excisional skin wounds
ACT peptide (n = 12) and control (n = 8) 5 mm-diameter excisional skin wounds
were generated on adult mice as described above. Quantitative assessments of
wound
closure rate, counts of regenerated hair follicles and tensile strength
measurements were then
undertaken on the wounded skin at time points up to 90 days following the
initial insult.
Relative to control wounds, closure was significantly enhanced within 24 hours
of peptide
treatment. Similarly, at 10 days, when most wounds were nearing completion of
closure, a
highly significant difference was still maintained such that ACT peptide-
treated wounds
were on average 43 % smaller than control wounds. At 10 days ACT peptide
wounds
showed a significant 3.2 fold increase in the number of regenerated hair
follicles per unit
area of the healed wound over control wounds.
Studies were undertaken of the mechanical properties of healed 5 mm diameter
excisional wounds at 1 month and 3 months following injury. For mechanical
property
measurements, the skin samples were obtained after sacrificing the animal and
evaluated
using a MTS 858 Mini Bionix (MTS Systems Corporation, MN, USA) equipped with a
5 kg
load cell. During measurement the skin sample was extended to break at a rate
of 0.5 mm/s.
Force and extension was measured at break. The tensile strength (stress) and
extension to
break (strain) was calculated as follows, Stress (N/mm2) = Force at break (N)
/ cross-
sectional area of sample (mm2). Strain (%) = [Increase in length at break
(mm)! Original
78
CA 02592285 2007-06-28
length (mm)] x 100. Stress and strain calculations for each wounded skin
sample was
normalized to a normal skin sample from a nearby area collected from the same
animal.
At 1 month, the stress (i.e., normalized force) required to break wounded skin
was
similar to that of control wounded skin. At 3 months, normalized stress to
break of peptide-
treated wound skin was on average double that of control wounded skin,
although the high
variance within the treatment group precluded significant mean separation from
the control.
This result demonstrates that the intrinsic tensile strength of peptide-
treated wounds was as
good or better than that of untreated wounds. Further, significant
improvements in
extensibility of peptide-treated wounds were found. The amount of strain
(i.e., extensibility)
required to break peptide-treated wounds was modestly improved over control
wounds at 1
month. At 3 months, peptide-treated wounds showed a more striking improvement,
increasing to a near normal 90 % of unwounded skin. By contrast, control
wounds at 3
months remained only 60 % as extensible as normal skin.
It is understood that the disclosed method and compositions are not limited to
the
particular methodology, protocols, and reagents described as these may vary.
It is also to be
understood that the terminology used herein is for the purpose of describing
particular
embodiments only, and is not intended to limit the scope of the present
invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a
", "an", and "the" include plural reference unless the context clearly
dictates otherwise.
Thus, for example, reference to "a polypeptide" includes a plurality of such
polyp eptides,
reference to "the polypeptide" is a reference to one or more polypeptides and
equivalents
thereof known to those skilled in the art, and so forth.
"Optional" or "optionally" means that the subsequently described event,
circumstance, or material may or may not occur or be present, and that the
description
includes instances where the event, circumstance, or material occurs or is
present and
instances where it does not occur or is not present.
Ranges may be expressed herein as from "about" one particular value, and/or to
"about" another particular value. When such a range is expressed, also
specifically
contemplated and considered disclosed is the range from the one particular
value and/or to
79
CA 02592285 2007-06-28
the other particular value unless the context specifically indicates
otherwise. Similarly,
when values are expressed as approximations, by use of the antecedent "about,"
it will be
understood that the particular value forms another, specifically contemplated
embodiment
that should be considered disclosed unless the context specifically indicates
otherwise. It
will be further understood that the endpoints of each of the ranges are
significant both in
relation to the other endpoint, and independently of the other endpoint unless
the context
specifically indicates otherwise. Finally, it should be understood that all of
the individual
values and sub-ranges of values contained within an explicitly disclosed range
are also
specifically contemplated and should be considered disclosed unless the
context specifically
indicates otherwise. The foregoing applies regardless of whether in particular
cases some or
all of these embodiments are explicitly disclosed.
Unless defined otherwise, all technical and scientific terms used herein have
the
same meanings as commonly understood by one of skill in the art to which the
disclosed
method and compositions belong. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or testing of
the present
method and compositions, the particularly useful methods, devices, and
materials are as
described. Publications cited herein and the material for which they are cited
are hereby
specifically incorporated by reference. Nothing herein is to be construed as
an admission
that the present invention is not entitled to antedate such disclosure by
virtue of prior
invention. No admission is made that any reference constitutes prior art. The
discussion of
references states what their authors assert, and applicants reserve the right
to challenge the
accuracy and pertinency of the cited documents. It will be clearly understood
that, although
a number of publications are referred to herein, such reference does not
constitute an
admission that any of these documents forms part of the common general
knowledge in the
art.
Throughout the description and claims of this specification, the word
"comprise" and
variations of the word, such as "comprising" and "comprises," means "including
but not
limited to," and is not intended to exclude, for example, other additives,
components,
integers or steps.
CA 02592285 2013-11-15
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G. Sequences
SEQ ID NO:1 (ACT 2)
PSSRASSRASSRPRPDDLEI
SEQ ID NO:2 (ACT 1)
RPRPDDLEI
SEQ ID NO:3 (ACT 3)
RPRPDDLEV
SEQ ID NO:4 (ACT 4)
RPRPDDVPV
84
CA 02592285 2007-06-28
SEQ ID NO:5 (ACT 5)
KARSDDLSV
SEQ ID NO:6
aga cct cgg cct gat gac ctg gag att
SEQ ID NO:7 (Antp)
RQPKIWFPNRRKPWKK
SEQ ID NO:8 (Antp/ ACT 2)
RQPKIWFPNRRKPWKKPSSRASSRASSRPRPDDLEI
SEQ ID NO:9 (Antp/ ACT 1)
RQPKIWFPNRRKPWKKRPRPDDLEI
SEQ ID NO:10 (Antp/ ACT 3)
RQPKIWFPNRRKPWKKRPRPDDLEV
SEQ ID NO:11 (Antp/ ACT 4)
RQPKIWFPNRRKPWKKRPRPDDVPV
SEQ ID NO:12 (Antp/ ACT 5)
RQPKIWFPNRRKPWKKKARSDDLSV
SEQ ID NO:13 (encodes polypeptide of SEQ ID NO 9)
cgg cag ccc aag atc tgg ttc ccc aac cgg cgg aag ccc tgg aag aag cgg ccc ggc
ccg acg acc tgg aga tc
SEQ ID NO:14 (HIV-Tat)
GRKKRRQRPPQ
SEQ ID NO:15 (Penetratin)
RQIKIWFQNRRMKWKK
SEQ ID NO:16 (Antp-3A)
RQIAIWFQNRRMKWAA
SEQ ID NO:17 (Tat)
RKKRRQRRR
SEQ ID NO:18 (Buforin II)
TRSSRAGLQFPVGRVHRLLRK
SEQ ID NO:19 (Transportan)
GWTLNSAGYLLGKINKALAALAKKIL
SEQ ID NO:20 (model amphipathic peptide)
KLALKLALKALKAALKLA
SEQ ID NO:21 (K-FGF)
AAVALLPAVLLALLAP
SEQ ID NO:22 (Ku70)
VPMLK- PMLKE
85
CA 02592285 2007-06-28
,
SEQ ID NO:23 (Prion)
MANLGYWLLALFVTMWTDVGLCKKRPKP
SEQ ID NO:24 (pVEC)
LLIILRRRIRKQAHAHSK
SEQ ID NO:25 (Pep-1)
KETVVWETVVWTEVVSQPKKKRKV
SEQ ID NO:26 (SynB1)
RGGRLSYSRRRFSTSTGR
SEQ ID NO:27 (Pep-7)
SDLWEMMMVSLACQY
SEQ ID NO:28 (HN-1)
TSPLNIHNGQKL
SEQ ID NO:29 (Chick alpha Cx43 ACT)
PSRASSRASSRPRPDDLEI
SEQ ID NO:30 (Human alpha Cx45)
GSNKSTASSKSPDPKNSVWI
SEQ ID NO:31 (Chick alpha Cx45)
GSNKSSASSKSGDGKNSVWI
SEQ ID: 32 (Human alpha Cx46)
GRASKASRASSGRARPEDLAI
SEQ ID: 33 (Human alpha Cx46.6)
GSASSRDGKTVWI
SEQ ID NO:34 (Chimp alpha Cx36)
PRVSVPNFGRTQSSDSAYV
SEQ ID NO:35 (Chick alpha Cx36)
PRMSMPNFGRTQSSDSAYV
SEQ ID NO:36 (Human alpha Cx47)
PRAGSEKGSASSRDGKITVW1
SEQ ID NO:37 (Human alpha Cx40)
GYHSDKRRLSKASSKARSDDLSV
SEQ ID NO:38 (Human alpha Cx50)
PLSRLSKASSRARSDDL'TV
SEQ ID NO:39 (Human alpha Cx59)
PNHVVSLTNNLIGRRVPTDLQI
SEQ ID NO:40 (Rat alpha Cx33)
PSCVSSSAVLTTICSSDQVVPVGLSSFYM
SEQ ID NO:41 (Sheep alpha Cx44)
86
CA 02592285 2007-06-28
=
GRSSKASKSSGGRARAADLAI
SEQ ID NO:42 (Human beta Cx26)
LCYLLIRYCSGKSKKPV
SEQ ID: 43 (Human alpha Cx37)
G QK PP SRPS SSAS K KQ*YV
SEQ ID 44: (conservative Cx43 variant)
SSRASSRASSRPRPDDLEV
SEQ ID 45: (conservative Cx43 variant)
RPKPDDLEI,
SEQ ID 46: (conservative Cx43 variant)
SSRASSRASSRPKPDDLEI,
SEQ ID 47: (conservative Cx43 variant)
RPKPDDLDI
SEQ ID 48: (conservative Cx43 variant)
SSRASSRASSRPRPDDLDI
SEQ ID 49: (conservative Cx43 variant)
SSRASTRASSRPRPDDLEI
SEQ ID 50: (conservative Cx43 variant)
RPRPEDLEI
SEQ ID 51: (conservative Cx43 variant)
SSRASSRASSRPRPEDLEI,
SEQ ID 52: (conservative Cx45 variant)
GDGKNSVWV
SEQ ID 53: (conservative Cx45 variant)
SKAGSNKSTASSKSGDGKNSVWV
SEQ ID 54: (conservative Cx37 variant)
GQKPPSRPSSSASKKLYV
SEQ ID NO: 55 (non-active control peptide)
RQPKIWFPNRRKPWKIELDDPRPR
SEQ ID NO:56 (HIV-Tat/ ACT 1)
GRKKRRQRPPQ RPRPDDLEI
SEQ ID NO:57 (Penetratin/ ACT 1)
RQIKIWFQNRRMKWKK RPRPDDLEI
SEQ ID NO:58 (Antp-3N ACT 1)
RQIAIWFQNRRMKWAA RPRPDDLEI
SEQ ID NO:59 (Tat/ ACT 1)
RKKRRQRRR RPRPDDLEI
87
CA 02592285 2007-06-28
SEQ ID NO:60 (Buforin II/ ACT 1)
TRSSRAGLQFPVGRVHRLLRK RPRPDDLEI
SEQ ID NO:61 (Transportan/ ACT 1)
GWTLNSAGYLLGKINKALAALAKKIL RPRPDDLEI
SEQ ID NO:62 (MAP/ ACT 1)
KLALKLALKALKAALKLA RPRPDDLEI
SEQ ID NO:63 (K-FGF/ ACT 1)
AAVALLPAVLLALLAP RPRPDDLEI
SEQ ID NO:64 (Ku70/ ACT 1)
VPMLKPMLKE RPRPDDLEI
SEQ ID NO:65(Prion/ ACT 1)
MANLGYWLLALFVTMWTDVGLCKKRPKP RPRPDDLEI
SEQ ID NO:66 (pVEC/ ACT 1)
LLIILRRRIRKQAHAHSK RPRPDDLEI
SEQ ID NO:67 (Pep-1/ ACT 1)
KETVVWETVVWTEWSQPKKKRKV RPRPDDLEI
SEQ ID NO:68 (SynB1/ ACT 1)
RGGRLSYSRRRFSTSTGR RPRPDDLEI
SEQ ID NO:69 (Pep-7/ ACT 1)
SDLWEMMMVSLACQY RPRPDDLEI
SEQ ID NO:70 (HN-1/ ACT 1)
TSPLNIHNGQKL RPRPDDLEI
SEQ ID NO: 72 ( 20 to 120 residues flanking amino acid 363 of human Cx43)
KGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVT
GDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDD
NQNSKKLAAGHELQPLAIVD
SEQ ID NO: 73 ( 20 to 120 residues flanking amino acid 362 of chick Cx43)
KTDPYSHSGTMSPSKDCGSPKYAYYNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASE
QNWANYSAEQNRMGQAGSTISNSHAQPFDFADEHQNTKKLASGHELQPLTIVDQRP
SEQ ID NO: 74 ( 20 to 120 residues flanking amino acid 377 of human Cx45)
LGFGTIRDSLNSKRRELEDPGAYNYPFTWNTPSAPPGYNIAVKPDQIQYTELSNAKIAYKQNKANTA
QEQQYGSHEENLPADLEALQREIRMAQERLDLAVQAYSHQNNPHGPREKKAKV
SEQ ID NO: 75 ( 20 to 120 residues flanking amino acid 375 of chick Cx45)
GFGTIRDTLNNKRKELEDSGTYNYPFTVVNTPSAPPGYNIAVKPDQMQYTELSNAKMAYKQNKANIA
QEQQYGSNEENIPADLENLQREIKVAQERLDMAIQAYNNQNNPGSSSREKKSKA.
SEQ ID NO: 76 ( 20 to 120 residues flanking amino acid 313 of human Cx37)
PYLVDCFVSRPTEKTIFIIFMLVVGLISLVLNLLELVHLLCRCLSRGMRARQGQDAPPTQGTSSDPYT
DQVFFYLPVGQGPSSPPCPTYNGLSSSEQNWANLTTEERLASSRPPLFLDPP
88
CA 02592285 2007-06-28
,
.
. SEQ ID NO: 77 ( 20 to 120 residues flanking amino acid 258 of rat Cx33)
CGSKEHGNRKMRGRLLLTYMASIFFKSVFEVAFLLIQWYLYGFTLSAVYICEQSPCPHRVDCFLSRP
TEKTIFILFMLVVSMVSFVLNVIELFYVLFKAIKNHLGNEKEEVYCNPVELQK.
SEQ ID NO: 78 (enhanced green fluorescent protein)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
SEQ ID NO:79 (ACT 2)
CCCTCCTCCCGGGCCTCCTCCCGGGCCTCCTCCCGGCCCCGGCCCGAC GACCTGGAGATC
SEQ ID NO:80(ACT 1)
CGGCCCCGGCCCGACGACCTGGAGATC
SEQ ID NO:81 (ACT 3)
CGGCCCCGGCCCGACGACCTGGAGGTG
SEQ ID NO:82 (ACT 4)
CGGCCCCGGCCCGACGACGTGCCCGTG
SEQ ID NO:83 (ACT 5)
AAGGCCCGGTCCGACGACCTGTCCGTG
SEQ ID NO:84 (Antp)
CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAG AAG
SEQ ID NO:85 (Antp/ ACT 2)
CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAG
AAGCCCTCCTCCCGGGCCTCCTCCCGGGCCTCCTCCCGGCCCCGGCCC
GACGACCTGGAGATC
SEQ ID NO:86 (Antp/ ACT 1)
CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGA
CGACCTGGAGATC
SEQ ID NO:87 (Antp/ ACT 3)
CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGA
CGACCTGGAGGTG
SEQ ID NO:88 (Antp/ ACT 4)
CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGCGGCCCCGGCCCGA
CGACGTGCCCGTG
SEQ ID NO:89 (Antp/ ACT 5)
CGGCAGCCCAAGATCTGGTTCCCCAACCGGCGGAAGCCCTGGAAGAAGAAGGCCCGGTCCGA
CGACCTGTCCGTG
SEQ ID NO:90 (Zebrafish alpha Cx43)
PCSRASSRMSSRARPDDLDV
SEQ ID NO:91 (Chick alpha Cx36)
PRVSVPNFGRTQSSDSAYV
89
