Note: Descriptions are shown in the official language in which they were submitted.
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The present invention is directed to adenoviral vectors having modified capsid
proteins which comprise heterologous ligands that improve and/or alter the
infectious
capability of the vector. Such ligands are capable of binding to target cells,
and their
inclusion into adenoviral vectors facilitates the binding and infectious
properties of the
vectors. The invention is also directed to compositions comprising the
adenoviral vectors
of the invention and methods for the use of these adenoviral vectors to
deliver transgenes
to target cells.
Adenovirus (Ad) is a nuclear DNA virus with a genome size of about 36 kb,
which has been well-characterized through studies in classical genetics and
molecular
biology. A detailed discussion of adenovirus is found in Shenk, T.,
"Adenoviridae and
their Replication", and Horwitz, M.S., "Adenoviruses", Chapters 67 and 6$,
respectively,
in ViroloQV, B.N. Fields et al., eds., 2nd edition, Raven Press, Ltd., New
York, 1996, and
reference therein is found to numerous aspects of adenovirus pathology,
epidemiology,
1 S structure, replication, genetics and classification.
In a simplified form, the adenoviral genome is classified into early (known as
E1-
E4) and late (known as L1-LS) transcriptionai units, referring to the
generation of two
temporal classes of viral proteins. The demarcation between these events is
viral DNA
replication.
The human adenoviruses are divided into numerous serotypes (approximately 47,
numbered accordingly and classified into 6 subgroups: A, B, C, D, E and F),
based upon
properties including hemagglutination of red blood cells, oncogenicity, DNA
and protein
compositions and relatedness, and antigenic relationships.
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Recombinant adenoviruses have several advantages for use as gene transfer
vectors, including tropism for both dividing and non-dividing cells, minimal
pathogenic
potential, ability to replicate to high titer for preparation of vector
stocks, and the
potential to carry large inserts (Berkner, K.L., Curr. Top. Micro. Immunol.
158:39-66,
S 1992; Jolly, D., Cancer Gene Therapy 1:51-64, 1994).
The cloning capacity of an adenovirus vector is proportional to the size of
the
adenovirus genome present in the vector. For example, a capacity of about 8 kb
can be
created from the deletion of certain regions of the virus genome dispensable
for virus
growth, e.g., E3, and the deletion of a genomic region such as E1 whose
function may be
restored in trans from 293 cells (Graham, F.L., J. Gen. Virol. 36:59-72, 1977)
or A549
cells (Imler et al., Gene Therapy 3:75-84, 1996). Such E1-deleted vectors are
rendered
replication-defective, which is desirable for the engineering of adenoviruses
for gene
transfer. The upper limit of vector DNA capacity for optimal carrying capacity
is about
105%-108% of the length of the wild-type genome. Further adenovirus genomic
modifications are possible in vector design using cell lines which supply
other viral gene
products in traps, e.g., complementation of E2a (Zhou et al., J. Virol.
70:7030-7038,
1996), complementation of E4 (Krougliak et al., Hum. Gene Ther. 6:1575-1586,
1995;
Wang et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX
(Caravokyri
et al., J. Virol. 69:6627-6633, 1995; Krougliak et al., Hum. Gene Ther. 6:1575-
1586,
1995). Maximal carrying capacity can be achieved using adenoviral vectors
deleted for
all viral coding sequences (allowed U.S. Patent Application No. 08/895,194;
Kochanek et
al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology
217:11-22,
1996).
Transgenes that have been expressed to date by adenoviral vectors include,
inter
alia, p53 (Wills et al., Human Gene Therapy 5:1079-188, 1994); dystrophin
(Vincent et
al., Nature Genetics 5:130-134, 1993; erythropoietin (Descamps et al., Human
Gene
Therapy 5:979-985, 1994; onnithine transcarbamylase (Stratford-Perricaudet et
al.,
Human Gene Therapy 1:241-256, 1990; We et al., J. Biol. Chem. 271;3639-3646,
1996;);
adenosine deaminase (Mitani et al., Human Gene Therapy 5:941-948, 1994);
interleukin-2 (Haddada et al., Human Gene Therapy 4:703-711, 1993); and al-
antitrypsin
(Jaffe et al., Nature Genetics 1:372-378, 1992); thrombopoietin (Ohwada et
al., Blood
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88:778-784, 1996); and cytosine deaminase (Ohwada et al., Hum. Gene Ther.
7:1567-
1576, 1996).
The particular tropism of adenoviruses for cells of the respiratory tract has
particular relevance to the use of adenovirus in gene therapy for cystic
fibrosis (CF),
which is the most common autosomal recessive disease in Caucasians. The
disease is
caused by the presence of one or more mutations in the gene that encodes a
protein
known as cystic fibrosis transmembrane conductance regulator (CFTR), and which
regulates the movement of ions (and therefore fluid) across the cell membrane
of
epithelial cells, including lung epithelial cells. Abnormal ion transport in
airway cells
IO leads to abnormal mucous secretion, inflammmation and infection, tisssue
damage, and
eventually death. Mutations in the CFTR gene that disturb the cAMP-regulated
CY
channel in airway epithelia result in pulmonary dysfunction (Zabner et al.,
Nature
Genetics 6:75-83, 1994). Adenovirus vectors engineered to carry the CFTR gene
have
been developed (Rich et al., Human Gene Therapy 4:461-476, 1993) and studies
have
shown the ability of these vectors to deliver CFTR to nasal epithelia of CF
patients
(Zabner et al., Cell 75:207-216, 1993), the airway epithelia of cotton rats
and primates
(Zabner et ai., Nature Genetics 6:75-83, 1994), and the respiratory epithelium
of CF
patients (Crystal et al., Nature Genetics 8:42-51, 1994). Recent studies have
shown that
administering an adenoviral vector containing a DNA encoding CFTR to airway
epithelial cells of CF patients can restore a functioning chloride ion channel
in the treated
epithelial cells (Zabner et al., J. Clin. Invest. 97:1504-151 l, 1996; U.S.
Patent No.
5,670,488 issued September 23, 1997).
Adenoviruses are nonenveloped, regular icosahedrons (having 20 triangular
surfaces and 12 vertices) that are about 65-80 nm in diameter. A protein
called fiber
projects from each of the vertices. The fiber protein is itself generally
composed of 3
identical polypeptide chains, although the length thereof can vary from
serotype to
serotype. The protein coat (capsid) is composed of 252 subunits (capsomeres),
of which
240 are hexons, and 12 are pentons. Each penton comprises a penton base, on
the surface
of the capsid, and a fiber protein projecting from the base. The Ad 2 penton
base protein,
for example, has been determined to be an 8 x 9 nm ring shaped complex
composed of 5
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identical protein subunits of 571 amino acids each. Adenovirus protein IX is
also located
on the surface of the viral capsid.
Current understanding of adenovirus-cell interactions suggests that adenovirus
utilizes two cellular receptors to attach to and, then, infect a target cell.
It has been
suggested that the fiber protein of an infecting adenovirus first binds with
high affinity to
a cellular receptor; subsequently, the viral penton base interacts with
cellular alpha-
integrins, leading to viral endocytosis. As presently understood, adenovirus
enters cells,
e.g., in the respiratory tract, by attaching via the fiber to a cell surface
receptor (known as
CAR for Coxsackie adenovirus receptor) on the cell membrane of the host cell.
The virus
thus attached to its receptor migrates into the cell, within the plasma
membrane to
clathrin-coated pits, which form endocytic vesicles or receptosomes (Shenk,
T.,
"Adenoviridae and Their Replication", in Vir~, 2nd ed., Fields et al., eds.,
Raven
Press, New York, 1996). The carboxy-terminus knob portion of the fiber protein
functions as the ligand that binds to its cellular receptor (Xia et al., Curr.
Top. Micro.
Immunol. 199:40-46, 1995; Xia et al., Structure 2:1259-1270, 1995; Henry et
al., J.Virol.
68:5239-5246, 1994; Roelvink et al., J.Virol. 70:7614-7621, 1996; Fender et
al., Virology
214:110-117, 1995).
It has been determined that alpha-integrins often recognize short amino acid
sequences on other cellular proteins for attachment proposes, including the
tripeptide
sequence Arg-Gly-Asp (abbreviated RGD). An RGD sequence is also found in the
penton base protein of adenovirus and is currently understood in the art to
mediate the
interaction of adenovirus with alpha-integrins (Mathias et al., J.Virol.
68:6811-6814,
1994; Wickham et al., J.Cell Biol. 127:257-264, 1994; Wickham et al., Cell
73:309-319,
1993; Goldman et al., J. Virol. 69:5951-5958, 1995). Once inside the cell,
viral particles
are transported to the nuclear membrane, where the viral DNA is released from
the virion
and enters the nucleus through the nuclear pores. Hexon proteins remain
associated with
the viral DNA in relatively intact particles up until the time of release of
the DNA into
the cell nucleus. Delivery of viral particles and viral DNA to a target cell
is, therefore,
largely dependent on the integrity of the individual capsid proteins.
There have been a number of attempts to modify the surface proteins of
adenoviral vectors in order to expand their infectious capacility and target
cell range.
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Modifications of the capsid proteins of adenovirus include those in PCT
International Application No. W097/20051, published June 5, 1997 and PCT
International Application No. W097/05266, published February 13, 1997.
Specific modifications to the penton protein include those in United States
Patent
No. 5,559,099, issued September 24, 1996, U.S. Patent No. 5,731,190 issued
March 24,
1998, and Wickham et al., Gene Ther. 2:750-756, 1995.
Specific modifications to the hexon protein of adenovirus include those in
Crompton et al., J. Gen Virol. 75:133-139, 1994, PCT International Publication
No.
W098/40509 published September 17, 1998, and PCT International Publication No.
W098/32842 published July 30, 1998.
Modifications of the adenovirus fiber protein include those in PCT
International
Application No. W096/26281 published August 29, 1996; United States Patent No.
5,543,328, issued August 6, 1996; Michael et al., Gene Ther. 2:660-668, 1995;
Douglas
et al., Nature Med. 14:1574-1578, 1996; Wickham et al., Nature Med. 14:1570-
1573,
1996; Gall et al., J.Virol. 70:2116-2123, 1996; Stevenson et al., J.Virol.
71:4782-4790,
1997; and Krasnykh et al., J.Virol. 70:6839-6846, 1996; PCT International
Application
No. W098/41618 published September 24, 1998; PCT International Application No.
W098/07877 published February 26, 1998; PCT International Application No.
W098/07865 published February 26, 1998; PCT International Application No.
W097/20575 published June 12, 1997; U.S. Patent No. 5,770,442 issued June 23,
1998;
and U.S. Patent No. 5,756,086 issued May 26, 1998.
Although adenoviral vectors are currently in clinical trials and have shown
the
ability to transfer genes to target cells and tissues for expression of the
delivered gene, a
need remains to improve the infection efficiency of these vectors in order to
further
improve their gene transfer capabilities and/or to optimize the infection of
specific target
cells. It would be desirable to identify specific ligands which can confer
infectious
capability to adenoviral vectors for specific target cells of interest, and to
provide
adenoviral vectors which comprise such ligands. The present invention
addresses this
goal.
nanr of the Invention
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The present invention is directed to adenoviral vectors having modified capsid
proteins which comprise heterologous ligands that improve and/or alter the
infectious
capability of the vectors. Such ligands are capable of binding to desired
target cells, and
their inclusion into adenoviral vectors facilitates the binding and infectious
properties of
these vectors. In a preferred embodiment, the ligands are peptides and the
target cells are
epithelial cells. The invention is also directed to compositions comprising
the adenoviral
vectors of the invention. Additional aspects of the invention include methods
for using
the adenoviral vectors of the invention to deliver transgenes to target cells.
Brief Description of th_e Dram
FigurelA shows substitutions in adenovirus hexon protein of heterologous
ligands. Figure 1B shows infection of 293 cells with adenoviral vectors with
modified
hexon proteins. Figure 1 C shows infection of CHO cells with adenoviral
vectors with
modified hexon proteins. Figure 1D shows a graph of transgene expression in
CHO cells
using adenoviral vectors with modified hexon proteins.
Figure 2A shows a schematic diagram of the adenovirus fiber protein. Figure 2B
shows the trimerization capacity of modified fiber proteins.
Figure 3A shows a flow diagram of the protocol used in the biopanning of human
airway epithelial cells. Figure 3B shows the consensus amino acid sequences of
peptide
ligands identified from the biopanning of the human airway epithelial cells.
Figure 4 shows graphs displaying the binding of phage displaying specific
peptides to specific cell types: A: normal human bronchial epithelial cells;
B: small
airway epithelial cells; C: HeLa cells; D: COS cells.
Figure SA shows cellular pseudo-stratification and ciliagenesis of
differentiated
normal human bronchial epithelial cells. Figure 5B shows the elution profile
of phage
displaying specific peptides and binding to normal human bronchial epithelial
cells on an
air-liquid interface. Figure SC shows the elution profile of phage displaying
specific
peptides and binding to differentiated monkey airway epithelial cells.
Figure 6A shows a graph of the elution profiles for phage displaying specific
peptides and binding to normal human bronchial epithelial cells on an air-
liquid interface.
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Figure 6B shows the results of the anti-phage antibody staining on normal
human
bronchial epithelial cells binding phage displaying specific peptides.
Figure 7 shows the transduction of well-differentiated human airway cells by
adenoviral vectors with modified hexon proteins.
Figure 8 shows the transduction of mouse lung cells by adenoviral vectors with
modified hexon proteins.
Figure 9 shows a plot of assay results from transduction of mice by adenoviral
vectors with modified hexon proteins.
Detailed Description of the Invention
The present invention is directed to adenoviral vectors having modified capsid
proteins which comprise heterologous ligands that improve and/or alter the
infectious
capability of such vectors. Such ligands are capable of binding to target
cells, and their
inclusion into adenoviral vectors facilitates the binding and infectious
properties of the
vectors. In a preferred embodiment, the ligands are peptides and the target
cells are
epithelial cells. The invention is also directed to the novel heterologous
ligands, to the
oligonucleotides encoding such molecules, and to complexes of a capsid protein
of the
invention and a cellular receptor which binds the heterologous ligand.
The capsid proteins of adenovirus are defined as the fiber, hexon, penton and
protein IX proteins. A heterologous ligand in an adenoviral vector of the
invention is
defined as a peptide or the amino acid sequence of such peptide which is not
native to the
adenovirus genome, or as a peptide or the amino acid sequence of such peptide
which is
native to the adenoviral genome but which is inserted into a heterologous site
in the
genome. A heterologous ligand of the invention is also defined as an amino
acid
sequence which substantially corresponds to the amino acid sequence of an
identified
ligand, or which is an analog or homolog of such ligand.
As used herein, the term "peptide" refers to an oligomer of at least two
contiguous
amino acids, linked together by a peptide bond., and not greater than fifty
amino acids.
As used herein, the term "polypeptide" refers to an oligomer of at least fifty
amino acids.
As used herein; "substantially corresponds" means an amino acid sequence of a
ligand having approximately 70% identity in amino acid sequence to a
heterologous
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ligand peptide or polypeptide, whether colinear or including gaps in the
parent sequence,
and which retain the functional capability of the parent peptide. Methods for
characterizing identity relationships among two or more amino acid sequences
can
include the use of algorithms (e.g., as decribed in Molecular Sequence
Comparison and
Alignment, in Nucleic Acid and Protein u~~P Anal;, Bishop, M. et al., eds.,
IRL
Press, Oxford 1987).
By "homolog" is meant the corresponding peptides or polypeptides from other
organisms, so long as the structural and functional properties of the peptides
are retained.
By "analog" is meant substitutions, rearrangements, deletions, truncations and
additions in the amino acid sequence of a heterologous ligand, so long as the
structural
and functional properties of the ligands are retained. Analogs also include
ligands which
contain additional amino acids added to either end of the peptides that do not
affect
biological activity, ~, the presence of inert sequences added to a functional
ligand
which are added to prevent degradation. In another embodiment, conservative
amino
acid substitutions can be introduced into a ligand provided that the
functional activity of
the ligand is retained.
The criticality of particular amino acid residues in a ligand of interest may
be
tested by altering or replacing the residue of interest. For example, the
requirement for a
cysteine residue at a particular site in the ligand, which can be involved in
the formation
of intramolecular or intermolecular disulfide bonds, can be tested by
mutagenesis of the
cysteine to another amino acid, for example, tyrosine, which cannot form such
a bond.
In one embodiment of the invention, peptide ligands which are known to bind to
target cells can be inserted into the capsid proteins of the adenoviral
vectors of the
invention. Such ligands include the following: Ad2/5 RGD (containing the RGD
sequence from adenovirus serotypes 2 and 5, HAIRGDTFA) (SEQ ID NO. 1) Adl7 RGD
(containing the RGD sequence from adenovirus serotype 17, GPARGDSSV) (SEQ ID
NO. 2) and the SV40 nuclear localization signal (SV44NLS) (PKKKRI~V) (SEQ ID
NO.
3) (Kalderon et al., Cell 39:499-509, 1984). Other known ligands which bind to
specific
receptors on target cells, including RGD sequences from other adenovirus
serotypes, or
ligands which are involved in nuclear entry pathways can be used to generate
adenoviral
vectors which are capable of infecting specific target cells. In one
embodiment of the
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invention, the RGD sequences normally found in the penton protein are inserted
into
either the fiber or hexon proteins of adenoviral vectors to enhance their
binding
capability. In a preferred embodiment of the invention, a capsid protein
comprises a
ligand having an Adl7 RGD sequence: GPARGDSSV (SEQ ID NO. 2)
In another embodiment of the invention, novel ligands which are capable of
binding to desired target cells can be inserted into adenoviral capsid
proteins. Such
ligands can be identified by, for example, phage biopanning techniques (Smith
et al.,
Science 228:1315-1317, 1985; Parmley et al., Gene 73:305-318, 1988; Scott et
al.,
Science 249:386-390, 1990), in which phage engineered to display specific
peptides on
their surface are incubated with desired target cells to select those phage
which bind to
the target cells. The peptide contained on the phage is identified and
sequenced, and is
characterized as a ligand for the particular cell type. Biopanning can be
performed by
using, for example, a phage library which displays surface peptides and
incubating such
phage with the desired target cells to identify those phage displaying
peptides which are
capable of binding to the cells. Phage libraries can be purchased from, for
example, New
England Biolabs (Beverly, MA) . Biopanning of target cells can be performed in
solution
on an air-liquid interface (ALI) . Cells grown on an ALI differentiate in a
pseudo-
stratified layer, with a histology resembling in vivo airway epithelia (Gray
et al., 1996,
Am. J. Respir. Cell Mol. Biol. 14:104-112; Yamaya et al., Am. J. Physiol.
262:L713-724,
1992).
Other methods to identify such peptides which bind to surface cellular
receptors
can be used, such as incubation of target cells with labelled peptides to
identify cells
which bind such peptides, or using peptides incubated with cells containing a
high
abundance of a known receptor in order to more readily isolate peptides which
bind to
such a receptor. Other methods, such as in vitro assays using a cellular
receptor bound to
a column, for example, to isolate peptide ligands, which can then be
incorporated into
adenoviral vectors, are known to those skilled in the art. Cells used in the
identification of
peptide ligands of interest to be inserted into the capsid proteins of
adenovirus can be
chosen with reference to the target cell or tissue of interest for infection.
For example,
where the desired target cells are epithelial cells, normal human bronchial
epithelial cells
(lVJiBE) or small airway epithelial cells (SAEC) can be used.
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Specific novel peptides of the invention which can be inserted into the capsid
proteins of the adenoviral vectors invention include the following:
SSS.10 TTDFYYALRALA SEQ ID NO.
4
SSS.14 TTDFYYALRALA SEQ ID NO.
5
SSS.8 LPKMASVQRNLA SEQ ID NO.
6
SSS.9 HETFYSMIRSLA SEQ ID NO.
7
SSS.S HDTFLYGLQRLV SEQ ID NO.
8
SSS.6 LTFDQTPLTAQI SEQ ID NO.
9
SSS.7 ITFNQTVTTSYM SEQ ID NO.
10
SSS.16 ETFSDPLAGSSS SEQ ID NO.
11
SSS.17 SDQLASPYSHPR SEQ ID NO.
12
polyK KGKGKGKGKGKG SEQ ID NO.
13
Preferred novel peptide ligands of the invention for insertion into adenoviral
capsid proteins are sss.l0 and sss.l7.
Other peptides which can be inserted into adenoviral capsid proteins are
within
the scope of the invention provided they function to enhance viral binding
and/ or
infectivity in target cells.
The peptide ligands of the invention can be inserted into the fiber, hexon,
penton
and protein IX proteins of an adenoviral vector, or a vector may contain any
combination
of such modifications. Preferred insertion sites for the heterologous ligands
of the
invention are the adenoviral fiber or hexon protein. In a preferred
embodiment, one or
more heterologous ligands are inserted into the knob region of a fiber protein
of an
adenoviral vector. The ligands can be inserted into the capsid proteins
without the
removal of endogenous amino acid sequences or, alternatively, may be inserted
in the
place of deleted amino acid sequences. Preferably, the heterologous ligands
are
substituted for wild-type sequence in the proteins in order to maintain the
conformational
integrity of the capsid protein. Determination of the length of a peptide
ligand for
insertion into a capsid protein is made with reference to the size of an
identified ligand,
the site of insertion, including three-dimensional analysis, and the desired
target cell.
Preferred sites in the hexon protein of an adenoviral vector for the insertion
of one
or more ligands to enhance infectivity are the hypervariable regions (1-7) in
the hexon
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protein (Crawford-Miksza et al., J. Virol. 70:1836-1844, 1996). In the most
preferred
embodiments, the ligands are inserted into loop 1 (hypervariable region 5)
and/or into
loop 2 (hypervariable region 7) (Crawford-Miksza et al., J. Virol. 70:1836-
1844, 1996) of
the hexon protein. Because each modification in the hexon protein is repeated
720 times,
the peptide ligand enhancement is geatly amplified.
Preferred sites in the fiber protein of an adenoviral vector for the insertion
of one
or more ligands to enhance infectivity are in the knob region of the protein
(see Zia et al.,
Structure 21:1259-1270, 1994), which is the carboxy terminus of the protein.
For
example, peptide ligands can be inserted into the conserved A-J regions, the
blade regions
of the fiber protein (such as the G, H, I and D regions), or any regions of
the knob which
mediate cellular interactions {see Xia et al., Curr. Top. Micro. Immunol. 199:
40-46,
1995; Xia et al., Structure 2: 1259-1270, 1995). A preferred site for the
insertion of a
peptide ligand of the invention is the G region.
The fiber protein can also be modified by altering the number of repeat
nucleotide
sequences in the shaft of the protein. Such modifications can be used, for
example, in
combination with hexon proteins containing the ligands of the invention. Where
the
hexon protein contains ligands of the invention, reduction of the fiber
protein shaft may
expose the ligand more readily for interaction with the cell surface and
enhance or
facilitate the infectivity of an adenoviral vector containing both
modifications.
Trimerization of the fiber protein, which is essential for infectivity, can
occur provided
that the first residue of the 22°d repeat of the fiber shaft is present
(Henry et al., J.Virol.
68:5239-5246, 1994).
The invention is fiuther directed to the cellular receptors for the
heterologus
ligands of the invention, and to the complexes formed between the ligands and
their
receptors. One skilled in the art can readily identify the receptors for the
peptides of the
invention using conventional techniques such as, for example, incubation of
labelled
peptides with cellular extracts to identify one or more proteins that bind to
the receptor or
to use a peptide containing a reactive group (for example, cysteine with a
free sulfhydryl
group) on a resin to isolate one or more receptors from a cellular extract.
Reverse genetic
techniques which are known to those skilled in the art can be used to identify
the genes
encoding the receptors so identified.
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The invention is also directed to adenoviral vectors which comprise a
heterologous DNA sequence of interest (transgene) operably linked to
expression control
sequences and fi~rther comprise one or more modified capsid proteins of the
invention.
This DNA sequence of interest can be characterized as a transgene. Specific
adenoviral
vectors into which the modified capsid proteins of the invention can be
engineered are
disclosed by Zabner et al., Cell 75 : 207, 1993; Zabner et al., J. Clin.
Invest. 6 : 1504,
1996; Armentano et al., J. Virol. 71:2408-2416, 1997; Scaria et al.; J. Virol.
72:7302,
1998; Kaplan et al., Human Gene Ther. 9:1469-1479, 1998; and U.S. Patent No.
5,670,488 issued September 23, 1997, U.S. Patent No. 5,707,618 issued January
13,
1998, and U.S. Patent No. 5,824,544, issued October 20, 1998, the disclosures
of which
are incorporated by reference. Adenoviral vectors of the invention may include
deletion
of the E1 region, partial or complete deletion of the E4 region, and deletions
within, for
example, the E2 and E3 regions. Adenoviral vectors which comprise a
heterologous
DNA/transgene of interest, and associated regulatory elements, flanked by the
adenoviral
inverted terminal repeats and packaging sequences, (as provided in allowed
U.S. Patent
Application Serial No. 08/895, 194) are also within the scope of the invention
as
candidates for the insertion of modified capsid proteins. The adenoviral
vectors of the
invention are preferably replication-defective, that is, they are incapable of
generating a
productive infection in the host cell.
In preferred embodiments, adenoviral vectors can also be constructed using
adenovirus serotypes from the well-studied group C adenoviruses, especially
Ad2 and
AdS. Adl7 is also a preferred semtype. However, the design of the adenoviral
vectors of
the invention using other group C or non-gmup C adenoviruses is also within
the scope of
the invention, including the design of chimeric adenviral vectors which
contain
nucleotide sequences from one or more serotypes. Within the scope of the
invention are
also, for example, chimeric vectors which contain a genome of a particular
serotype and
one or more capsid proteins from other serotypes, such as for example, those
disclosed in
allowed U.S. Application Serial No. 08/752,760 and in PCT Application
PCT/US97/21494, filed November 20, 1997. Adenoviral vectors which are chimeric
for
the capsid proteins are also within the scope of the invention, such as where
the fiber and
hexon proteins are from different serotypes. In another embodiment, a
particular capsid
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protein may itself be a chimera, such as a fiber protein which has a modular
composition
such that the. tail, shaft and knob regions may be derived from one or more
serotypes. In
one preferred embodiment of the invention, an adenoviral vector comprises a
fiber protein
which has a tail and knob region from serotype Ad2 and a shaft region from
serotype
Adl7.
In order to construct the adenoviral vectors of the invention, reference may
be
made to the substantial body of literature on how such vectors may be
designed,
constructed and propagated using techniques from molecular biology and
microbiology
that are well-known to the skilled artisan. For example, the skilled artisan
can use the
standard techniques of molecular biology to engineer a heterologous
DNA/transgene
operably linked to appropriate regulatory elements into a backbone vector
genome and to
engineer a ligand into a capsid protein (Berkner, K.L., Curr. Top. Micro.
Immunol.
158:39-66, 1992). For example, a plasmid containing a transgene and any
regulatory
elements of the invention inserted into an adenovirus gnomic fragment can be
co-
transfected with a linearized viral genome derived from an adenoviral vector
of interest
into a recipient cell under conditions whereby homologous recombination occurs
between
the genomic fragment and the virus. Preferably, a transgene and any regulatory
elements
are engineered into the site of an E 1 deletion. As a result, the transgene is
inserted into
the adenoviral genome at the site in which it was cloned into the plasmid,
creating a
recombinant adenoviral vector. The adenoviral vectors can also be constructed
using
standard ligation techniques, for example, by engineering a desired
restriction site into a
capsid protein, allowing for the insertion of a desired oligonucleotide
encoding a peptide
ligand of interest. Peptides can also be synthesized by standard techniques of
protein or
peptide synthesis, and may be composed of linear or cyclic peptides.
Construction of the adenoviral vectors can be based on adenovirus DNA sequence
information widely available in the field, e.g., nucleic acid sequence
databases such as
GenBank.
Preparation of replication-defective adenoviral vector stocks can be
accomplished
using cell lines that complement viral genes deleted from the vector, e.g.,
293 or A549
cells containing the deleted adenovirus E1 genomic sequences. HER3 cells
(human
embryonic retinoblasts transformed by Ad 12), or vK2-20 cells can also be
used. After
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amplification of plaques in suitable complementing cell lines, the viruses can
be
recovered by freeze-thawing and subsequently purified using cesium chloride
centrifugation. Alternatively, virus purification can be performed using
chromatographic
techniques, e.g., as set forth in International Application No.
PCT/US96/13872, filed
August 30, 1996, incorporated herein by reference.
Titers of replication-defective adenoviral vector stocks can be determined by
plaque formation in a complementing cell line, e.g., 293 cells. End-point
dilution using
an antibody to the adenoviral hexon protein may be used to quantitate virus
production or
infection efficiency of target cells (Armentano et al., Hum. Gene Ther. 6:1343-
1353,
1995, incorporated herein by reference).
In another embodiment of the invention, the adenoviral vectors containing
modified capsid proteins further comprise nucleotide sequences coding for one
or more
transgenes. A transgene is identified as a gene which is exogenously provided
to a cell
by any method of gene transfer. Transgenes which can be delivered and
expressed from
an adenoviral vector of the invention include, but are not limited to, those
encoding
enzymes, blood derivatives, hormones, lymphokines such as the interleukins and
interferons, coagulants, growth factors, neurotransmitters, tumor suppressors,
apoliproteins, antigens, and antibodies, and other biologically active
proteins. Specific
transgenes which may be encoded by the adenoviral vectors of the invention
include, but
are not limited to, cystic fibrosis transmembraile regulator (CFTR),
dystrophin,
glucocerebrosidase, tumor necrosis factor, p53, p21, herpes simplex thymidine
kinase and
gancyclovir, retinoblastoma (Rb), and adenosine deaminase (ADA). Transgenes
encoding antisense molecules or ribozymes are also within the scope of the
invention.
The vectors may contain one or more transgenes under the control of one or
more
regulatory elements.
In addition to containing the DNA sequences encoding one or more transgenes,
the adenoviral vectors of the invention may contain any expression control
sequences
such as a promoter or enhancer, a polyadenylation element, and any other
regulatory
elements that may be used to modulate or increase expression, all of which are
operably
linked in order to allow expression of the transgene. Viral or non-viral
promoters can be
operably linked to a transgene in an adenovirai vector, including the CMV
promoter or
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functional variants thereof. The use of any expression control sequences, or
regulatory
elements, which facilitate expression of the transgene is within the scope of
the invention.
Such sequences or elements may be capable of generating tissue-specific
expression or be
susceptible to induction by exogenous agents or stimuli. Preferred regulatory
elements in
the adenoviral vectors of the invention include the K18, K14, human [i-actin,
BOS (EF-
1 a), ubiquitin B and mucin promoters, the CMV enhancer/promoter, CMV
enhancer/ElA promoter, hybrid intron (HI) (Yew et al., Hum. Gene Ther. 8:575-
584,
1997) and a-globin stability element (aSE). In a particularly preferred
embodiment of
the invention, the K18 promoter and the a-globin stability element are used as
regulatory
elements for expression of a transgene in an adenoviral vector which comprises
one or
more modified capsid proteins according to the invention.
Infection of target cells by the adenoviral vectors of the invention may also
be
facilitated by the use of cationic molecules, such as cationic lipids as
disclosed in PCT
Publication No. W096/18372, published 3une 20, 1996, incorporated herein by
reference.
Adenoviral vectors complexed with cationic molecules are also described in PCT
Publication No.W098/22144, published May 28, 1998, incorporated herein by
reference.
Cationic amphiphiles have a chemical structure which encompasses both polar
and non-polar domains so that the molecule can simultaneously facilitate entry
across a
lipid membrane with its non-polar domain while its cationic polar domain
attaches to a
biologically useful molecule to be transported across the membrane.
Cationic amphiphiles which may be used to form complexes with the adenoviral
vectors of the invention include, but are not limited to, cationic lipids,
such as DOTMA
(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987) (N-[1-(2,3-
dioletloxy)propyl)-N,N,N-trimethylammonium chloride); DOGS
(dioctadecylamidoglycylspermine) (Behr et al., Proc. Natl. Acad. Sci. USA
86:6982-
6986, 1989); DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium
bromide) (Felgner et al., J. Biol. Chem. 269:2550-2561, 1994; and DC-chol (3B
[N-N',
N'-dimethylaminoethane) -carbamoyl] cholesterol) (U.S. Patent No. 5, 283,185
to Epand
et al.). The use of other cationic amphiphiles recognized in the art or which
come to be
discovered is within the scope of the invention.
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In preferred embodiments of the invention, the cationic amphiphiles useful to
complex with and facilitate transfer of the vectors of the invention are those
lipids which
are described in PCT Publication No. W096/18372, published June 20, 1996, and
U.S.
Patent No. 5,650;096, both incorporated herein by reference. Preferred
cationic
amphiphiles described herein to be used in the delivery of the plasmids and/or
viruses are
GL-53, GL-67, GL-75, GL-87 and GL-89, including protonated, partially
protonated, and
deprotonated forms thereof. Further embodiments include the use of non-T-
shaped
amphiphiles as described on pp. 22-23 of the aforementioned PCT application,
including
protonated, partially protonated and deprotonated forms thereof. Most
preferably, the
cationic amphiphile which can be used to deliver the vectors of the invention
is N4-
spermine cholesteryl carbamate (GL-67).
In the formulation of compositions comprising the adenoviral vectors of the
invention, one or more cationic amphiphiles may be formulated with neutral co-
lipids
such as dileoylphosphatidylethanolamine (DOPE) to facilitate delivery of the
vectors into
a cell. Other co-lipids which may be used in these complexes include, but are
not limited
to, diphytanoylphosphatidylethanolamine, lyso-phosphatidylethanolamines, other
phosphatidylethanolamines, phosphatidylcholines, lyso-phosphatidylcholines and
cholesterol. A preferred molar ratio of cationic amphiphile to colipid is 1:1.
However, it
is within the scope of the invention to vary this ratio, including also over a
considerable
range. In a preferred embodiment of the invention, the cationic amphiphile GL-
67 and
the neutral co-lipid DOPE are combined in a 1:2 molar ratio, respectively,
before
complexing with an adenoviral vector for delivery to a cell.
In the formulation of complexes containing a cationic amphiphile with an
adenoviral vector, a preferred range of 10' - 10'° infectious units of
virus may be
combined with a range of 104 - 106 cationic amphiphile molecules/viral
particle.
Assays which determine the binding properties of a modified capsid protein or
a
ligand of the invention can be performed using in vitro assays in which the
protein or
peptide of interest is incubated with target cells of interest and binding to
the cells is
measured. Biochemical properties relative to infectivity can be assayed, for
example,
modified fiber proteins can be assayed for their ability to trimerize,
essential for infection,
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using standard assays for detection of a high molecular weight protein
homotrimer by
nondenaturing gel electrophoresis.
The infection efficiency of the adenoviral vectors of the invention containing
one
or more modified capsid proteins may be assayed by standard techniques to
determine the
infection of target cells. Such methods include, but are not limited to,
plaque formation,
end-point dilution using, for example, an antibody to the adenovira.l hexon
protein, and
cell binding assays using radiolabelled virus, or expression of a transgene
which is
delivered by the virus. Imprpved infection efficiency may be characterized as
an increase
in infection of at least an order of magnitude with reference to a control
virus.
Where an adenoviral vector of the invention encodes a marker or other
transgene,
relevant molecular assays to determine expression of the gene include the
measurement
of transgene mRNA, by, for example, Northern blot, S 1 analysis or reverse
transcription-
polymerase chain reaction (RT-PCR). The presence of a protein encoded by a
transgene
may be detected by Western blot, immunoprecipitation, immunocytochemistry, or
other
techniques known to those skilled in the art. Marker-specific assays can also
be used,
such as X-gal staining of cells infected with an adenoviral vector encoding (3
galactosidase or a chemiluminescence assay of (3-gal expression using
commerical kits,
such as GalactolightTM, manufactured by Tropix, Bedford, MA..
Specific cell lines which can be used to assess the infection efficiency of
the
adenoviral vectors of the invention include cells which are normally
susceptible to
adenoviral infection as well as those that are poorly infected or refractory
to wild-type
adenoviral infection. For example, Chinese hamster ovary (CHO) cells, which
are poorly
infected by wild-type adenovirus, can be used to test the adenoviral vectors
of the
invention containing modified capsid proteins in order to determine whether
the
heterologous ligand facilitates adenovirus binding to a target cell to provide
infection
enhancement and efficiency. Other cells can be chosen depending on the
particular target
cell type sought to be infected with the adenoviral vectors. For example,
where epithelial
cells are the target cells of interest, normal human bronchial epithelial
cells (hTHBE) or
small airway epithelial cells (SAEC) can be used. Specific enhancement of the
infection
of a specific cell type can be determined by reference to a non-epithelial
cell type, for
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example. Other cell lines suitable for assaying the vectors of the invention
include HeLa,
HWEC, other established cell lines, as well as primary cells.
In order to determine infection efficiency and transgene expression in vivo
using
the vectors and compositions of the invention, animal models may be
particularly
relevant in order to assess transgene expression against a background of
potential host
immune response. Such a model may be chosen with reference to such parameters
as
ease of delivery, identity of transgene, relevant molecular assays, and
assessment of
clinical status. Where the transgene encodes a protein whose lack is
associated with a
particular disease state, an animal model which is representative of the
disease state may
optimally be used in order to assess a specific phenotypic result and clinical
improvement. However, it is also possible that particular adenoviral vectors
of the
invention display enhanced infection efficiency only in human model systems,
e.g., using
primary cell cultures, tissue explants, or permanent cell lines. In such
circumstances
where there is no animal model system available in which to model the
infection
efficiency of an adenoviral vector with respect to human cells, reference to
art-recognized
human cell culture models will be most relevant and definitive.
Relevant animals in which the adenoviral vectors may be assayed include, but
are
not limited to, mice, rats, monkeys, and rabbits. Suitable mouse strains in
which the
vectors may be tested include, but are not limited to, C3H, C57B1/6 (wild-type
and nude)
and Balb/c (available from Taconic Farms, Genmantown, New York).
Where it is desirable to assess the host immune response to vector
administration,
testing in immunocompetent and immunodeficient animals may be compared in
order to
define specific adverse responses generated by the immune system. The use of
immunodeficient animals, e.g., nude mice, may be used to characterize vector
performance and persistence of transgene expression, independent of an
acquired host
response.
In a particular embodiment where the transgene encodes human cystic fibrosis
transmembrane conductance regulator protein (CFTR) which is administered to
the
respiratory epithelium of test animals, expression of human CFTR may be
assayed in the
lungs of relevant animal models, for example, C57B1/6 or Balb/c mice, cotton
rats, or
Rhesus monkeys. Molecular markers, which may used to determine expression,
include
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the measurement of CFTR mRNA, by, for example, Northern blot, S 1 analysis or
RT-
PCR. The presence of the CFTR protein may be detected by Western blot,
immunoprecipitation, immunocytochemistry, or other techniques known to those
skilled
in the art. Such assays may also be used in tissue culture where cells
deficient in a
functional CFTR protein and into which the adenoviral vectors have been
introduced may
be assessed to determine the presence of functional chloride ion channels -
indicative of
the presence of a functional CFTR molecule.
The adenoviral vectors of the invention have a number of in vivo and in vitro
utilities. The vectors can be used to transfer a normal copy of a transgene
encoding a
biologically active protein to target cells in order to remedy a deficient or
dysfunctional
protein, or to provide a protein not normally found in the cell but of
interest with respect
to a specific phenotype. The vectors can be used to transfer marked transgenes
(e.g.,
containing nucleotide alterations) which allow for distinguishing expression
levels of a
transduced gene from the levels of an endogenous gene. The adenoviral vectors
can also
be used to define the mechanism of specific viral protein-cellular protein
interactions that
are mediated by specific virus surface protein sequences. The vectors can also
be used to
optimize infection efficiency of specific target cells by adenoviral vectors
by engineering
specific peptide ligands relevant to target cells of interest into one or more
of the capsid
proteins. Where it is desirable to use an adenoviral vector for gene transfer
to cancer cells
in an individual, an adenoviral vector can be chosen which selectively infects
the specific
type of target cancer cell and avoids promiscuous infection. Where primary
cells are
isolated from a tumor in an individual requiring gene transfer, the cells may
be tested
against a panel of adenoviral vectors to select a vector with optimal
infection efficiency
for gene delivery. The vectors can fiuther be used to transfer tumor antigens
to dendritic
cells which can then be delivered to an individual to elicit an anti-tumor
immune
response. The adenoviral vectors can also be used to evade undesirable immune
responses to particular adenovirus serotypes or recombinant constructs which
compromise the gene transfer capability of adenoviral vectors.
The present invention is further directed to compositions containing the
adenoviral vectors of the invention which can be administered in an amount
effective to
deliver one or more desired transgenes to the cells of an individual in need
of such
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molecules and cause expression of a transgene encoding a biologically active
protein to
achieve a specific phenotypic result. The cationic amphiphile-virus complexes
may be
formulated into compositions for administration to an individual in need of
the delivery
of the transgenes.
The compositions can include physiologically acceptable carriers, including
any
relevant solvents. As used herein, "physiologically acceptable carrier"
includes any and
all solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents, and the like. Except insofar as any conventional
media or
agent is incompatible with the active ingredient, its use in the compositions
is
contemplated. The compositions containing the adenoviral vectors having capsid
proteins comprising a heterologous ligand of the invention can also be
formulated into
dry powder complexes for administration.
Routes of administration for the compositions containing the adenoviral
vectors
having capsid proteins comprising a heterologous ligand of the invention
include
1 S conventional and physiologically acceptable routes such as direct delivery
to a target
organ or tissue, intranasal, intravenous, intramuscular, subcutaneous,
intradermal, oral
and other parenteral routes of administration.
The invention is further directed to methods for using the compositions
containing
the adenoviral vectors of the invention in vivo or ex vivo applications in
which it is
desirable to deliver one or more transgenes into cells such that the transgene
produces a
biologically active protein for a normal biological or phenotypic effect. In
vivo
applications involve the direct administration of one ore more adenoviral
vectors
formulated into a composition to the cells of an individual. Ex vivo
applications involve
the transfer of a composition containing the adenoviral vectors directly to
autologous
cells which are maintained in vitro, followed by readministration of the
transduced cells
to a recipient.
Dosage of the adenoviral vector having capsid proteins comprising a
heterologous
ligand of the invention to be administered to an individual for expression of
a transgene
encoding a biologically active protein and to achieve a specific phenotypic
result is
determined with reference to various parameters, including the condition to be
treated, the
age, weight and clinical status of the individual, and the particular
molecular defect
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requiring the provision of a biologically active protein. The dosage is
preferably chosen
so that administration causes a specific phenotypic result, as measured by
molecular
assays or clinical markers. For example, determination of the infection
efficiency of an
adenoviral vector containing the CFTR transgene which is administered to an
individual
can be performed by molecular assays including the measurement of CFTR mRNA,
by,
for example, Northern blot, S 1 or RT-PCR analysis or the measurement of the
CFTR
protein as detected by Western blot, immunoprecipitation, immunocytochemistry,
or
other techniques known to those skilled in the art. Relevant clinical studies
which could
be used to assess phenotypic results from delivery of the CFTR transgene
include PFT
assessment of lung function and radiological evaluation of the lung.
Demonstration of
the delivery of a transgene encoding CFTR can also be demonstrated by
detecting the
presence of a functional chloride channel in cells of an individual with
cystic fibrosis to
whom the vector containing the transgene has been administered (Zabner et al.,
J. Clin.
Invest. 97:1504-1511, 1996). Transgene expression in other disease states can
be assayed
analogously, using the specific clinical parameters most relevant to the
condition.
Dosages of an adenoviral vector of the invention which are effective to
provide
expression of a transgene encoding a biologically active protein and achieve a
specific
phenotypic result range from approximately 10g infectious units (LU.) to 10"
LU. for
humans.
It is especially advantageous to formulate parenteral compositions in dosage
unit
form for ease of administration and uniformity of dosage. Dosage unit form as
used
herein refers to physically discrete units suited as unitary dosages for the
subjects to be
treated, each unit containing a predetermined quantity of active ingredient
calculated to
produce the specific phenotypic effect in association with the required
physiologically
acceptable carrier. The specification for the novel dosage unit forms of the
invention are
dictated by and directly depend on the unique characteristics of the
adenoviral vector and
the limitations inherent in the art of compounding. The principal active
ingredient (the
modified adenoviral vector) is compounded for convenient and effective
administration in
effective amounts with the physiologically acceptable carrier in dosage unit
form as
discussed above.
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Maximum benefit and achievement of a specific phenotypic result firm
administration of the adenoviral vectors of the invention may require repeated
administration. Such repeated administration may involve the use of the same
adenoviral
vector, or, alternatively, may involve the use of different adenoviral vectors
which are
rotated in order to alter viral antigen expression and decrease host immune
response.
The practice of the invention employs, unless otherwise indicated,
conventional
techniques of protein chemistry, molecular virology, microbiology, recombinant
DNA
technology, and pharmacology, which are within the skill of the art. Such
techniques are
explained fully in the literature. See, e.g.. Current Protocols in Molecul r
Rinln~v,
Ausubel et aL, eds., John Wiley & Sons, Inc., New York, 1995, and Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, PA, 1985.
The invention is fiwther illustrated by the following specific examples which
are
not intended in any way to limit the scope of the invention.
EXAMPLE 1: Adenoviral vectors with modified
hexon proteins with ligand enhancement
Ligands with known interaction for a cellular receptor or a nuclear entry
pathway
were substituted into the hypervariable domain of hexon loop 1: the RGD
sequences from
adenovirus types 2 and 5 (Ad2/5 RGD) (SEQ ID NO 1) or type 17 (Adl7 RGD) (SEQ
ID
NO. 2), as well as the basic stretch of amino acids within SV40 large T
antigen that
targets that protein to the nucleus (SV40 NLS) (SEQ ID NO. 3) (Figure lA).
Modified
hexon proteins were incorporated into adenoviral vectors expressing (3ga1 and
used for
infections of both established and primary cells.
Hexon proteins were modified by the insertion of oligonucleotides encoding the
peptides into newly created restriction enzyme sites within the respective
sequences. An
AatII site was created in substitution for wild-type nucleotides 19680 through
19740 by
changing the flanking nucleotides at the S' end, TACCTC, to GACGTC and at the
3' end,
GATGTA, to GACGTC. Oligonucleotides encoding specific amino acids were then
inserted into the AatII site, creating a modified hexon protein with in-frame
protein
substitutions within loop 1.
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293 cells (in DMEM) and CHO cells (in F12-Ham's media) were infected at
subconfluency with Ad2-~igal-4 (Armentano et al., J.Virol. 71:2408-2416, 1997)
or an
identical vector except for the substitution of RGD or SV40NLS peptide
sequences
within hexon loop 1. 293 cells were infected overnight at a multiplicity of
infection
S (MOI) of O.S. 24 hours post-infection, the cells were stained for ~i-gal
activity and
photographed. CHO cells were infected for 4 hours at an MOI of S0. 48 hours
following
infection, the cells were stained for ~i-gal activity and photographed.
Figure lA): Peptides substituted into the hypervariable domain of hexon loop 1
include the RGD sequences from adenovirus types 2 and S (Ad2/S RGD) or type 17
(Adl7 RGD), as well as the basic stretch of amino acids within SV40 large T
antigen that
targets that protein to the nucleus (SV40 NLS). The amino acid substitutions
within the
hexon protein are underlined. Figure 1B): 293 cells were infected overnight
with
Ad2/bgal-4 (A), Ad2/(3ga1/hex.iriod.Ad2/SRGD (B), Ad2/(3ga1/hex.mod.AdI7RGD
{C),
or Ad2[3ga1/hex.mod.SV40NLS (D) at an MOI = O.S. Cells were photographed 24
hours
1 S post-infection. Figure 1 C): CHO cells were infected for 4 hours with
Ad2/~gal-4 (A),
Ad2/~igal/hex.mod.Ad2/SRGD (B), Ad2/(3gal/hex.mod.AdI7RGD (C), or
Ad2(3ga1/hex.mod.SV40NLS (D) at an MOI = S0. Cells were photographed 48 hours
post-infection. Cells from similarly infected dishes were quantitated for
(3ga1 expression
by luminometer. Each bar in the graph represents the average of 3 separate
assays on
CHO cells from one dish (Figure 1D).
The results indicate that adenoviral vectors modified by the insertion of RGD
or
SV40NLS peptides in loop 1 of the hexon protein can infect cells with
differential
efficiency, as a function of the specific peptide ligand inserted into the
hexon protein.
EXAMPLE 2: Modification of the adenovirus fiber knob
2S by incorporation of a novel ligand
Oligonucleotides encoding a peptide linker (PGSASGSASGSP) (SEQ ID NO. 20)
and a new enzyme site (AatII) were inserted just upstream of the translation
termination
site of adenovirus DNA encoding the fiber protein (Figure 2A).
Oligonucleotides
encoding specific amino acids were then inserted into the AatII site, creating
a fiber knob
with in-frame protein additions just upstream of the translation termination
site. The
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oligonucleotide additions are flanked by AatII sites, thus adding two amino
acids (DV)
upstream and downstream of the inserted peptides. The carboxy-terminus of
fiber knob
was modified by the addition of a linker (-PGSASGSASGSP-) and ligand (sss.l0
or
sss.17).
Modified fiber knob proteins, from the first residue of the 22°d repeat
of the shaft
through the stop site, were cloned into the Ndel-BamHI site of pET-15b
(Novagen).
Each protein was expressed from the T7 promoter in a rabbit reticulocyte
lysate
(Promega). 1 ul of each translation reaction was added to a standard protein
loading
buffer with (+) or without (-) DTT as a denaturing reagant and with (+) or
without {-)
heating. Protein trimerization capacity was verified by the migration of the
protein at a
high molecular weight (2-3 times the size of the monomer), under nondenaturing
conditions, on a 12% polyacrylamide gel.
The results show that novel ligands, having high affinity for human epithelial
cells, can be incorporated into the fiber carboxy terminus, while allowing the
capacity of
the knob to trimerize to be retained (Figure 2B). Lanes 1 and 2, pET-15b
vector; lanes 3
and 4, fiber knob/sss.l7; lanes 5 and 6, fiber knob/sss. 10; lanes 7 and 8,
wild-type fiber
knob; M, marker.
EXAMPLE 3: Identification of novel binding peptides
Primary human airway epithelial (NHBE) cells were purchased from Clonetics
(San Diego, CA). Frozen cells were spun down gently (1000 rpm for 5' at room
temperature) in media or HBSS, then resuspended in blocking buffer for
biopanning.
Differentiated, ciliated airway epithelial cells on an air-liquid interface
(ALI) were
created according to the protocol of Gray et al (Am. J. Respir. Cell Mol.
Biol. 14:104-
112, 1996). Cell growth medium was supplemented as recommended by the
supplier,
with the following modifications: 25ng/ml hEGF, 5x10'8 M retinoic acid and 0.5
mg/ml
BSA were added to the media of cells growing on plastic, while 0.5 ng/ml hEGF,
SxlO-g
M retinoic acid, and 1.5 mg/ml BSA were added to the media of cells growing in
transwells. Cells on plastic were grown in BEGM medium; cells in transwells
were
grown in a 1:1 mixture of DMEM (low glucose):BEBM.
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Rat tail collagen type 1 (Collaborative Biomedical Products, Waltham, MA) was
diluted to 3 mg/ml with 0.02N acetic acid. 100 ~cl was added to the upper
chamber of
transwells (Costar transwell clear) in 24-well plates. The plates were then
placed,
uncovered, in an ammonium vapor chamber for 3' to cross-link the collagen
matrix.
Upper and lower chambers of the wells were rinsed with 3 changes of sterile
water over 2
- 3 hours. Water was replaced by unsupplemented DMEM for 24 hours. Prior to
cell
seeding, inserts were equilibrated for 2 hours at room temperature with
DMEM:BEGM/ 10%FBS.
Primary NHBE cells were seeded on the collagen gels at 0.5 - 1.0 x 105
cells/cm2
in DMEM:BEGM. Cells were grown submerged until confluency, with media changes
24 hours following seeding and every 48 hours subsequently: For the first 24
hours
following seeding, DMEM:BEGM/10%FBS was left in the bottom chamber.
Subsequently, DMEM:BEGM was added to both upper and lower chambers. The ALI
was created by removing the media from the upper chamber upon cell confluency.
Media
in the lower chamber (basal surface of cells) was changed daily thereafter.
For cells on ALIs, blocking buffer was added to both the top and bottom
chambers for 30'. Block buffer in the top chamber was then replaced with phage
in 100 ,ul
block buffer. Following phage binding, the inserts were transferred to
separate 50 ml
polypropylene tubes, where they were washed 3 successive times with 10 ml
block
buffer. Phage were eluted by adding 200 ul elution buffer directly to the
inner well
chambers.
Phage biopanning. A phage library, displaying linear dodecapeptides fused to
protein III, was purchased from New England Biolabs. For biopanning cells in
solution,
10" to 10" phage and 106 cells were blocked separately in 200 ,ul blocking
buffer (3%
BSA, 0.1% hydrolysate casein, 0.02% azide in HBSS without Mg** or Ca**) for
30' with
gentle rocking at room temperature. The cells and phage were then combined
(400 ,ul
total) and incubated for 1 hour at room temperature, with gentle rocking. The
cells bound
with phage were spun down at 2000 rpm for 3' in a microcentrifuge, then washed
with 3
successive 10 ml rinses of block buffer. Phage were eluted from the cell
pellet with 200
,ul of 0.12M glycine, pH2.0/0.5% BSA for 5' at room temperature. Debris was
removed
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by a quick centrifugation. The elution solution was neutralized by the
addition of 2 mls
SOmM Tris, pH 8Ø
After 3 rounds of biopanning in solution or on ALIs (Figure 3A), eluted phage
were used to infect bacterial cells (Supplier's protocol, New England Biolabs)
for single
plague isolations, DNA extractions, and sequencing. 20 plaques from each
infection
were sequenced in the region corresponding to the 5' end of gene III, the site
of the
insertions in the library. The results indicate that 3 rounds of biopanning of
primary
NHBE cells in solution led to the isolation of phage with similar amino acid
sequences
(see underlined amino acids). Figure 3B shows the consensus amino acid
sequneces from
peptide Iigands identified from phage which bound the epithelial cells.
Sequence identity
among various peptides are shown as underlined amino acids. Only the phage
from the
solution biopanning showed the consensus sequences as shown here.
EXAMPLE 4: Affinity profiles of phage for different cell types
NHBE and SAEC cells for solution binding to phage were used directly from
fibzen cultures. HeLa and COS cells were grown on plastic (DMEM medium),
trypsinized, spun down in media, and resuspended in block buffer for binding
to phage.
Phage binding was as described in Figure 3, with the exception that the phage
used for
these studies (sss.l0, sss.6, Ad2/SRGD, and S3-21) are monoclonal phage.
Primary human bronchial epithelial NHBE (Figure 4A), primary small airway
epithelial cells SAEC (Figure 4B), transformed cervical carcinoma cells HeLa
(Figure
4C) or transformed African green monkey kidney fibroblast cells COS (Figure
4D) cells
in solution were bound with phage displaying sss.l0 or sss.6 peptides, an
Ad2/5 RGD
ligand, a Fab fragment with high affinity for many cell types (S3-21), or a
wild-type
phage filament. Bar graphs represent the affinity of each monoclonal phage, as
measured
by acid elution and subsequent bacterial infection by the phage.
The results show that phage displaying peptides with high affinity for NHBE
cells
show a similar binding profile for SAEC. Cells of a different tissue from the
same or
different species (HeLa, and COS, respectively) bind these phage with
different relative
efficiencies.
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EXAMPLE 5: Affinity of phage for differentiated
ciliated NHBE cells and monkey airway cells
Monkey airway epithelial cells were isolated by a standard primary cell
isolation
procedure. In brief summary, airways were dissected and rinsed with DMEM.
Lumen of
airways were flushed with DMEM, flushed with 0.1 % protease, and submerged in
protease overnight. Airways were then flushed with MEM/10%FBS. The recovered
washes were spun down at 4°C for 15' at 1000 rpm. The cell pellet was
resuspended in
MEM/10%FBS, and the cells were seeded onto plastic in a 24-well dish.
The air-liquid interfaces were as described in Figure 3. Phage binding and
elution
was also performed as described in Figure 3, with the exception that the phage
are
monoclonal.
Primary NHBE cells were seeded on collagen gels in plastic inserts of a 24-
well
dish, grown to confluence while submerged in media, then transferred to an
air/liquid
interface. Cellular pseudo-stratification and ciliagenesis occured at 14 - 21
days post-
seeding (Figure SA). Separate inserts were bound with phage displaying high
affinity
peptides (sss.l0, sss.8, sss.l6) or wild-type filaments. Each bar in the
graphs represents
the elution profile of phage bound to a separate collagen insert (Figure SB).
Differentiated, ciliated monkey airway epithelial cells (from proteolytically
digested airways) were seeded in plastic wells, then bound with phage
displaying
peptides (sss.l0, sss.8) or a Fab fragment (N3-14) with high affinity for
human airway
epithelial cells, or with wild-type phage. Wells without cells ("none") were
also bound
with phage as a control for non-specific binding to plastic (Figure SC).
The results show that phage displaying peptides with high affinity for NHBE
cells
in solution also show high affinities for both human and monkey
differentiated, ciliated
airway epithelial cells (Figures SB and SC).
EXAMPLE 6: Affinity of phage for NHBE cells on ALI
Differentiated, ciliated NHBE cells were bound with monoclonal phage using the
biopanning protocol described above. Following washes, inserts were fixed at
4°C for
30' in 2% paraformaldehyde, 0.2% gluteraldehyde in PBS. Following 3-5 rinses
in cold
PBS, cells were blocked with 2% BSA, 1% fish gelatin, 10% horse serum in PBS
for 1
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hour at room temperature. Primary antibody (anti-M13, Pharmacia) was added
overnight
at 4°C. Following 3-5 washes with cold PBS, secondary FITC-labelled
antibody was
added for 1 hour at 37°C. After 3-5 washes with cold PBS, cells were
again fixed. The
inserts were mounted on slides for photography. 2 /cg/ml DAPI was added for
S visualization of the nuclei.
Figure 6A shows a graph of the elution profiles for each phage. These results
show that sss.l0 and sss.l7 (the two phage with the highest peptide
affinities), and N3-14
(a phage displaying a Fab fragment with high affinity for human epithelial
cells} were
bound to ALI inserts. The inserts were subsequently washed, fixed, and bound
with an
anti-phage primary antibody followed by a FITC-labelled secondary antibody.
Figure 6B
shows the results of the antibody staining on inserts binding phage displaying
specific
peptides: N3-14, sss.l0, sss.l7 and wild type control. These results
illustrate that phage
displaying peptides isolated by biopanning on NHBE cells bind directly and
with
specificity to differentiated airway epithelial cells.
EXAMPLE 7: Transduction of well-differentiated human airway
epithelial cells on air-liquid interfaces by adenoviruses
containing modified capsid proteins
Airway epithelial cells were obtained from normal lung donors. Cells were
isolated by enzyme digestion as previously described (Zabner et al.,
J.CIin.Invest.
100:1144-1149, 1997). Freshly isolated cells were seeded at a density of S x
105
cellslcm2 onto collagen-coated permeable membranes (0.6 cm2/Millipore-
Inserts). The
cells were maintained at 37°C in a humidified atmosphere of 7% COZ and
air. Twenty-
four hours after plating, the mucosal media was removed and the cells were
allowed to
grow at the air-liquid interface. The culture medium was a mixture of 49%
DMEM, 49%
Ham's F12 and 2% Ultraser G (Sepracor Inc:, Marlborough, PA). Penicillin 100
U/ml
and streptomycin 100 ug/ml were added to the media.
The airway epithelia were then cultured for 14 days at the air-liquid
interface.
The cells were exposed to 50 MOI of the modified viruses in 50 ul of PBS for
30 min,
and then rinsed twice with PBS. Seventy-two hours later, [i-galactosidase
expression was
measured using a commercially available galactocyte assay using AMPGD (3-(4-
methoxyspiro [1,2-dioxethane-3,2'-tricyclo-[3.3.1.13'] decan]-4-yl) phenyl-~3-
D-
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galactopyranoside) (GalactolightTM) assay (Tropix, Bedford, MA). Briefly,
after rinsing
with PBS, cells were removed from filters by incubation with 120 ul lysis
buffer ( 25 mM
Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-
tetraacetic acid; 10% glycerol; and 1% Triton X-100) for 15 min. Light
emission was
quantified in a luminometer (Analytical Luminescence Laboratory, San Diego,
CA) and
expressed as light units (LU).
The results are shown in Figure 7. Substitution of the sss.l7 peptide in hexon
loop 1 increased transduction of well-differentiated human airway cells 2-
fold, in
comparison to wild-type vector (Ad2/(3-gal-4). Substitution with the Adl7 RGD
peptide
in the hexon protein increased transduction efficiency 2.5-fold.
EXAMPLE 8: Transduction Efl~iciency of Mouse Airways
with Hexon-Modified Adenoviral Vectors
Balb/c mice were instilled with 5 x 108 infectious units (ILn of each
adenoviral
vector. At 3 days post-instillation, mice were sacrificed to determine ~i-gal
expression in
the lungs by X-gal staining and AMPGD analysis, as described in Example 7. The
results
are expressed in relative light units per microgram protein (RLU/~,g).
Substitution of the sss.l7 peptide in the hexon loop 1 of an adenoviral
vecvtor
increases transduction efficiency 2.4-fold, in comparison to wild-type vector
(Ad2/~i-gal
4), as shown in Figure 8. Substitution with the AdI7RGD peptide in hexon loop
1
increases transduction efllciency 2.5-fold. The results from assays on
individual animals
and calculation of the mean result from all assays in shown in Figure 9.
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1
SEQUENCE LISTING
<110> Romanczuk, Helen
Armentano, Donna
O'Riordan, Catherine E.
<120> ADENOVIRAL VECTORS WITH MODIFIED CAPSID
PROTEINS
<130> 31366-PCT
<150> 60/071,674
<151> 1998-O1-16
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2
<210> 4
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His Glu Thr Phe Tyr Ser Met Ile Arg Ser Leu Ala
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4
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