U.S. patent application number 14/326293 was filed with the patent office on 2015-01-01 for methods of gene delivery using capsid-modified raav expression systems.
The applicant listed for this patent is University of Florida Research Foundation, Inc.. Invention is credited to Nicholas Muzyczka, Shaun R. Opie, Kenneth H. Warrington.
Application Number | 20150005369 14/326293 |
Document ID | / |
Family ID | 32030574 |
Filed Date | 2015-01-01 |
United States Patent
Application |
20150005369 |
Kind Code |
A1 |
Muzyczka; Nicholas ; et
al. |
January 1, 2015 |
METHODS OF GENE DELIVERY USING CAPSID-MODIFIED RAAV EXPRESSION
SYSTEMS
Abstract
Disclosed are methods of gene delivery using capsid-modified
recombinant adeno-associated viral (rAAV) vectors. Exemplary
methods are provided employing vectors that have altered affinity
for heparin or heparin sulfate, as well as vectors, expression
systems, and rAAV virions that lack functional VP2 protein
expression, but are nevertheless, fully virulent. Also provided by
the invention are methods employing the rAAV vector-based
compositions, virus particles, host cells, and pharmaceutical
formulations in the expression of selected therapeutic proteins,
polypeptides, peptides, antisense oligonucleotides and/or ribozymes
in selected mammals, including organs, tissues, and human host
cells.
Inventors: |
Muzyczka; Nicholas;
(Gainesville, FL) ; Opie; Shaun R.; (Phoenix,
AZ) ; Warrington; Kenneth H.; (Gainesville,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Florida Research Foundation, Inc. |
Gainesville |
FL |
US |
|
|
Family ID: |
32030574 |
Appl. No.: |
14/326293 |
Filed: |
July 8, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10513059 |
Oct 2, 2005 |
8802080 |
|
|
PCT/US03/13583 |
May 1, 2003 |
|
|
|
14326293 |
|
|
|
|
60377315 |
May 1, 2002 |
|
|
|
Current U.S.
Class: |
514/44R ;
435/350; 435/351; 435/354; 435/363; 435/366; 435/368; 435/369;
435/370; 435/371; 435/375 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 15/86 20130101; C12N 2810/50 20130101; C12N 2810/858 20130101;
A61K 48/005 20130101; C12N 2750/14143 20130101; C12N 2750/14122
20130101; C12N 2750/14145 20130101 |
Class at
Publication: |
514/44.R ;
435/350; 435/351; 435/354; 435/363; 435/366; 435/368; 435/369;
435/370; 435/371; 435/375 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
Nos. HL59412 and HL51811 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A method for targeting a selected therapeutic agent to a
mammalian cell, the method comprising providing to the cell an
effective amount of a composition comprising: (a) an rAAV vector
system that comprises or that encodes a selected therapeutic agent,
and that further comprises: (i) a first expression vector
comprising a coding sequence of a first AAV capsid protein under
control of an expression control sequence; and (ii) a second
expression vector comprising coding sequences for a second AAV
capsid protein under control of an expression control sequence and
a third AAV capsid protein under control of an expression control
sequence; wherein the first and the second expression vectors are
not on the same nucleic acid segment, and wherein the first, the
second, and the third capsid proteins are distinct capsid proteins
selected from the group consisting of an AAV2 Vp1 protein, an AAV2
Vp2 protein and an AAV2 Vp3 protein, and further wherein the
composition is modified 1) by a mutation in the coding sequence for
the Vp2 protein such that the coding sequence for Vp2 does not
express a functional AAV Vp2 capsid protein and/or 2) by at least
one mutation such that binding to HPSG is altered, impaired, or
prevented, wherein the at least one mutation is an
arginine-to-alanine mutation, or an arginine-to-lysine mutation at
an amino acid residue corresponding to R487, R585, or R588 of an
AAV2 capsid protein; or (b) an infectious rAAV virion that
comprises or that encodes a selected therapeutic agent, and that
further comprises: (i) a first expression vector comprising a
coding sequence of a first AAV capsid protein under control of an
expression control sequence; and (ii) a second expression vector
comprising coding sequences for a second AAV capsid protein under
control of an expression control sequence, and a third AAV capsid
protein under control of an expression control sequence; wherein
the first and the second expression vectors are not on the same
nucleic acid segment, and wherein the first, the second, and the
third capsid proteins are distinct capsid proteins selected from
the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein
and an AAV2 Vp3 protein, and further wherein the composition is
modified 1) by a mutation in the coding sequence for the Vp2
protein such that the coding sequence for Vp2 does not express a
functional AAV Vp2 capsid protein and/or 2) by at least one
mutation such that binding to HPSG is altered, impaired, or
prevented, wherein the at least one mutation is an
arginine-to-alanine mutation, or an arginine-to-lysine mutation at
an amino acid residue corresponding to R487, R585, or R588 of an
AAV2 capsid protein.
2. The method of claim 1, wherein the selected therapeutic agent
comprises a ribozyme, an antisense molecule, a peptide, a
polypeptide, a protein, an antibody or an antigen binding fragment
thereof, or any combination thereof.
3. The method of claim 2, wherein the selected therapeutic agent is
a protein or a polypeptide selected from the group consisting of an
adrenergic agonist, an anti-apoptosis factor, an apoptosis
inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an
erythropoietic agent, a glutamic acid decarboxylase, a
glycoprotein, a growth factor, a growth factor receptor, a hormone,
a hormone receptor, an interferon, an interleukin, an interleukin
receptor, a kinase, a kinase inhibitor, a nerve growth factor, a
netrin, a neuroactive peptide, a neuroactive peptide receptor, a
neurogenic factor, a neurogenic factor receptor, a neuropilin, a
neurotrophic factor, a neurotrophin, a neurotrophin receptor, an
N-methyl-D-aspartate antagonist, a plexin, a protease, a protease
inhibitor, a protein decarboxylase, a protein kinase, a protein
kinase inhibitor, a proteolytic protein, a proteolytic protein
inhibitor, a semaphorin, a semaphorin receptor, a serotonin
transport protein, a serotonin uptake inhibitor, a serotonin
receptor, a serpin, a serpin receptor, and a tumor suppressor.
4. The method of claim 3, wherein the protein or polypeptide is
selected from the group consisting of BDNF, CNTF, CSF, EGF, FGF,
G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF,
TGF, TGF-B2, TNF, VEGF, prolactin, somatotropin, XIAP1, IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and
IL-18.
5. The method of claim 1, wherein the mammalian cell is a human, a
primate, a murine, a feline, a canine, a porcine, an ovine, a
bovine, an equine, an epine, a caprine, or a lupine cell.
6. The method of claim 5, wherein the mammalian cell is a human
endothelial cell, a human vascular cell, a human epithelial cell, a
human liver cell, a human lung cell, a human cardiac cell, a human
pancreatic cell, a human renal cell, a human muscle cell, a human
bone cell, a human neural cell, a human blood cell, or a human
brain cell.
7. The method of claim 1, wherein the composition further comprises
a liposome, a lipid, a lipid complex, a microsphere, a
microparticle, a nanosphere, a nanoparticle, or any combination
thereof.
8. A method for treating or ameliorating one or more symptoms of a
disease, a dysfunction, or a deficiency in a mammal, the method
comprising: administering to a mammal in need thereof, an effective
amount of a composition comprising: (a) an rAAV vector system that
comprises or that encodes a selected therapeutic agent, and that
further comprises: (i) a first expression vector comprising a
coding sequence of a first AAV capsid protein under control of an
expression control sequence; and (ii) a second expression vector
comprising coding sequences for a second AAV capsid protein under
control of an expression control sequence and a third AAV capsid
protein under control of an expression control sequence; wherein
the first and the second expression vectors are not on the same
nucleic acid segment, and wherein the first, the second, and the
third capsid proteins are distinct capsid proteins selected from
the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein
and an AAV2 Vp3 protein, and further wherein the composition is
modified 1) by a mutation in the coding sequence for the Vp2
protein such that the coding sequence for Vp2 does not express a
functional AAV Vp2 capsid protein and/or 2) by at least one
mutation such that binding to HPSG is altered, impaired, or
prevented, wherein the at least one mutation is an
arginine-to-alanine mutation, or an arginine-to-lysine mutation at
an amino acid residue corresponding to R487, R585, or R588 of an
AAV2 capsid protein; or (b) an infectious rAAV virion that
comprises or that encodes a selected therapeutic agent, and that
further comprises: (i) a first expression vector comprising a
coding sequence of a first AAV capsid protein under control of an
expression control sequence; and (ii) a second expression vector
comprising coding sequences for a second AAV capsid protein under
control of an expression control sequence, and a third AAV capsid
protein under control of an expression control sequence; wherein
the first and the second expression vectors are not on the same
nucleic acid segment, and wherein the first, the second, and the
third capsid proteins are distinct capsid proteins selected from
the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein
and an AAV2 Vp3 protein, and further wherein the composition is
modified 1) by a mutation in the coding sequence for the Vp2
protein such that the coding sequence for Vp2 does not express a
functional AAV Vp2 capsid protein and/or 2) by at least one
mutation such that binding to HPSG is altered, impaired, or
prevented, wherein the at least one mutation is an
arginine-to-alanine mutation, or an arginine-to-lysine mutation at
an amino acid residue corresponding to R487, R585, or R588 of an
AAV2 capsid protein; in an amount and for a time sufficient to
treat or to ameliorate the one or more symptoms of the disease, the
dysfunction, or the deficiency in the mammal.
9. The method of claim 8, wherein the composition is administered
to the mammal intra-muscularly, intravenously, subcutaneously,
intrathecally, intraperitoneally, or by direct injection into one
or more organs or tissues of the mammal.
10. The method of claim 9, wherein the one or more organs or
tissues are selected from the group consisting of pancreas, liver,
heart, lung, brain, kidney, joint, and muscle.
11. The method of claim 8, wherein the rAAV vector system or the
infectious rAAV virion further comprises a third distinct
expression vector that comprises an expression cassette flanked by
AAV2 terminal repeat sequences.
12. The method of claim 11, wherein the expression cassette
comprises a first polynucleotide that comprises a first nucleic
acid segment that encodes a selected therapeutic agent.
13. The method of claim 12, wherein the selected therapeutic agent
is a peptide, a polypeptide, a protein, a catalytic RNA molecule, a
ribozyme, an antisense oligonucleotide, or an antisense
polynucleotide.
14. The method of claim 12, wherein the first polynucleotide
further comprises a promoter operably linked to the first nucleic
acid segment, wherein the promoter controls expression of the
selected therapeutic agent in a mammalian cell.
15. The method of claim 14, wherein the promoter is a heterologous
promoter, a tissue-specific promoter, a constitutive promoter, or
an inducible promoter.
16. The method of claim 12, wherein the first polynucleotide
further comprises an enhancer sequence operably linked to the first
nucleic acid segment.
17. The method of claim 16, wherein the enhancer sequence comprises
a CMV enhancer, a synthetic enhancer, a liver-specific enhancer, an
vascular-specific enhancer, a brain-specific enhancer, a neural
cell-specific enhancer, a lung-specific enhancer, a muscle-specific
enhancer, a kidney-specific enhancer, a pancreas-specific enhancer,
or an islet cell-specific enhancer.
18. The method of claim 12, wherein the first polynucleotide
further comprises a post-transcriptional regulatory sequence or a
polyadenylation signal operably linked to the first nucleic acid
segment.
19. The method of claim 18, wherein the post-transcriptional
regulatory sequence is obtained from a woodchuck hepatitis virus
post-transcription regulatory element, or wherein the
polyadenylation signal is obtained from a bovine growth hormone
gene.
20. A method for targeting an AAV virion or viral particle to a
mammalian cell that comprises a cell-surface receptor, the method
comprising: providing to a population of mammalian cells a
recombinant adeno-associated viral expression system comprising:
(i) a first expression vector comprising a coding sequence of a
first AAV capsid protein under control of an expression control
sequence; and (ii) a second expression vector comprising coding
sequences for a second AAV capsid protein under control of an
expression control sequence and a third AAV capsid protein under
control of an expression control sequence; wherein the first and
the second expression vectors are not on the same nucleic acid
segment, and wherein the first, the second, and the third capsid
proteins are distinct capsid proteins selected from the group
consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2
Vp3 protein, and further wherein the composition is modified 1) by
a mutation in the coding sequence for the Vp2 protein such that the
coding sequence for Vp2 does not express a functional AAV Vp2
capsid protein and/or 2) by at least one mutation such that binding
to HPSG is altered, impaired, or prevented, wherein the at least
one mutation is an arginine-to-alanine mutation, or an
arginine-to-lysine mutation at an amino acid residue corresponding
to R487, R585, or R588 of an AAV2 capsid protein; in an amount and
for a time effective to target the virion or the viral particle to
one or more cells of the population that express the cell-surface
receptor.
21. The method of claim 20, wherein the rAAV vector system further
comprises a third distinct expression vector that comprises an
expression cassette flanked by AAV2 terminal repeat sequences,
wherein the expression cassette comprises a first polynucleotide
that comprises a first nucleic acid segment that encodes a selected
therapeutic agent operably linked to a promoter that controls
expression of the selected therapeutic agent in a mammalian
cell.
22. The method of claim 21, wherein the selected therapeutic agent
is a peptide, a polypeptide, a protein, a catalytic RNA molecule, a
ribozyme, an antisense oligonucleotide, or an antisense
polynucleotide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 10/513,059, filed Oct. 2, 2005 (allowed; Atty.
Dkt. No. 36689.32); which was a U.S. .sctn.371 nationalization of
PCT Intl. Pat. Appl. No. PCT/US03/13583, filed May 1, 2003
(nationalized); which claims priority to U.S. Prov. Pat. Appl. No.
60/377,315, filed May 1, 2002 (expired); the contents of each of
which is specifically incorporated herein in its entirety by
express reference thereto.
NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT
[0003] Not Applicable.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention relates generally to the fields of
molecular biology and virology, and in particular, to the
development of gene delivery vehicles. The invention provides
improved recombinant adeno-associated virus (rAAV) vectors that
while deleted for VP2, are still able to form infectious virion
particles, as well as other AAV vector compositions useful in
expressing a variety of nucleic acid segments, including those
encoding therapeutic proteins polypeptides, peptides, antisense
oligonucleotides, and ribozyme constructs, in various gene therapy
regimens. Methods are also provided for preparing and using these
modified rAAV-based vector constructs in a variety of viral-based
gene therapies, and in particular, treatment and prevention of
human diseases using conventional gene therapy approaches. The
invention also provides multicomponent vector systems which may be
used to assess the relative efficiency and infectivity of a variety
of AAV particles having mutated, or deleted capsid proteins.
[0006] 2. Description of Related Art
[0007] Major advances in the field of gene therapy have been
achieved by using viruses to deliver therapeutic genetic material.
The adeno-associated virus (AAV) has attracted considerable
attention as a highly effective viral vector for gene therapy due
to its low immunogenicity and ability to effectively transduce
non-dividing cells. AAV has been shown to infect a variety of cell
and tissue types by using heparin sulfate proteoglycan (HSPG) as
its primary cellular receptor. The natural tropism of AAV for the
abundantly expressed HSPG presents a challenge to specifically
targeting particular cell populations. For safety and targeting
considerations it is highly desirable to have a vector that cannot
infect its natural host cell types.
SUMMARY OF THE INVENTION
[0008] The present invention overcomes these and other limitations
inherent in the prior art by providing new rAAV-based genetic
constructs that encode one or more mammalian therapeutic
polypeptides for the prevention, treatment, and/or amelioration of
various disorders resulting from a deficiency in one or more of
such polypeptides. In particular, the invention provides AAV-based
genetic constructs encoding one or more mammalian therapeutic
proteins, polypeptides, peptides, antisense oligonucleotides, and
ribozymes, as well as variants, and/or active fragments thereof,
for use in the treatment and prophylaxis of a variety of conditions
and mammalian diseases and disorders.
[0009] Current AAV2 targeting strategies involve inserting DNA
sequences that code for specific receptor ligands within the capsid
open reading frame of the pIM45 plasmid. While this approach has
identified surface positions capable of tolerating peptide
insertions, there are certain limitations. Because the three capsid
proteins share the same open reading frame and stop codon, the
amino acid sequence of the major capsid protein, VP3, and any
peptide ligands inserted in this region of the open reading frame,
are contained within the 2 larger and significantly less abundant
capsid proteins, VP1 and VP2.
[0010] In order to target peptide ligands to a specific capsid
protein, the inventors have investigated an alternative method for
the production of recombinant AAV2 vectors. By mutating the capsid
proteins' start codons the inventors have generated pIM45 plasmids
that only express one capsid protein: pIM45-VP1, pIM45-VP2
(acg/atg), and pIM45-VP3. Such plasmids can be complemented with
plasmids that express the remaining 2 capsid proteins (pIM45-VP2,3,
pIM45-VP1,3, and pIM45-VP1,2; respectively) in order to produce
viable recombinant AAV2 vectors. Interestingly, the plasmid,
pIM45-VP1,3, is also capable of producing infectious virions in the
absence of VP2 expression. Expression of the capsid proteins in
this manner allows for the genetic modification of a specific
capsid protein across its entire sequence. As a result, more
control of the position and number of expressed peptide insertions
is obtained in producing recombinant AAV2 vectors. This system
allows for the production of novel targeted recombinant AAV2
vectors containing significantly larger peptide insertions in an
individual capsid protein without disruption of the remaining
capsid structure.
[0011] In one embodiment, the invention concerns rAAV vectors that
comprise a nucleic acid segment modified to express functional VP1
and VP3 capsid proteins substantially in the absence of functional
VP2 protein. Surprisingly, the inventors have shown that such a
vector can produce an infectious virion in the absence of exogenous
VP2 protein.
[0012] The lack or substantial absence of functional VP2 protein
may be the result of at least a first mutation in the capsid gene
sequence region that comprises the VP2 start codon, or
alternatively in the VP2 start codon itself. An exemplary vector
described herein is pIM45-VP1,3.
[0013] In another embodiment, the invention concerns rAAV vectors
that comprise a nucleic acid segment modified to express functional
VP1 and VP2 capsid proteins substantially in the absence of
functional VP3 protein. Although such vector cannot produce an
infectious virion in the absence of exogenous VP3 protein, if a
second helper vector that encodes a functional VP3 protein is
employed to coinfect cells with this vector, infectious virions can
be obtained.
[0014] The lack or substantial absence of functional VP3 protein
may be the result of at least a first mutation in the capsid gene
sequence region that comprises the VP3 start codon, or
alternatively in the VP3 start codon itself. An exemplary vector
described herein is pIM45-VP1,2.
[0015] In a third embodiment, the invention concerns rAAV vectors
that comprise a nucleic acid segment modified to express functional
VP2 and VP3 capsid proteins substantially in the absence of
functional VP1 protein. Although such vector cannot produce an
infectious virion in the absence of exogenous VP1 protein, if a
second helper vector that encodes a functional VP1 protein is
employed to coinfect cells with this vector, infectious virions can
be obtained.
[0016] The lack or substantial absence of functional VP1 protein
may be the result of at least a first mutation in the capsid gene
sequence region that comprises the VP1 start codon, or
alternatively in the VP1 start codon itself. An exemplary vector
described herein is pIM45-VP2,3.
[0017] A yet further embodiment of the invention is an expression
vector that expresses an rAAV capsid protein selected from the
group consisting of VP1, VP2, and VP3, each in the absence of
substantially any other rAAV protein, such as the other capsid
proteins or helper functions.
[0018] This expression vector may comprise, for example, a mutation
at position 1 of the cap gene, a mutation at position 138 of the
cap gene, or a mutation at position 203 of the cap gene. Exemplary
such vectors provided herein are pIM45-VP1, pIM45-VP2, or
pIM45-VP3, which produce substantially a single VP1, VP2, or VP3
protein, respectively.
[0019] Another embodiment of the invention is an expression vector
that expresses: (a) rAAV capsid proteins VP1 and VP2 in the absence
of substantial amounts of VP3 protein; (b) rAAV capsid proteins VP1
and VP3 in the absence of substantial amounts of VP2 protein; or
(c) rAAV capsid proteins VP2 and VP3 in the absence of substantial
amounts of VP1 protein.
[0020] Such vector typically comprises: (a) at least one mutation
in the start codon of the VP1 protein and at least one mutation in
the start codon of the VP2 protein; (b) at least one mutation in
the start codon of the VP1 protein and at least one mutation in the
start codon of the VP3 protein; or (c) at least one mutation in the
start codon of the VP2 protein and at least one mutation in the
start codon of the VP3 capsid protein.
[0021] For example, the vector may comprise: (a) at least one
mutation at position 1 and at least one mutation at position 138 of
the cap gene, (b) at least one mutation at position 1 and at least
one mutation at position 203 of the cap gene; or (c) at least one
mutation at position 138 and at least one mutation at position 203
of the cap gene. Vectors pIM45-VP1,2; pIM45-VP1,3; and pIM45-VP2,3
described herein, are representative examples of each of such
vectors, respectively.
[0022] The invention also provides in an important embodiment, an
rAAV expression system substantially lacking in expression of VP2
protein. This VP2-free system comprises:(a) at least a first rAAV
vector comprising at least a first heterologous nucleic acid
segment inserted into the capsid sequence region, with the segment
encoding at least a first heterologous peptide; and (b) at least a
second expression vector that expresses functional VP1 and VP3
capsid proteins in the absence of substantial quantities of VP2
protein, or at least a second and a third expression vector that
separately express functional VP1 and VP3 capsid proteins, each of
these second and third plasmids expressing a single VP1 or VP3
protein, both in the absence of substantial amounts of VP2
protein.
[0023] For example, the system will preferably comprise at least a
first rAAV vector that substantially lacks VP2 expression. Such
expression systems will give rise to infectious virions, so long as
the helper plasmids provide sufficient exogenous VP1 and VP3
protein to permit the rAAV vector to form the capsid.
[0024] In one embodiment, when it is desirable to "target"
particular cells, cell surfaces, or cell surface ligands or
receptors, it may be desirable to alter the sequence of the capsid
gene through the addition of one or more relatively short nucleic
acid segments that encode at least 1 or more targeting peptides
that, when these heterologous peptides are expressed on the surface
of an rAAV virion comprising the vector, the peptide sequence
contained within the altered capsid protein will permit the
selective targeting of the rAAV virions comprising them to one or
more specific types of cells, cell surfaces, or cell surface
receptors when the particles are used to transfect a plurality or
population of such host cells. The inventors contemplate that the
exploitation of such targeting peptide sequences, when expressed on
the surface of the rAAV virions as contained within the capsid
proteins, may be critical in localizing, enhancing, improving, or
increasing the specificity of the rAAV virions for a particular
cell type, or may even be useful in permitting transduction of
cells or cell types that previously were not appropriate host cells
for AAV infection. Such methods could be particularly desirable in
altering the native affinity of one or more of the various known
serotypes of AAV to one or more host cells not previously capable
of efficient transfection by one or more particular serotypes. For
example, by appropriate insertion of one or more peptide epitopes,
ligands, or recognition sequences, an rAAV serotype 1 vector may be
able to efficiently transfect a cell line not readily transfected
by wild-type rAAV1 vectors. Likewise, an rAAV serotype 2 vector may
be sufficiently modified by addition of appropriate targeting
ligands to effectively transfect one or more cell lines, cells
types, tissues, or organs, not previously capable of efficient
transfection using the unmodified wild-type rAAV2 vector.
[0025] As such, preferred embodiments include those VP2-free rAAV
expression systems, wherein at least a first peptide inserted into
one or more of the capsid protein sequences, permits the rAAV
virion to transfect a specific organ tissue, or host cell, with a
higher efficiency than an unmodified rAAV vector.
[0026] The VP2-free rAAV expression systems of the invention may
utilize any rAAV vector, including those of serotypes 1, 2, 3, 4,
5, or 6, and may employ at least two helper plasmids such as
pIM45-VP1, pIM45-VP2, or pIM45-VP3, as the second and third
expression vectors required in the system to provide exogenous VP1,
VP2, and/or VP3 as may be required for efficient virion formation
by the rAAV vectors. When only a second helper plasmid is desired,
a single vector may be employed such as, for example, pIM45-VP1,3.
Alternatively, so long as at least VP1 and VP3 are provided to the
system, either on a single plasmid, each on separate plasmids, or
by exogenous supplementation of one or both of the purified
protein(s) themselves, a fully functional, fully virulent rAAV
virion may be reconstituted from the disclosed expression system,
either in the presence of functional VP2 protein, or alternatively,
substantially in the absence of any endogenously- or
exogenously-provided VP2 protein.
[0027] When the use of such vectors is contemplated for
introduction of one or more exogenous proteins, polypeptides,
peptides, ribozymes, and/or antisense oligonucleotides, to a
particular cell transfected with the vector, one may employ the
rAAV vectors or the VP2-free rAAV expression systems disclosed
herein by genetically modifying the vectors to further comprise at
least a first exogenous polynucleotide operably positioned
downstream and under the control of at least a first heterologous
promoter that expresses the polynucleotide in a cell comprising the
vector to produce the encoded peptide, protein, polypeptide,
ribozyme, or antisense oligonucleotide. Such constructs may employ
heterologous promoters that are constitutive, inducible, or even
cell-specific promoters. Exemplary such promoters include, but are
not limited to, a CMV promoter, a .beta.-actin promoter, a hybrid
CMV promoter, a hybrid .beta.-actin promoter, an EF1 promoter, a
U1a promoter, a U1b promoter, a Tet-inducible promoter and a
VP16-LexA promoter.
[0028] The vectors or expression systems may also further comprise
one or more enhancers, regulatory elements, transcriptional
elements, to alter or effect transcription of the heterologous gene
cloned in the rAAV vectors. For example, the rAAV vectors of the
present invention may further comprise at least a first CMV
enhancer, a synthetic enhancer, or a cell- or tissue-specific
enhancer. The exogenous polynucleotide may also further comprise
one or more intron sequences.
[0029] In other aspects, the invention concerns methods for
altering, reducing, or eliminating, the binding of particular rAAV
vectors for particular ligands. In an illustrative embodiment, the
invention provides rAAV vectors that have altered affinity for
heparin, heparin sulfate, and heparin sulfate proteoglycan. This
vector comprises at least a first mutation in the capsid gene,
wherein the mutation substantially reduces or eliminates the
affinity of a viral particle comprising the vector for binding to
heparin, heparin sulfate, or heparin sulfate proteoglycan.
Preferably, these rAAV vectors comprise one or more Arginine to
Alanine mutations, and particularly one or more Arginine to Alanine
mutations at position 585 or position 588 of the capsid polypeptide
sequence. In rAAV vectors comprising either a single R585A or R588A
mutation, or a double mutant comprising both the R585A and the
R588A mutations, affinity for heparin sulfate binding by the vector
was eliminated. Such vectors are therefore important when one
wishes to design improved rAAV vectors that comprise particular
capsid protein mutations that either have increased or reduced
affinity for one or more particular ligands.
[0030] In all aspects of the invention, the exogenous
polynucleotides that are comprised within one or more of the
improved rAAV vectors disclosed herein will be of mammalian origin,
with human, murine, porcine, bovine, ovine, feline, canine, equine,
epine, caprine, and lupine polynucleotides being particularly
preferred.
[0031] As described above, the exogenous polynucleotide will
preferably encode one or more proteins, polypeptides, peptides,
ribozymes, or antisense oligonucleotides, or a combination of
these. In fact, the exogenous polynucleotide may encode two or more
such molecules, or a plurality of such molecules as may be desired.
When combinational gene therapies are desired, two or more
different molecules may be produced from a single rAAV expression
system, or alternatively, a selected host cell may be transfected
with two or more unique rAAV expression systems, each of which will
provide unique heterologous polynucleotides encoding at least two
different such molecules.
[0032] In other embodiment, the invention also concerns the
disclosed rAAV vectors comprised within an infectious
adeno-associated viral particle, comprised within one or more
pharmaceutical vehicles, and may be formulated for administration
to a mammal such as a human for therapeutic, and/or prophylactic
gene therapy regimens. Such vectors may also be provided in
pharmaceutical formulations that are acceptable for veterinary
administration to selected livestock, domesticated animals, pets,
and the like.
[0033] The invention also concerns host cells that comprise the
disclosed rAAV vectors and expression systems, particularly
mammalian host cells, with human host cells being particularly
preferred.
[0034] Compositions comprising one or more of the disclosed rAAV
vectors, expression systems, infectious AAV particles, host cells
also form part of the present invention, and particularly those
compositions that further comprise at least a first
pharmaceutically-acceptable excipient for use in the manufacture of
medicaments and methods involving therapeutic administration of
such rAAV vectors. Such pharmaceutical compositions may optionally
further comprise liposomes, a lipid, a lipid complex; or the rAAV
vectors may be comprised within a microsphere or a nanoparticle.
Pharmaceutical formulations suitable for intramuscular,
intravenous, or direct injection into an organ or tissue of a human
are particularly preferred.
[0035] Other aspects of the invention concern recombinant
adeno-associated virus virion particles, compositions, and host
cells that comprise one or more of the AAV vectors disclosed
herein, such as for example pharmaceutical formulations of the
vectors intended for administration to a mammal through suitable
means, such as, by intramuscular, intravenous, or direct injection
to cells, tissues, or organs of a selected mammal. Typically, such
compositions may be formulated with pharmaceutically-acceptable
excipients as described hereinbelow, and may comprise one or more
liposomes, lipids, lipid complexes, microspheres or nanoparticle
formulations to facilitate administration to the selected organs,
tissues, and cells for which therapy is desired.
[0036] Kits comprising one or more of the disclosed vectors,
virions, host cells, viral particles or compositions; and (ii)
instructions for using the kit in therapeutic, diagnostic, or
clinical embodiments also represent preferred aspects of the
present disclosure. Such kits may further comprise one or more
reagents, restriction enzymes, peptides, therapeutics,
pharmaceutical compounds, or means for delivery of the compositions
to host cells, or to an animal, such as syringes, injectables, and
the like. Such kits may be therapeutic kits for treating or
ameliorating the symptoms of particular diseases, and will
typically comprise one or more of the modified AAV vector
constructs, expression systems, virion particles, or therapeutic
compositions described herein, and instructions for using the
kit.
[0037] Another important aspect of the present invention concerns
methods of use of the disclosed vectors, virions, expression
systems, compositions, and host cells described herein in the
preparation of medicaments for treating or ameliorating the
symptoms of various polypeptide deficiencies in a mammal. Such
methods generally involve administration to a mammal, or human in
need thereof, one or more of the disclosed vectors, virions, host
cells, or compositions, in an amount and for a time sufficient to
treat or ameliorate the symptoms of such a deficiency in the
affected mammal. The methods may also encompass prophylactic
treatment of animals suspected of having such conditions, or
administration of such compositions to those animals at risk for
developing such conditions either following diagnosis, or prior to
the onset of symptoms.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to the following description taken in
conjunction with the accompanying drawings, in which like reference
numerals identify like elements, and in which:
[0039] FIG. 1A-1, FIG. 1A-2, FIG. 1B-1, FIG. 1B-2, FIG. 1C-1, and
FIG. 1C-2 show generation of plasmids that express two capsid
proteins through missense mutation of individual capsid protein
start codons. FIG. 1A shows mutations required to eliminate VP1 and
VP2 expression. Immunoblot of whole cell lysates using B1 antibody
that recognizes all three capsids following transfection of
plasmids, pIM45(lane 1); pIM45-VP2,3 (lane 2); pIM45-VP1,3 (lane
3); and pIM45-M203L (lane 4). Note: lane 4 is the initial attempt
to produce plasmid that expresses only VP1 and VP2. Further
mutations are required. FIG. 1B-1 and FIG. 1B-2 show mutations
required to eliminate VP3 expression. Immunoblot of whole cell
lysates using B1 antibody that recognizes all three capsid
following transfection of pIM45 (lane 1); pIM45-M203L (lane 2);
pIM45-M203,211L (lane 3); pIM45-M203,211,235L (lane 4). Note,
pIM45-M203,211,235L is designated pIM45-VP1,2. FIG. 1C-1 and FIG.
1C-2 show alternative mutation used to eliminate VP3 expression
while maximizing expression of VP2 protein. Immunoblot of whole
cell lysates using B1 antibody that recognizes all three capsid
proteins following transfection of pIM45 (lane 1) and pIM45-VP1,2A
(lane 3) in which the start codon for VP2 protein is changed from
ACG to ATG.
[0040] FIG. 2A and FIG. 2B show generation of plasmids that express
a single capsid protein. Immunoblot of whole cell lysates using B1
antibody that recognizes all three capsid proteins following
transfection of pIM45 (lane 1); pIM45-VP1 (lane 2); pIM45-VP2 (lane
3) pIM45-VP2A (lane 4); and pIM45-VP3 (lane 5);
[0041] FIG. 3A-1, FIG. 3A-2, FIG. 3B-1, FIG. 3B-2, FIG. 3C-1 and
FIG. 3C-2 show production and purification of rAAV2-like particles
that lack expression of specific capsid proteins. FIG. 3A-1 and
FIG. 3A-2 show analysis of effects of missense mutations required
to eliminate VP3 expression. FIG. 3A-1 shows immunoblot using B1
antibody that recognizes all three capsid proteins of purified
particle stocks from pIM45 (lane 1); pIM45-M203L (lane 2);
pIM45-M211L (lane 3); pIM45-M235L (lane 4), pIM45-M203,211,235
(lane 5). FIG. 3A-2 shows dot blot autoradiograph of DNA extracted
from same particle stocks. Aliquots from an iodixinal step gradient
were with incubated with DNAseI, inactivated with EDTA, digested
with proteinase K, phenol:chloroform extracted, and precipitated
with ethanol. DNA was transferred to nitrocellulose and probed with
radiolabelled GFP probe. FIG. 3B-1 and FIG. 3B-2 show analysis of
effects of eliminating a single capsid on the production and
purification of virus particles. FIG. 3B-1 shows immunoblot using
B1 antibody that recognizes all three capsid proteins of purified
particle stocks from pIM45 (lane 1); pIM45-VP1,2 (lane 2);
pIM45-VP1,3 (lane 3); and pIM45-VP2,3 (lane 4). FIG. 3B-2 shows dot
blot autoradiograph of DNA extracted from same particle stocks.
Aliquots from an iodixinal step gradient were with incubated with
DNAse I, inactivated with EDTA, digested with proteinase K,
phenol:chloroform extracted, and precipitated with ethanol. DNA was
transferred to nitrocellulose and probed with radiolabelled GFP
probe (FIG. 3C-1 and FIG. 3C-2);
[0042] FIG. 4 shows complementation capsid plasmid groups employed
to produce viable rAAV2 particle preparations. Group VP0 is a
control group consisting of pIM45 and pIM45-VP0 (all capsid
expression eliminated). Group VP1 is group consisting of pIM45-VP1
and pIM45-VP2,3 in which expression of VP1 is isolated. Group
VP2/VP2A is group consisting of pIM45-VP2 or pIM45-VP2A and
pIM45-VP1,3 in which expression of VP2 is isolated, and in case of
pIM45-VP2A, VP2 expression is maximized. Group VP3 is group
consisting of pIM45-VP3 and pIM45-VP1,2 in which expression of VP3
is isolated. Isolation of specific capsid proteins allows genetic
modification of the isolated capsid without further modifying
remaining capsids. Alternatively, genetic modification of two
capsids can be accomplished without further modification of
remaining capsid. These groups are cotransfected with pXX6 (Ad
helper functions) and pTR-UF5 (terminal repeats flanking expression
cassette with CMV promoter driving expression of GFP) to produce
rAAV vectors;
[0043] FIG. 5A-1, FIG. 5A-2, FIG. 5B-1 and FIG. 5B-2 show
production and purification of rAAV2-like particles from
complementation groups described in FIG. 4. FIG. 5A-1 shows
immunoblot using B1 antibody that recognizes all three capsid
proteins of purified particle stocks from Group VP0(lane 1); Group
VP1 (lane 2); Group VP2 (lane 3); Group VP2A (lane 4); and Group
VP3 (lane 5). Note lane 4 shows production of particle stock with
increased level of VP2 protein in resultant particles composed of
all three capsid proteins. FIG. 5A-2 shows dot blot autoradiograph
of DNA extracted from same particle stocks. Aliquots from an
iodixinal step gradient were with incubated with DNAse I,
inactivated with EDTA, digested with proteinase K,
phenol:chloroform extracted, and precipitated with ethanol. DNA was
transferred to nitrocellulose and probed with radiolabelled GFP
probe. FIG. 5B-1 shows immunoblot using B1 antibody that recognizes
all three capsid proteins of purified particle stocks from
transfection of pIM45-VP2A and pIM45-VP3 showing production of
rAAV2-like particles composed of VP2 and VP3 with increased VP2
levels relative to VP3. FIG. 5B-2 shows dot blot autoradiograph of
DNA extracted from same particle stocks. Aliquots from an iodixinal
step gradient were with incubated with DNAseI, inactivated with
EDTA, digested with proteinase K, phenol:chloroform extracted, and
precipitated with ethanol. DNA was transferred to nitrocellulose
and probed with radiolabelled GFP probe;
[0044] FIG. 6A, FIG. 6B-1, FIG. 6B-2, FIG. 6C-1, and FIG. 6C-2
depict production of rAAV2-like particles with large peptide
insertions in VP1 and VP2 capsid proteins. FIG. 6A shows production
scheme for insertion of large peptides in VP1 and VP2 (top)
involves insertion of peptide immediately after amino acid 138 in a
plasmid that expresses only VP1 and VP2 (pIM45-VP1,2A) and
complementing this plasmid with plasmid, pIM45-VP3, to produce
particles. Production scheme for insertion of large peptides only
in VP2 (bottom) involves insertion of peptide immediately after
amino acid 138 in a plasmid that expresses only VP2 (pIM45-VP2A)
and complementing this plasmid with plasmid, pIM45-VP1,3 to produce
particles. FIG. 6B-1 and FIG. 6B-2 show immunoblot of purified
rAAV2-like particles produced by above production schemes with
protein, leptin, inserted in VP1 and VP2 or only in VP2. FIG. 6B-1
shows immunoblot probed with antibody recognizing all three capsids
proteins. FIG. 6B-2 shows immunoblot probed with antibody
recognizing inserted peptide, leptin. Both panels: Lane 1: pIM45;
Lane 2: pIM45-VP1,2A-Leptin/pIM45-VP3; Lane 3:
pIM45-VP2A-Leptin/pIM45-VP1,3; Lane 4: pIM45-VP3 only; and Lane 5:
pIM45-VP1,3 only. FIG. 6C-1 and FIG. 6C-2 show immunoblot of
purified rAAV2-like particles produced by above production schemes
with protein, GFP, inserted in VP1 and VP2 or only in VP2. FIG.
6C-1 shows immunoblot probed with antibody recognizing all three
capsids proteins. FIG. 6C-2 shows immunoblot probed with antibody
recognizing inserted peptide, GFP. Both panels: Lane 1: pIM45; Lane
2: pIM45-VP1,2A-GFP/pIM45-VP3; Lane 3: pIM45-VP2A-GFP/pIM45-VP1,3;
Lane 4: pIM45-VP3 only; and Lane 5: pIM45-VP1,3 only;
[0045] FIG. 7 shows Western blot of iodixanol virus stocks. Equal
volumes of virus stock were separated by 10% SDS-PAGE and analyzed
by Western blot using the B1 antibody;
[0046] FIG. 8 shows heparin-agarose binding profiles of mutant
capsids. Approximately 5.times.10.sup.10 particles were applied to
500 .mu.L of heparin-agarose affinity matrix at a NaCl
concentration of 100 mM, washed extensively with the loading
buffer, and bound capsids were eluted with 2 M NaCl. Pooled
fractions were denatured and slot blotted onto nitrocellulose for
immunodetection with mAb B1. For each mutant, L is the total amount
of iodixanol purified virus that was loaded onto the heparin
agarose column; FT is the total virus that flowed through the
column, W is the wash; E is the eluate;
[0047] FIG. 9A and FIG. 9B show production and purification of AAV
serotypes. FIG. 9A shows equivalent amounts of iodixanol purified
AAV1, AAV2 and AAV5 were separated by 10% PAGE and analyzed by
Western blot using the B1 antibody. FIG. 9B shows heparin-agarose
binding properties of AAV2, AAV1 and AAV5. Abbreviations are the
same as FIG. 8;
[0048] FIG. 10 shows particle-to-infectivity ratios of mutants
relative to wild type. The particle-to-infectivity ratio for each
mutant was calculated by dividing the average genomic titer by the
average green cell assay titer (Table 2). The P/I ratio of each
mutant was then normalized to wild type by dividing the P/I of each
mutant by the P/I of wild type rAAV2, and the log.sub.10 value of
the ratio was plotted. Wild type, therefore, equals one, and is
indicated by the dashed line. Hatched bars, mutant viruses with
infectivity comparable to wild type; gray bars, mutant viruses that
are heparin binding deficient; White bars, mutant viruses with an
undetermined block to infectivity; Asterisks indicate those mutants
for which no green cells were scored. For these mutants the green
cell assay titer used was the limit of detection in the assay.
Thus, the log difference is a minimum estimate.
[0049] FIG. 11 shows GFP transduction ability of mutants in HeLa
C12 cells. Cells were infected with wild type rAAV or mutant virus
at an MOI=500 genomic particles and an Ad5 MOI=10 pfu per cell.
Twenty-four hrs post infection cells were fixed with 2%
paraformaldehyde and the number of GFP positive cells was
determined by FACS analysis;
[0050] FIG. 12A and FIG. 12B show binding and uptake of rAAV2 and
R585A/R588A genomes in Hela C12 cells. FIG. 12A shows 10.sup.6
cells were infected with rAAV2 or R585/R588A at an M01=100 or 1000
genome containing particles per cell, respectively. At the
indicated times, infection media was removed and saved. The cells
were washed and harvested, and Hirt DNA was extracted from both the
infection media and the cell pellet. Southern analysis was
performed using an [.alpha.-.sup.32P]-dATP labeled GFP probe. FIG.
12B shows the percent bound/internalized DNA was calculated by
dividing the total DNA present in both the media and the cell
pellet by the amount bound/internalized for each time point. The
average of three determinations is shown. Error bars indicate a
standard deviation;
[0051] FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D show modifying the
heparin binding properties of AAV5. FIG. 13A shows alignment of
AAV2 amino acid residues 585 through 590 (RGNRQA; SEQ ID NO:1) to
residues predicted by amino acid alignment to be structurally
equivalent in AAV5 (SNSNLP; SEQ ID NO:2). FIG. 13B shows Western
blot of iodixanol virus stocks. Equal volumes of virus were
separated by 10% SDS-PAGE and analyzed by Western blot using the B1
antibody. FIG. 13C shows novel heparin binding properties of
AAV5-HS. Heparin-agarose binding was performed as described in FIG.
8. See FIG. 8 for abbreviations. FIG. 13D shows the log of the
particle-to-infectivity ratio of the rAAV5 variants normalized to
wild type rAAV2 as described in FIG. 10;
[0052] FIG. 14 shows an immunoslotblot of total capsid protein from
novel production system following standard purification procedures.
Immunoslotblot was probed with anti-VP1,2,3 monoclonal antibody.
Lane 1: pIM45/pIM45-VPO; Lane 2: pIM45-VP1/pIM45-VP2,3; Lane 3:
pIM45-VP2acg/pIM45-VP1,3; Lane 4: pIM45-VP2atg/pIM45-VP1,3; and
Lane 5: pIM45-VP3/pIM45-VP1,2;
[0053] FIG. 15 shows a dot blot autoradiograph of DNA extracted
from pTR-UF5 and system plasmid combinations. Numbering scheme is
the same as described in FIG. 14. Equal volume aliquots from an
iodixinol step gradient were with incubated with DNAseI,
inactivated with EDTA, digested with proteinase K,
phenol:chloroform extracted, and precipitated with ethanol. DNA was
transferred to nitrocellulose and probed with radiolabeled GFP
probe;
[0054] FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D show the in vivo
transduction ability of recombinant AAV vectors produced using
various system components. GFP fluorescence microscopy was
performed on Hela C12 infected at an MOI of 1000 genomes/cell 24
hrs post infection.
[0055] FIG. 17A and FIG. 17B show the Immunoblot and dot blot
autoradiograph of virions produced from pTR-UF5; pIM45-VP1,2;
pIM45-VP1,3; and pIM45-VP2,3 plasmids following standard
purification protocols. The capsid proteins VP1, VP2, and VP3 are
indicated. No virions were obtained in 40% iodixanol fraction from
plasmid pIM45-VP1,2;
[0056] FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show the in vivo
transduction ability of recombinant AAV vectors containing only two
capsid proteins. GFP fluorescence microscopy was performed on Hela
C12/24 hrs post infection;
[0057] FIG. 19 depicts an immunoblot of protein fractions collected
from iodixinol purified passed over a heparin-agarose column.
Immunoblot was probed with anti-VP1,2,3 monoclonal antibody. C:
5.sup.10 virus particles loaded directly onto blot, FT: flowthrough
fraction, W: wash fraction, and E: 2 M NaCl fraction;
[0058] FIG. 20 shows a dot blot autoradiograph of DNA extracted
from pTR-UF5 and rAAV R585A, R588A. Equal volume aliquots from an
iodixinal step gradient were with incubated with DNAseI,
inactivated with EDTA, digested with proteinase K,
phenol:chloroform extracted, and precipitated with ethanol. DNA was
transferred to nitrocellulose and probed with radiolabeled GFP
probe;
[0059] FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D show the in vivo
transduction ability of pTR-UF5 and R585A, R588A. GFP fluorescence
microscopy was performed on Hela C12 and HEK 293 cells infected at
an MOI of 1000 genomes/cell 24 hrs post infection;
[0060] FIG. 22 shows a slot blot autoradiograph of an in vivo DNA
tracking time-course experiment of pTR-UF5, rAAV R585A, R588A.
Media and cells infected with pTR-UF5 and rAAV R585A, R588A were
collected at 1, 4, and 20 hrs post infection. Hirt DNA was
extracted, transferred to nitrocellulose and probed with a
radiolabeled GFP probe; and
[0061] FIG. 23 shows a schematic diagram of the pIM45 vector
showing the rep and cap sequences.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0062] Illustrative embodiments of the invention are described
below. In the interest of clarity, not all features of an actual
implementation are described in this specification. It will of
course be appreciated that in the development of any such actual
embodiment, numerous implementation-specific decisions must be made
to achieve the developers' specific goals, such as compliance with
system-related and business-related constraints, which will vary
from one implementation to another. Moreover, it will be
appreciated that such a development effort might be complex and
time-consuming, but would nevertheless be a routine undertaking for
those of ordinary skill in the art having the benefit of this
disclosure.
[0063] rAAV Type 2
[0064] The adeno-associated virus type 2 (AAV2) is a small,
non-enveloped parvovirus that has received considerable attention
as a gene therapy vector (see, e.g., Muzyczka and Berns, 2001). The
capsid has a diameter of approximately 20 nm formed by an
icosahedral lattice with T=1 symmetry (60 structurally equivalent
subunits). In purified virions, three structural proteins, VP1,
VP2, and VP3 with molecular masses of 87, 73, and 62 Kda,
respectively, are present in a molar ratio of 1:1:18 (Buller and
Rose, 1978). mRNAs encoding capsid proteins are synthesized from a
single open reading frame and use alternative splicing and start
codons to produce three VP proteins that share an identical 532
carboxyl-terminal amino acid domain (Becerra et al., 1988; Becerra
et al., 1985), with VP2 and VP3 containing successive amino
terminal truncations of VP1.
[0065] The atomic structure of the AAV2 capsid has been determined
to a resolution of 3.5 angstroms (Xie et al., 2002). In this model,
sixty copies of VP3 minus 14 amino terminal residues are present in
an icosahedral arrangement. The VP3 protein contains 8
anti-parallel .beta.-strands that adopt a barreloid structure
similar to capsid proteins of other non-enveloped viruses. Loops of
variable length connect the interior .beta.-barrel scaffold and
extend outwards to form the capsid surface. Cryo-electron
microscopy of empty AAV2 particles generated a surface density map
that described holes, spikes and canyon features similar to those
found in other parvoviruses (Kronenberg et al., 2001). Before the
crystal structure was available, several alternative methods were
investigated in an attempt to localize specific regions of the
capsid. Neutralizing antibody screening of peptide sequences
derived from VP1 found multiple antigenic determinants distributed
on the capsid exterior in both linear and conformation dependent
forms (Moskalenko et al., 2000). Computer modeling of AAV structure
based on the known atomic structure of the related canine
parvovirus coupled with genetic modification of the capsid
identified several positions that were on the surface of the capsid
and could tolerate insertions and substitutions (Grifman et al.,
2001; Nicklin et al., 2001; Rabinowitz et al., 1999; Ried et al.,
2002; Shi et al., 2001; Wu et al., 2000; Yang et al., 1998).
[0066] Cell membrane binding and entry initiates all viral
infections. Non-enveloped viruses rely on membrane bound
extracellular receptors for attachment to the cell membrane. AAV2
has evolved a dynamic and multistep infectious entry pathway that
utilizes the abundantly expressed heparan sulfate proteoglycan
(HSPG) as its primary target (Summerford and Samulski, 1998). Two
co-receptors, .alpha.V.beta.5 integrin and basic fibroblast growth
factor receptor (bFGFR) have been identified, which act as
secondary receptors that may stabilize virus attachment or
participate during internalization (Duan et al., 1999; Qing et al.,
1999; Summerford et al., 1999). HSPG is a macromolecule expressed
by many cell types and is a component of the extracellular matrix
of most tissues (see, e.g., Hileman et al., 1998; Mull9oy and
Linhardt, 2001). Attached to the core protein are glycosaminoglycan
side chains heparin and heparin sulfate (HS). These carbohydrate
polymers are formed by disaccharide repeats consisting of
alternating N-acetylglucosamine and iduronic acid residues in a
.alpha.1,4 linkage. The saccharides can be modified by N-sulfation
as well as 2-O and 6-O-sulfation to impart a dense overall negative
charge at physiological pH. As a result, HS interacts with an
extensive range of proteins primarily by electrostatic attraction
between the electron dense sulfate groups and a cluster of
positively charged amino acids. Two linear consensus-binding
sequences, XBBXBX and XBBBXXBX, and a conformation dependent
sequence, TXXBXXTBXXXTBB, (where B is any basic amino acids
including His, Lys or Arg and X is any hydropathic amino acid and T
is a turn) have been reported (Hileman et al., 1998). Although HSPG
is thought to participate in attachment during the infectious
process of numerous human viruses (Liu and Thorp, 2002),
information about the molecular mechanisms of these interactions is
limited. A report describing the atomic structure of the foot and
mouth disease virus co-crystallized with a HS pentasaccharide is
available and serves as the only model defined at the atomic level
that describes the molecular interaction between a non-enveloped
icosahedral virus and HS (Fry et al., 1999).
[0067] Several laboratories have attempted to retarget AAV vectors
to non-permissive cell types by inserting sequences coding for
short foreign peptides into VP3. Interestingly, insertions at
position 587, including an L14 integrin binding peptide, a myc tag,
an IgG binding domain truncation of protein A and an endothelial
cell targeting peptide, abolished the natural heparin binding
ability of virus capsids with these alterations (Girod et al.,
1999; Grifman et al., 2001; Nicklin et al., 2001; Ried et al.,
2002; Shi et al., 2001). Similarly, an alanine repeat insertion at
position 509, an L14 peptide insertion at position 520, a
hemagluttinin tag insertion at positions 522 and 591, and peptides
derived from the human luteinizng hormone receptor and the bovine
papilloma virus at inserted positions 520 and 584, respectively,
have been reported to disrupt heparin binding (Shi et al., 2001; Wu
et al., 2000). Curiously, alanine substitutions of acidic residues
between 561 and 565 also reduced heparin binding, suggesting that
nearby basic residues were affected (Wu et al., 2000). Finally, a
substitution mutation of two arginines and a glutamine at positions
585, 588, and 587, respectively, binds poorly to heparin-agarose
(Wu et al., 2000). Taken together, these genetic modifications
suggested two potential heparin-binding loci that cluster between
positions 509-522 and 561-591.
[0068] rAAV Capsid Proteins
[0069] Supramolecular assembly of 60 individual capsid protein
subunits into a non-enveloped, T-1 icosahedral lattice capable of
protecting a 4.7 kb single-stranded DNA genome is a critical step
in the life-cycle of the helper-dependent human parvovirus,
adeno-associated virus2 (AAV2). The mature 20 nm diameter AAV2
particle is composed of three structural proteins designated VP1,
VP2, and VP3 (molecular masses of 87, 73, and 62 kDa respectively)
in a ratio of 1:1:18. Based on its symmetry and these molecular
weight estimates, of the 60 capsid proteins comprising the
particle, three are VP1 proteins, three are VP2 proteins, and
fifty-four are VP3 proteins. The employment of three structural
proteins makes AAV serotypes unique among parvoviruses, as all
others known package their genomes within icosahedral particles
composed of only two capsid proteins. The anti-parallel
.beta.-strand barreloid arrangement of these 60 capsid proteins
results in a particle with a defined tropism that is highly
resistant to degradation.
[0070] The AAV2 genome contains two large open reading frames
(ORF), rep and cap, flanked by inverted terminal repeats. The AAV2
capsid proteins are produced in an overlapping fashion from the cap
ORF; arising through alternative mRNA splicing of the transcript
(initiated at the p40 promoter), with subsequent alternative
translational start codon usage. A common stop codon is employed
for all three capsid proteins. Correct capsid protein stoichiometry
is maintained by translating VP1 from the 2.4 KB mRNA, while VP2
and VP3 arise from the 2.3-kB mRNA using a weaker ACG start codon
for VP2 protein production with resultant read-through translation
for the production of the VP3 protein. Differing only in the length
of their N-terminus, these proteins are produced such that the
amino acid sequence of VP3 is contained within the significantly
less abundant and longer VP1 and VP2 proteins. As such, VP1's
unique 137 amino acid N-terminal extension of VP2 contains a
phospholipase enzymatic activity important for viral infectivity.
Similarly, VP2 extends the N-terminus of VP3 by 64 amino acids with
this VP1/VP2 overlap region possessing a putative nuclear
localization signal (NLS) involved in the nuclear translocation of
VP2. The VP3 region common to all three capsid proteins contains
the critical .beta.-barrel structural motifs characteristic of all
parvoviruses and particle surface loops involved in determining
viral tropism.
[0071] While specific activities have been attributed to regions of
an individual AAV2 capsid protein, the role of each capsid protein
in the structural formation of the particle is less clear. Early
studies in which all AAV2 capsid expression was eliminated revealed
that capsid protein expression is required for the accumulation of
single stranded genomes. It follows that AAV2 particle assembly
occurs within the nucleus and a putative NLS for VP2 has been
localized to the VP1/VP2 overlap region in transfected COS
cells.
[0072] In the absence of VP1 expression, this study suggested a
major role of VP2 is the nuclear localization of VP3. However,
since VP1 was deleted in this study, one cannot rule out that VP1
has the ability to nuclear localize VP3. Site-directed missense
mutagenesis of the individual capsid proteins' start codons
suggested that infectious particles are obtained only when all
three capsid proteins are present. In contrast, later genetic
analysis demonstrated that in the absence of VP1, VP2 and VP3 are
able to encapsidate progeny genomes. Similarly, in vitro assembly
of purified individual AAV capsid proteins demonstrated that VP2
and VP3 could form an AAV2-like particle. Baculovirus expression of
the AAV2 capsid proteins within SF9 cells suggests an absolute
requirement for VP2, although this study failed to eliminate
VP3-like fragments produced by the VP2-baculovirus. However, it is
feasible that studies of AAV2 assembly in baculovirus have subtle
differences with particles assembled in mammalian cells.
[0073] An examination of the assembly process of the related
autonomous canine parvovirus, CPV, in baculovirus observed
significantly more aggregation of capsid proteins in insect cells.
In addition, the results of the baculovirus and NLS studies have
the caveat that they were performed in the absence of AAV2 Rep
proteins, Ad helper gene functions, and a replicating AAV2 genome.
Furthermore, the p40 promoter in these studies does not control
AAV2 capsid protein expression, resulting in altered stoichiometry
of the available capsid protein pool. Indeed, the above concerns
seem warranted, as a recent insertional mutagenesis study of the
AAV2 cap ORF, using standard AAV2 production protocols, reported
the purification of an AAV2-like particle composed of only the VP3
protein. Therefore, despite the uncertainty of the precise role of
VP1 and VP2 in particle formation, the evidence thus far suggests
that the VP3 protein is absolutely required for the formation of an
AAV2 particle. Finally, co localization studies of AAV2 assembly in
293 cells demonstrated an interaction of AAV2 Rep and capsid
proteins with Ad proteins and the replicating genome in the
nucleus, thus, supporting a current model of AAV2 assembly which
proposes nucleoplasmic formation of empty particles with subsequent
maturation of the particle as a result of Rep 52/40 mediated
translocation of capsid protein associated single stranded genomes
into the preformed particles.
[0074] Genetic Modification of rAAV Capsid Proteins
[0075] Great interest in the assembly, structure, and mutability of
the AAV2 particle results from its promise as a recombinant gene
delivery vehicle (rAAV2) in vivo. Essential to the clinical
development of rAAV2 vectors for gene therapy is the ability to
target specific tissue types. Manipulation of the rAAV2 particle in
order to control its cellular receptor interactions is essential
for vector targeting. The feasibility of various targeting
strategies based on AAV cap ORF mutagenesis is currently an area of
active investigation. A better understanding of the AAV2 particle
surface architecture through systematic scanning-alanine and
insertional mutagenesis of the AAV cap ORF and recent publication
of the AAV2 crystal structure has identified several amino acid
regions on the surface of the particle that tolerate sequence
alteration without loss of capsid stability or integrity.
[0076] However, small changes in charge, sequence, and/or position
of the mutation can result in dramatic changes in the mutant
particle phenotype. One limitation in sequence mutation of the
overlapping cap ORF is that mutation of only one capsid protein
across its entire sequence is currently not possible. The full
potential in manipulation of the particle is not reached with
direct alteration of regions of capsid overlap. Predicted surface
regions of capsid overlap leading to defective phenotypes upon
mutagenesis may allow production of viable particles if such
mutations were only in one or two of the capsid proteins. An
additional degree of flexibility in modifying the rAAV2 particle
would result from the ability to mutate the entire coding region of
a specific capsid protein without altering the remaining two capsid
proteins. Indeed, while mutations in the C-terminus of the VP3
region have been reported to be completely defective in particle
formation following insertion of HA and 6.times.His tags into the
overlapping cap ORF, a recent report focusing on the purification
of rAAV2 particles demonstrated that the C-terminus of VP3 is
capable of accepting a 6.times.His tag if VP1 and VP2 are not
altered. This rAAV2 production strategy involved expressing VP1 and
VP2 from one construct, and expressing the VP3-6.times.His fusion
protein from a CMV promoter in a second plasmid. In the absence of
the isolation of a specific capsid protein's expression, the
N-terminal 137 amino acids of VP1 are the only region of the cap
ORF where mutations are restricted to a single capsid protein.
Successful insertions within this region have included HA and
serpin. The VP1/VP2 overlap region (amino acid 138-202) also has
been receptive to sequence modification. Insertions in this region
have included HA, serpin and luetinizing hormone receptor ligand
sequences immediately following amino acid 138 in the cap ORF.
[0077] The success of inserting sequences to the VP1 and VP1/VP2
regions may be due in part to less disruption of the integrity of
the particle compared to insertion in the VP3 region of capsid
overlap (amino acid 203-735). It is important to note that these
mutant particles would require further mutation of the putative
heparin-binding motif to restrict infection to the target cell. Not
surprisingly, since it is the longest region of capsid protein
overlap, contains many critical structural motifs, and targeting
sequences in this region have 60 representatives in the rAAV
particle, mutations in the VP3 region of the AAV2 cap ORF have
resulted in the highest number of defective phenotypes. Yet, one
location within the VP3 region has received much attention for the
successful insertion of small targeting sequences in all three
capsid proteins (amino acid 587). One major advantage of targeting
insertions to this position is that the resultant mutant particle
also has lost the ability to bind its native receptor. Viable
mutations in the VP3 region of the cap ORF have been restricted in
size (<30 amino acids).
[0078] One caveat of creating genetically-targeted rAAV2 particles,
is the consideration that many cell surface receptors have ligands
whose coding sequence are much larger than those successfully
inserted directly into the overlapping cap ORF. Due to the modest
size of this ORF (.about.2 kB), the insertion of larger peptide
sequences into the capsid coding sequences may result in serious
disruption of splicing, read-through translation, capsid structure
and/or stability. The insertion of large sequences into the rAAV2
particle have been limited to a study involving the fusion of the
CD34 single chain antibody coding sequence with the N-termini of
the individual capsid proteins following isolation of their
expression to separate CMV promoters. Viable CD34-retargeted rAAV
particles of extremely low titer were produced only when this
fusion was to VP2 protein, and co-expression of wild-type VP2
protein was required. Nonetheless, the fusion of large peptide
sequences to the N-terminus of VP2 does not interfere with the
incorporation of this capsid protein into the rAAV2 particle.
[0079] Wild-Type AAV2 Binds to Heparan Sulfate Proteoglycan
[0080] The adeno-associated virus type-2 (AAV2) uses heparan
sulfate proteoglycan (HSPG) as its primary cellular receptor. In
order to identify amino acids within the capsid of AAV2 that
contribute to HSPG association, biochemical information about
heparin/heparin sulfate (HS), AAV serotype protein sequence
alignments, and data from previous capsid studies was used to
select residues for mutagenesis. In the present invention,
charged-to-alanine substitution mutagenesis was performed on
individual and combinations of basic residues for the production
and purification of recombinant viruses that contained a GFP
reporter gene cassette. Intact capsids were assayed for their
ability to bind to heparin-agarose in vitro and virions that
packaged DNA were assayed for their ability to transduce normally
permissive cell lines. It was found that mutation of arginine
residues at position 585 or 588 eliminated binding to
heparin-agarose. Mutation of residues R484, R487, and K532 showed
partial binding to heparin-agarose. A general correlation between
heparin-agarose binding and infectivity was observed as measured by
GFP transduction; however, a subset of mutants that partially bound
heparin-agarose (R484A and K532A) was completely non-infectious,
suggesting that they had additional blocks to infectivity that were
unrelated to heparin binding. Conservative mutation of positions
R585 and R588 to lysine slightly reduced heparin-agarose binding,
and had comparable effects on infectivity. Substitution of AAV2
residues 585 through 590 into a location predicted to be
structurally equivalent in AAV5 generated a hybrid virus that bound
to heparin-agarose efficiently, was able to package DNA, but was
non-infectious. Taken together, these suggest that residues R585
and R588 are primarily responsible for heparin sulfate binding and
mutation of these residues has little effect on other aspects of
the viral life cycle.
[0081] Computer modeling using the AAV2 VP3 atomic coordinates
revealed that residues which contribute to heparin binding form a
cluster of five basic amino acids on the surface of each three-fold
axis of symmetry related spike. Three other kinds of mutants were
found as well. Mutants, R459A, H509A and H526A/K527A bound heparin
as well as wild type but were defective for transduction. Another
mutant, H358A, was defective for capsid assembly. Finally, a mutant
R459A produced significantly lower levels of full capsids,
suggesting a packaging defect.
[0082] Pharmaceutical Compositions
[0083] The genetic constructs of the present invention may be
prepared in a variety of compositions, and may also be formulated
in appropriate pharmaceutical vehicles for administration to human
or animal subjects. The AAV molecules of the present invention and
compositions comprising them provide new and useful therapeutics
for the treatment, control, and amelioration of symptoms of a
variety of disorders. Moreover, pharmaceutical compositions
comprising one or more of the nucleic acid compounds disclosed
herein, provide significant advantages over existing conventional
therapies--namely, (1) their reduced side effects, (2) their
increased efficacy for prolonged periods of time, (3) their ability
to increase patient compliance due to their ability to provide
therapeutic effects following as little as a single administration
of the selected therapeutic AAV composition to affected
individuals. Exemplary pharmaceutical compositions and methods for
their administration are discussed in significant detail
hereinbelow.
[0084] The invention also provides compositions comprising one or
more of the disclosed vectors, expression systems, virions, viral
particles; or mammalian cells. As described hereinbelow, such
compositions may further comprise a pharmaceutical excipient,
buffer, or diluent, and may be formulated for administration to an
animal, and particularly a human being. Such compositions may
further optionally comprise a liposome, a lipid, a lipid complex, a
microsphere, a microparticle, a nanosphere, or a nanoparticle, or
may be otherwise formulated for administration to the cells,
tissues, organs, or body of a mammal in need thereof. Such
compositions may be formulated for use in therapy, such as for
example, in the amelioration, prevention, or treatment of
conditions such as peptide deficiency, polypeptide deficiency,
tumor, cancer or other malignant growth, neurological dysfunction,
autoimmune diseases, lupus, cardiovascular disease, pulmonary
disease, ischemia, stroke, cerebrovascular accidents, diabetes and
diseases of the pancreas, neural diseases, including Alzheimer's,
Huntington's, Tay-Sach's, and Parkinson's diseases, memory loss,
trauma, motor impairment, and the like, as well as biliary, renal
or hepatic disease or dysfunction, as well as musculoskeletal
diseases including, for example, arthritis, cystic fibrosis (CF),
amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS),
muscular dystrophy (MD), and such like, to name only a few.
[0085] In certain embodiments, the present invention concerns
formulation of one or more of the rAAV compositions disclosed
herein in pharmaceutically acceptable solutions for administration
to a cell or an animal, either alone or in combination with one or
more other modalities of therapy, and in particular, for therapy of
human cells, tissues, and diseases affecting man.
[0086] It will also be understood that, if desired, nucleic acid
segments, RNA, DNA or PNA compositions that express one or more of
therapeutic gene products may be administered in combination with
other agents as well, such as, e.g., proteins or polypeptides or
various pharmaceutically-active agents, including one or more
systemic or topical administrations of therapeutic polypeptides,
biologically active fragments, or variants thereof. In fact, there
is virtually no limit to other components that may also be
included, given that the additional agents do not cause a
significant adverse effect upon contact with the target cells or
host tissues. The rAAV compositions may thus be delivered along
with various other agents as required in the particular instance.
Such compositions may be purified from host cells or other
biological sources, or alternatively may be chemically synthesized
as described herein. Likewise, such compositions may further
comprise substituted or derivatized RNA, DNA, or PNA
compositions.
[0087] Formulation of pharmaceutically-acceptable excipients and
carrier solutions is well-known to those of skill in the art, as is
the development of suitable dosing and treatment regimens for using
the particular compositions described herein in a variety of
treatment regimens, including e.g., oral, parenteral, intravenous,
intranasal, and intramuscular administration and formulation.
[0088] Typically, these formulations may contain at least about
0.1% of the active compound or more, although the percentage of the
active ingredient(s) may, of course, be varied and may conveniently
be between about 1 or 2% and about 70% or 80% or more of the weight
or volume of the total formulation. Naturally, the amount of active
compound(s) in each therapeutically-useful composition may be
prepared is such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
[0089] In certain circumstances it will be desirable to deliver the
AAV vector-based therapeutic constructs in suitably formulated
pharmaceutical compositions disclosed herein either subcutaneously,
intraocularly, intravitreally, parenterally, intravenously,
intracerebroventricularly, intramuscularly, intrathecally, orally,
intraperitoneally, by oral or nasal inhalation, or by direct
injection to one or more neural cells, nervous tissues, or even by
direct injection or administration to the brain, CNS or to the
peripheral nervous system. The methods of administration may also
include those modalities as described in U.S. Pat. No. 5,543,158;
U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363 (each of which
is specifically incorporated herein in its entirety by express
reference thereto). Solutions of the active compounds as freebase
or pharmacologically acceptable salts may be prepared in sterile
water and may also suitably mixed with one or more surfactants,
such as hydroxypropylcellulose. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0090] The pharmaceutical forms of the AAV-based viral compositions
suitable for injectable use include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions (U.S. Pat. No.
5,466,468, specifically incorporated herein by reference in its
entirety). In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and/or vegetable oils. Proper
fluidity may be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial ad antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0091] For administration of an injectable aqueous solution, for
example, the solution may be suitably buffered, if necessary, and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, a sterile
aqueous medium that can be employed will be known to those of skill
in the art in light of the present disclosure. For example, one
dosage may be dissolved in 1 mL of isotonic NaCl solution and
either added to 1000 mL of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
and the general safety and purity standards as required by FDA
Office of Biologics standards.
[0092] Sterile injectable solutions are prepared by incorporating
the active AAV vector-delivered therapeutic polypeptide-encoding
DNA fragments in the required amount in the appropriate solvent
with several of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0093] The AAV vector compositions disclosed herein may also be
formulated in a neutral or salt form. Pharmaceutically-acceptable
salts include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions,
drug-release capsules, and the like.
[0094] As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0095] The phrase "pharmaceutically-acceptable" refers to molecular
entities and compositions that do not produce an allergic or
similar untoward reaction when administered to a human, and in
particular, when administered to the human eye. The preparation of
an aqueous composition that contains a protein as an active
ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
[0096] The amount of AAV compositions and time of administration of
such compositions will be within the purview of the skilled artisan
having benefit of the present teachings. It is likely, however,
that the administration of therapeutically-effective amounts of the
disclosed compositions may be achieved by a single administration,
such as for example, a single injection of sufficient numbers of
infectious particles to provide therapeutic benefit to the patient
undergoing such treatment. Alternatively, in some circumstances, it
may be desirable to provide multiple, or successive administrations
of the AAV vector compositions, either over a relatively short, or
a relatively prolonged period of time, as may be determined by the
medical practitioner overseeing the administration of such
compositions. For example, the number of infectious particles
administered to a mammal may be on the order of about 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, or
even higher, infectious particles/mL given either as a single dose,
or divided into two or more administrations as may be required to
achieve therapy of the particular disease or disorder being
treated. In fact, in certain embodiments, it may be desirable to
administer two or more different AAV vector compositions, either
alone, or in combination with one or more other therapeutic drugs
to achieve the desired effects of a particular therapy regimen.
[0097] Liposome-, Nanocapsule-, and Microparticle-Mediated
Delivery
[0098] In certain embodiments, the inventors contemplate the use of
liposomes, nanocapsules, microparticles, microspheres, lipid
particles, vesicles, and the like, for the introduction of the
compositions of the present invention into suitable host cells. In
particular, the rAAV vector delivered gene therapy compositions of
the present invention may be formulated for delivery either
encapsulated in a lipid particle, a liposome, a vesicle, a
nanosphere, or a nanoparticle or the like.
[0099] Such formulations may be preferred for the introduction of
pharmaceutically acceptable formulations of the nucleic acids or
the rAAV constructs disclosed herein. The formation and use of
liposomes is generally known to those of skill in the art (see for
example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998; which
describes the use of liposomes and nanocapsules in the targeted
antibiotic therapy for intracellular bacterial infections and
diseases). Recently, liposomes were developed with improved serum
stability and circulation half-times (Gabizon and Papahadjopoulos,
1988; Allen and Choun, 1987; and U.S. Pat. No. 5,741,516, each of
which is specifically incorporated herein in its entirety by
express reference thereto). Further, various methods of liposome
and liposome like preparations as potential drug carriers have been
reviewed (Takakura, 1998; Chandran et al., 1997; Margalit, 1995;
U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No.
5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587,
each of which is specifically incorporated herein in its entirety
by express reference thereto).
[0100] Liposomes have been used successfully with a number of cell
types that are normally resistant to transfection by other
procedures including T cell suspensions, primary hepatocyte
cultures and PC 12 cells (Renneisen et al., 1990; Muller et al.,
1990). In addition, liposomes are free of the DNA length
constraints that are typical of viral-based delivery systems.
Liposomes have been used effectively to introduce genes, drugs
(Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al.,
1989; Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et
al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumi et al.,
1990b), viruses (Faller and Baltimore, 1984), transcription factors
and allosteric effectors (Nicolau and Gersonde, 1979) into a
variety of cultured cell lines and animals. In addition, several
successful clinical trails examining the effectiveness of
liposome-mediated drug delivery have been completed
(Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier et al.,
1988). Furthermore, several studies suggest that the use of
liposomes is not associated with autoimmune responses, toxicity or
gonadal localization after systemic delivery (Mori and Fukatsu,
1992).
[0101] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 .ANG.,
containing an aqueous solution in the core.
[0102] Liposomes bear resemblance to cellular membranes and are
contemplated for use in connection with the present invention as
carriers for the peptide compositions. They are widely suitable as
both water- and lipid-soluble substances can be entrapped, i.e. in
the aqueous spaces and within the bilayer itself, respectively. It
is possible that the drug-bearing liposomes may even be employed
for site-specific delivery of active agents by selectively
modifying the liposomal formulation.
[0103] In addition to the teachings of Couvreur et al. (1977;
1988), the following information may be utilized in generating
liposomal formulations. Phospholipids can form a variety of
structures other than liposomes when dispersed in water, depending
on the molar ratio of lipid to water. At low ratios the liposome is
the preferred structure. The physical characteristics of liposomes
depend on pH, ionic strength and the presence of divalent cations.
Liposomes can show low permeability to ionic and polar substances,
but at elevated temperatures undergo a phase transition which
markedly alters their permeability. The phase transition involves a
change from a closely packed, ordered structure, known as the gel
state, to a loosely packed, less-ordered structure, known as the
fluid state. This occurs at a characteristic phase-transition
temperature and results in an increase in permeability to ions,
sugars and drugs.
[0104] In addition to temperature, exposure to proteins can alter
the permeability of liposomes. Certain soluble proteins, such as
cytochrome c, bind, deform and penetrate the bilayer, thereby
causing changes in permeability. Cholesterol inhibits this
penetration of proteins, apparently by packing the phospholipids
more tightly. It is contemplated that the most useful liposome
formations for antibiotic and inhibitor delivery will contain
cholesterol.
[0105] The ability to trap solutes varies between different types
of liposomes. For example, MLVs are moderately efficient at
trapping solutes, but SUVs are extremely inefficient. SUVs offer
the advantage of homogeneity and reproducibility in size
distribution, however, and a compromise between size and trapping
efficiency is offered by large unilamellar vesicles (LUVs). These
are prepared by ether evaporation and are three to four times more
efficient at solute entrapment than MLVs.
[0106] In addition to liposome characteristics, an important
determinant in entrapping compounds is the physicochemical
properties of the compound itself. Polar compounds are trapped in
the aqueous spaces and nonpolar compounds bind to the lipid bilayer
of the vesicle. Polar compounds are released through permeation or
when the bilayer is broken, but nonpolar compounds remain
affiliated with the bilayer unless it is disrupted by temperature
or exposure to lipoproteins. Both types show maximum efflux rates
at the phase transition temperature.
[0107] Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. It often
is difficult to determine which mechanism is operative and more
than one may operate at the same time.
[0108] The fate and disposition of intravenously injected liposomes
depend on their physical properties, such as size, fluidity, and
surface charge. They may persist in tissues for hrs or days,
depending on their composition, and half lives in the blood range
from min to several hrs. Larger liposomes, such as MLVs and LUVs,
are taken up rapidly by phagocytic cells of the reticuloendothelial
system, but physiology of the circulatory system restrains the exit
of such large species at most sites. They can exit only in places
where large openings or pores exist in the capillary endothelium,
such as the sinusoids of the liver or spleen. Thus, these organs
are the predominate site of uptake. On the other hand, SUVs show a
broader tissue distribution but still are sequestered highly in the
liver and spleen. In general, this in vivo behavior limits the
potential targeting of liposomes to only those organs and tissues
accessible to their large size. These include the blood, liver,
spleen, bone marrow, and lymphoid organs.
[0109] Targeting is generally not a limitation in terms of the
present invention. However, should specific targeting be desired,
methods are available for this to be accomplished. Antibodies may
be used to bind to the liposome surface and to direct the antibody
and its drug contents to specific antigenic receptors located on a
particular cell-type surface. Carbohydrate determinants
(glycoprotein or glycolipid cell-surface components that play a
role in cell-cell recognition, interaction and adhesion) may also
be used as recognition sites as they have potential in directing
liposomes to particular cell types. Mostly, it is contemplated that
intravenous injection of liposomal preparations would be used, but
other routes of administration are also conceivable.
[0110] Alternatively, the invention provides for pharmaceutically
acceptable nanocapsule formulations of the AAV vector-based
polynucleotide compositions of the present invention. Nanocapsules
can generally entrap compounds in a stable and reproducible way
(Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998;
Douglas et al., 1987). To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
nm) should be designed using polymers able to be degraded in vivo.
Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these
requirements are contemplated for use in the present invention.
Such particles may be are easily made, as described (Couvreur et
al., 1980; Couvreur, 1988; zur Muhlen et al., 1998; Zambaux et al.
1998; Pinto-Alphandry et al., 1995 and U.S. Pat. No. 5,145,684,
each of which is specifically incorporated herein in its entirety
by express reference thereto).
[0111] Additional Modes of Delivery
[0112] In addition to the methods of delivery described above, the
following techniques are also contemplated as alternative methods
of delivering the disclosed rAAV vector based polynucleotide
compositions to a target cell or animal. Sonophoresis (i.e.,
ultrasound) has been used and described in U.S. Pat. No. 5,656,016
(specifically incorporated herein in its entirety by express
reference thereto) as a device for enhancing the rate and efficacy
of drug permeation into and through the circulatory system. Other
drug delivery alternatives contemplated are intraosseous injection
(U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No.
5,797,898), ophthalmic formulations (Bourlais et al., 1998),
transdermal matrices (U.S. Pat. No. 5,770,219 and U.S. Pat. No.
5,783,208) and feedback-controlled delivery (U.S. Pat. No.
5,697,899), each of which is specifically incorporated herein in
its entirety by express reference thereto.
[0113] Promoters and Enhancers
[0114] Recombinant AAV vectors form important aspects of the
present invention. The term "expression vector or construct" means
any type of genetic construct containing a nucleic acid in which
part or all of the nucleic acid encoding sequence is capable of
being transcribed. In preferred embodiments, expression only
includes transcription of the nucleic acid, for example, to
generate a biologically-active therapeutic peptides, polypeptides,
proteins, antisense molecules, or catalytic RNA ribozymes from a
transcribed gene.
[0115] Particularly useful vectors are contemplated to be those
vectors in which the nucleic acid segment to be transcribed is
positioned under the transcriptional control of a promoter. A
"promoter" refers to a DNA sequence recognized by the synthetic
machinery of the cell, or introduced synthetic machinery, required
to initiate the specific transcription of a gene. The phrases
"operatively positioned," "under control" or "under transcriptional
control" means that the promoter is in the correct location and
orientation in relation to the nucleic acid to control RNA
polymerase initiation and expression of the gene.
[0116] In preferred embodiments, it is contemplated that certain
advantages will be gained by positioning the coding DNA segment
under the control of a recombinant, or heterologous, promoter. As
used herein, a recombinant or heterologous promoter is intended to
refer to a promoter that is not normally associated with an
cytokine or serpin-encoding gene in its natural environment. Such
promoters may include promoters normally associated with other
genes, and/or promoters isolated from any bacterial, viral,
eukaryotic, or mammalian cell.
[0117] Naturally, it will be important to employ a promoter that
effectively directs the expression of the serpin or
cytokine-encoding DNA segment in the cell type, organism, or even
animal, chosen for expression. The use of promoter and cell type
combinations for protein expression is generally known to those of
skill in the art of molecular biology, for example, see Sambrook et
al. (1989), incorporated herein by reference. The promoters
employed may be constitutive, or inducible, and can be used under
the appropriate conditions to direct high-level expression of the
introduced DNA segment, or the promoters may direct tissue- or
cell-specific expression of the therapeutic constructs, such as,
for example, an islet cell- or pancreas-specific promoter such as
the insulin promoter.
[0118] At least one module in a promoter functions to position the
start site for RNA synthesis. The best-known example of this is the
TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0119] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 by upstream of the start site, although a number of
promoters have been shown to contain functional elements downstream
of the start site as well. The spacing between promoter elements
frequently is flexible, so that promoter function is preserved when
elements are inverted or moved relative to one another. In the tk
promoter, the spacing between promoter elements can be increased to
50 by apart before activity begins to decline. Depending on the
promoter, it appears that individual elements can function either
co-operatively or independently to activate transcription.
[0120] The particular promoter that is employed to control the
expression of a nucleic acid is not believed to be critical, so
long as it is capable of expressing the serpin or
cytokine-polypeptide encoding nucleic acid segment in the targeted
cell. Thus, where a human cell is targeted, it is preferable to
position the nucleic acid coding region adjacent to and under the
control of a promoter that is capable of being expressed in a human
cell. Generally speaking, such a promoter might include either a
human or viral promoter, such as a CMV or an HSV promoter. In
certain aspects of the invention, .beta.-actin, and in particular,
chicken .beta.-actin promoters have been shown to be particularly
preferred for certain embodiments of the invention.
[0121] In various other embodiments, the human cytomegalovirus
(CMV) immediate early gene promoter, the SV40 early promoter and
the Rous sarcoma virus long terminal repeat can be used to obtain
high-level expression of transgenes. The use of other viral or
mammalian cellular or bacterial phage promoters that are well known
in the art to achieve expression of a transgene is contemplated as
well, provided that the levels of expression are sufficient for a
given purpose. A variety of promoter elements have been described
in Tables 1 and 2 that may be employed, in the context of the
present invention, to regulate the expression of the present serpin
or cytokine-encoding nucleic acid segments comprised within the
recombinant AAV vectors of the present invention.
[0122] Enhancers were originally detected as genetic elements that
increased transcription from a promoter located at a distant
position on the same molecule of DNA. This ability to act over a
large distance had little precedent in classic studies of
prokaryotic transcriptional regulation. Subsequent work showed that
regions of DNA with enhancer activity are organized much like
promoters. That is, they are composed of many individual elements,
each of which binds to one or more transcriptional proteins.
[0123] The basic distinction between enhancers and promoters is
operational. An enhancer region as a whole must be able to
stimulate transcription at a distance; this need not be true of a
promoter region or its component elements. On the other hand, a
promoter must have one or more elements that direct initiation of
RNA synthesis at a particular site and in a particular orientation,
whereas enhancers lack these specificities. Promoters and enhancers
are often overlapping and contiguous, often seeming to have a very
similar modular organization.
[0124] Additionally any promoter/enhancer combination (as per the
Eukaryotic Promoter Data Base EPDB) could also be used to drive
expression. Use of a T3, T7 or SP6 cytoplasmic expression system is
another possible embodiment. Eukaryotic cells can support
cytoplasmic transcription from certain bacterial promoters if the
appropriate bacterial polymerase is provided, either as part of the
delivery complex or as an additional genetic expression
construct.
TABLE-US-00001 TABLE 1 ILLUSTRATIVE PROMOTER AND ENHANCER ELEMENTS
PROMOTER/ENHANCER REFERENCE Immunoglobulin Heavy Chain Banerji et
al., 1983; Gilles et al., 1983; Grosschedl and Baltimore, 1985;
Atchinson and Perry, 1986, 1987; Imler et al., 1987; Weinberger et
al., 1984; Kiledjian et al., 1988; Porton et al.; 1990
Immunoglobulin Light Chain Queen and Baltimore, 1983; Picard and
Schaffner, 1984 T-Cell Receptor Luria et al., 1987; Winoto and
Baltimore, 1989; Redondo et al.; 1990 HLA DQ a and DQ .beta.
Sullivan and Peterlin, 1987 .beta.-Interferon Goodbourn et al.,
1986; Fujita et al., 1987; Goodbourn and Maniatis, 1988
Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene et
al., 1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC
Class II HLA-Dra Sherman et al., 1989 .beta.-Actin Kawamoto et al.,
1988; Ng et al.; 1989 Muscle Creatine Kinase Jaynes et al., 1988;
Horlick and Benfield, 1989; Johnson et al., 1989 Prealbumin
(Transthyretin) Costa et al., 1988 Elastase I Omitz et al., 1987
Metallothionein Karin et al., 1987; Culotta and Hamer, 1989
Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin Gene
Pinkert et al., 1987; Tronche et al., 1989, 1990
.alpha.-Fetoprotein Godbout et al., 1988; Campere and Tilghman,
1989 t-Globin Bodine and Ley, 1987; Perez-Stable and Constantini,
1990 .beta.-Globin Trudel and Constantini, 1987 e-fos Cohen et al.,
1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlund
et al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990
(NCAM) .alpha..sub.1-Antitrypain Latimer et al., 1990 H2B (TH2B)
Histone Hwang et al., 1990 Mouse or Type I Collagen Ripe et al.,
1989 Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and
GRP78) Rat Growth Hormone Larsen et al., 1986 Human Serum Amyloid A
(SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey et al., 1989
Platelet-Derived Growth Factor Pech et al., 1989 Duchenne Muscular
Dystrophy Klamut et al., 1990 SV40 Banerji et al., 1981; Moreau et
al., 1981; Sleigh and Lockett, 1985; Firak and Subramanian, 1986;
Herr and Clarke, 1986; Imbra and Karin, 1986; Kadesch and Berg,
1986; Wang and Calame, 1986; Ondek et al., 1987; Kuhl et al., 1987;
Schaffner et al., 1988 Polyoma Swartzendruber and Lehman, 1975;
Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al.,
1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al.,
1986; Satake et al., 1988; Campbell and Villarreal, 1988
Retroviruses Kriegler and Botchan, 1982, 1983; Levinson et al.,
1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;
Miksicek et al., 1986; Celander and Haseltine, 1987; Thiesen et
al., 1988; Celander et al., 1988; Chol et al., 1988; Reisman and
Rotter, 1989 Papilloma Virus Campo et al., 1983; Lusky et al.,
1983; Spandidos and Wilkie, 1983; Spalholz et al., 1985; Lusky and
Botchan, 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et
al., 1987; Stephens and Hentschel, 1987 Hepatitis B Virus Bulla and
Siddiqui, 1986; Jameel and Siddiqui, 1986; Shaul and Ben-Levy,
1987; Spandau and Lee, 1988; Vannice and Levinson, 1988 Human
Immunodeficiency Virus Muesing et al., 1987; Hauber and Cullan,
1988; Jakobovits et al., 1988; Feng and Holland, 1988; Takebe et
al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et
al., 1989; Sharp and Marciniak, 1989; Braddock et al., 1989
Cytomegalovirus Weber et al., 1984; Boshart et al., 1985; Foecking
and Hofstetter, 1986 Gibbon Ape Leukemia Virus Holbrook et al.,
1987; Quinn et al., 1989
TABLE-US-00002 TABLE 2 INDUCIBLE ELEMENTS ELEMENT INDUCER
REFERENCES MT II Phorbol Ester (TFA) Palmiter et al., 1982; Heavy
metals Haslinger and Karin, 1985; Searle et al., 1985; Stuart et
al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al.,
1987b; McNeall et al., 1989 MMTV (mouse mammary tumor virus)
Glucocorticoids Huang et al., 1981; Lee et al., 1981; Majors and
Varmus, 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et
al., 1985; Sakai et al., 1988 .beta.-Interferon poly(rI)x Tavernier
et al., 1983 poly(rc) Adenovirus 5 E2 Ela Imperiale and Nevins,
1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a
Stromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol
Ester (TPA) Angel et al., 1987b Murine MX Gene Interferon,
Newcastle Disease Virus GRP78 Gene A23187 Resendez et al., 1988
.alpha.-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin Serum
Rittling et al., 1989 MHC Class I Gene H-2.kappa.b Interferon
Blanar et al., 1989 HSP70 Ela, SV40 Large T Antigen Taylor et al.,
1989; Taylor and Kingston, 1990a, b Proliferin Phorbol Ester-TPA
Mordacq and Linzer, 1989 Tumor Necrosis Factor FMA Hensel et al.,
1989 Thyroid Stimulating Hormone .alpha. Gene Thyroid Hormone
Chatterjee et al., 1989
[0125] As used herein, the terms "engineered" and "recombinant"
cells are intended to refer to a cell into which an exogenous DNA
segment, such as DNA segment that leads to the transcription of a
biologically-active serpin or cytokine polypeptide or a ribozyme
specific for such a biologically-active serpin or cytokine
polypeptide product, has been introduced. Therefore, engineered
cells are distinguishable from naturally occurring cells, which do
not contain a recombinantly introduced exogenous DNA segment.
Engineered cells are thus cells having DNA segment introduced
through the hand of man.
[0126] To express a biologically-active serpin or cytokine encoding
gene in accordance with the present invention one would prepare an
rAAV expression vector that comprises a biologically-active serpin
or cytokine polypeptide-encoding nucleic acid segment under the
control of one or more promoters. To bring a sequence "under the
control of" a promoter, one positions the 5'-end of the
transcription initiation site of the transcriptional reading frame
generally between about 1 and about 50 nucleotides "downstream" of
(i.e., 3' of) the chosen promoter. The "upstream" promoter
stimulates transcription of the DNA and promotes expression of the
encoded polypeptide. This is the meaning of "recombinant
expression" in this context. Particularly preferred recombinant
vector constructs are those that comprise an rAAV vector. Such
vectors are described in detail herein.
[0127] Mutagenesis and Preparation of Modified Nucleotide
Compositions
[0128] In certain embodiments, it may be desirable to prepared
modified nucleotide compositions, such as, for example, in the
generation of the nucleic acid segments that encode either parts of
the AAV vector itself, or the promoter, or even the therapeutic
gene delivered by such rAAV vectors. Various means exist in the
art, and are routinely employed by the artisan to generate modified
nucleotide compositions.
[0129] Site-specific mutagenesis is a technique useful in the
preparation and testing of sequence variants by introducing one or
more nucleotide sequence changes into the DNA. Site-specific
mutagenesis allows the production of mutants through the use of
specific oligonucleotide sequences which encode the DNA sequence of
the desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 17
to 25 nucleotides in length is preferred, with about 5 to 10
residues on both sides of the junction of the sequence being
altered.
[0130] In general, the technique of site-specific mutagenesis is
well known in the art. As will be appreciated, the technique
typically employs a bacteriophage vector that exists in both a
single stranded and double stranded form. Typical vectors useful in
site-directed mutagenesis include vectors such as the M13 phage.
These phage vectors are commercially available and their use is
generally well known to those skilled in the art. Double stranded
plasmids are also routinely employed in site directed mutagenesis,
which eliminates the step of transferring the gene of interest from
a phage to a plasmid.
[0131] In general, site-directed mutagenesis is performed by first
obtaining a single-stranded vector, or melting of two strands of a
double stranded vector that includes within its sequence a DNA
sequence encoding the desired ribozyme or other nucleic acid
construct. An oligonucleotide primer bearing the desired mutated
sequence is synthetically prepared. This primer is then annealed
with the single-stranded DNA preparation, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli cells, and clones are
selected that include recombinant vectors bearing the mutated
sequence arrangement.
[0132] The preparation of sequence variants of the selected nucleic
acid sequences using site-directed mutagenesis is provided as a
means of producing potentially useful species and is not meant to
be limiting, as there are other ways in which sequence variants may
be obtained. For example, recombinant vectors encoding the desired
gene may be treated with mutagenic agents, such as hydroxylamine,
to obtain sequence variants.
[0133] Nucleic Acid Amplification
[0134] In certain embodiments, it may be necessary to employ one or
more nucleic acid amplification techniques to produce the nucleic
acid segments of the present invention. Various methods are
well-known to artisans in the field, including for example, those
techniques described herein:
[0135] Nucleic acid, used as a template for amplification, may be
isolated from cells contained in the biological sample according to
standard methodologies (Sambrook et al., 1989). The nucleic acid
may be genomic DNA or fractionated or whole cell RNA. Where RNA is
used, it may be desired to convert the RNA to a complementary DNA.
In one embodiment, the RNA is whole cell RNA and is used directly
as the template for amplification.
[0136] Pairs of primers that selectively hybridize to nucleic acids
corresponding to the ribozymes or conserved flanking regions are
contacted with the isolated nucleic acid under conditions that
permit selective hybridization. The term "primer", as defined
herein, is meant to encompass any nucleic acid that is capable of
priming the synthesis of a nascent nucleic acid in a
template-dependent process. Typically, primers are oligonucleotides
from ten to twenty base pairs in length, but longer sequences can
be employed. Primers may be provided in double-stranded or
single-stranded form, although the single-stranded form is
preferred.
[0137] Once hybridized, the nucleic acid:primer complex is
contacted with one or more enzymes that facilitate
template-dependent nucleic acid synthesis. Multiple rounds of
amplification, also referred to as "cycles," are conducted until a
sufficient amount of amplification product is produced.
[0138] Next, the amplification product is detected. In certain
applications, the detection may be performed by visual means.
Alternatively, the detection may involve indirect identification of
the product via chemiluminescence, radioactive scintigraphy of
incorporated radiolabel or fluorescent label or even via a system
using electrical or thermal impulse signals (e.g., Affymax
technology).
[0139] A number of template dependent processes are available to
amplify the marker sequences present in a given template sample.
One of the best-known amplification methods is the polymerase chain
reaction (referred to as PCR.TM.), which is described in detail in
U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No.
4,800,159 (each of which is specifically incorporated herein in its
entirety by express reference thereto).
[0140] Briefly, in PCR.TM., two primer sequences are prepared that
are complementary to regions on opposite complementary strands of
the marker sequence. An excess of deoxynucleoside triphosphates is
added to a reaction mixture along with a DNA polymerase, e.g., Taq
polymerase. If the marker sequence is present in a sample, the
primers will bind to the marker and the polymerase will cause the
primers to be extended along the marker sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
marker to form reaction products, excess primers will bind to the
marker and to the reaction products and the process is
repeated.
[0141] A reverse transcriptase PCR.TM. amplification procedure may
be performed in order to quantify the amount of mRNA amplified.
Methods of reverse transcribing RNA into cDNA are well known and
described (Sambrook et al., 1989). Alternative methods for reverse
transcription utilize thermostable, RNA-dependent DNA polymerases.
These methods are described in Int. Pat. Appl. Publ. No. WO
90/07641 (specifically incorporated herein in its entirety by
express reference thereto). Polymerase chain reaction methodologies
are well known in the art.
[0142] Another method for amplification is the ligase chain
reaction ("LCR"), disclosed in EP Appl. No. 320308 (specifically
incorporated herein in its entirety by express reference thereto).
In LCR, two complementary probe pairs are prepared, and in the
presence of the target sequence, each pair will bind to opposite
complementary strands of the target such that they abut. In the
presence of a ligase, the two probe pairs will link to form a
single unit. By temperature cycling, as in PCR.TM., bound ligated
units dissociate from the target and then serve as "target
sequences" for ligation of excess probe pairs. U.S. Pat. No.
4,883,750 (specifically incorporated herein in its entirety by
express reference thereto) describes a method similar to LCR for
binding probe pairs to a target sequence.
[0143] Q.beta. Replicase (Q.beta.R), described in Int. Pat. Appl.
No. PCT/US87/00880 (specifically incorporated herein in its
entirety by express reference thereto), may also be used as still
another amplification method in the present invention. In this
method, a replicative sequence of RNA that has a region
complementary to that of a target is added to a sample in the
presence of an RNA polymerase. The polymerase will copy the
replicative sequence that can then be detected.
[0144] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[.alpha.-thio]-triphosphates in one strand of a restriction site
may also be useful in the amplification of nucleic acids in the
present invention.
[0145] Strand Displacement Amplification (SDA), described in U.S.
Pat. Nos. 5,455,166, 5,648,211, 5,712,124 and 5,744,311 (each of
which is specifically incorporated herein in its entirety by
express reference thereto), is another method of carrying out
isothermal amplification of nucleic acids which involves multiple
rounds of strand displacement and synthesis, i.e., nick
translation. A similar method, called Repair Chain Reaction (RCR),
involves annealing several probes throughout a region targeted for
amplification, followed by a repair reaction in which only two of
the four bases are present. The other two bases can be added as
biotinylated derivatives for easy detection. A similar approach is
used in SDA. Target specific sequences can also be detected using a
cyclic probe reaction (CPR). In CPR, a probe having 3' and 5'
sequences of non-specific DNA, and a middle sequence of specific
RNA is hybridized to DNA that is present in a sample. Upon
hybridization, the reaction is treated with RNase H, and the
products of the probe identified as distinctive products that are
released after digestion. The original template is annealed to
another cycling probe and the reaction is repeated.
[0146] Still another amplification methods described in GB
Application No. 2 202 328, and in Int. Pat. Appl. No.
PCT/US89/01025 (each of which is specifically incorporated herein
in its entirety by express reference thereto), may be used in
accordance with the present invention. In the former application,
"modified" primers are used in a PCR.TM.-like, template- and
enzyme-dependent synthesis. The primers may be modified by labeling
with a capture moiety (e.g., biotin) and/or a detector moiety
(e.g., enzyme). In the latter application, an excess of labeled
probes is added to a sample. In the presence of the target
sequence, the probe binds and is cleaved catalytically. After
cleavage, the target sequence is released intact to be bound by
excess probe. Cleavage of the labeled probe signals the presence of
the target sequence.
[0147] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR Gingeras et al.,
PCT Intl. Pat. Appl. Publ. No. WO 88/10315 (specifically
incorporated herein in its entirety by express reference thereto).
In NASBA, the nucleic acids can be prepared for amplification by
standard phenol/chloroform extraction, heat denaturation of a
clinical sample, treatment with lysis buffer and minispin columns
for isolation of DNA and RNA or guanidinium chloride extraction of
RNA. These amplification techniques involve annealing a primer that
has target specific sequences. Following polymerization, DNA/RNA
hybrids are digested with RNase H while double stranded DNA
molecules are heat denatured again. In either case the single
stranded DNA is made fully double stranded by addition of second
target specific primer, followed by polymerization. The
double-stranded DNA molecules are then multiply transcribed by an
RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction,
the RNAs are reverse transcribed into single stranded DNA, which is
then converted to double-stranded DNA, and then transcribed once
again with an RNA polymerase such as T7 or SP6. The resulting
products, whether truncated or complete, indicate target specific
sequences.
[0148] Davey et al., EPA No. 329 822 (specifically incorporated
herein in its entirety by express reference thereto) disclose a
nucleic acid amplification process involving cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA (dsDNA), which may be used in accordance with
the present invention. The ssRNA is a template for a first primer
oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from the
resulting DNA:RNA duplex by the action of ribonuclease H(RNase H,
an RNase specific for RNA in duplex with either DNA or RNA). The
resultant ssDNA is a template for a second primer, which also
includes the sequences of an RNA polymerase promoter (exemplified
by T7 RNA polymerase) 5' to its homology to the template. This
primer is then extended by DNA polymerase (exemplified by the large
"Klenow" fragment of E. coli DNA polymerase I), resulting in a
double-stranded DNA ("dsDNA") molecule, having a sequence identical
to that of the original RNA between the primers and having
additionally, at one end, a promoter sequence. This promoter
sequence can be used by the appropriate RNA polymerase to make many
RNA copies of the DNA. These copies can then re-enter the cycle
leading to very swift amplification. With proper choice of enzymes,
this amplification can be done isothermally without addition of
enzymes at each cycle. Because of the cyclical nature of this
process, the starting sequence can be chosen to be in the form of
either DNA or RNA.
[0149] Miller et al., Int. Pat. Appl. Publ. No. WO 89/06700
(specifically incorporated herein in its entirety by express
reference thereto) disclose a nucleic acid sequence amplification
scheme based on the hybridization of a promoter/primer sequence to
a target single-stranded DNA ("ssDNA") followed by transcription of
many RNA copies of the sequence. This scheme is not cyclic, i.e.,
new templates are not produced from the resultant RNA transcripts.
Other amplification methods include "RACE" and "one-sided PCR.TM."
(Frohman, 1990; specifically incorporated herein in its entirety by
express reference thereto).
[0150] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide," thereby amplifying the
di-oligonucleotide, may also be used in the amplification step of
the present invention.
[0151] Following any amplification, it may be desirable to separate
the amplification product from the template and the excess primer
for the purpose of determining whether specific amplification has
occurred. In one embodiment, amplification products are separated
by agarose, agarose-acrylamide or polyacrylamide gel
electrophoresis using standard methods (see e.g., Sambrook et al.,
1989).
[0152] Alternatively, chromatographic techniques may be employed to
effect separation. There are many kinds of chromatography which may
be used in the present invention: adsorption, partition,
ion-exchange and molecular sieve, and many specialized techniques
for using them including column, paper, thin-layer and gas
chromatography.
[0153] Amplification products must be visualized in order to
confirm amplification of the marker sequences. One typical
visualization method involves staining of a gel with ethidium
bromide and visualization under UV light. Alternatively, if the
amplification products are integrally labeled with radio- or
fluorometrically-labeled nucleotides, the amplification products
can then be exposed to x-ray film or visualized under the
appropriate stimulating spectra, following separation.
[0154] In one embodiment, visualization is achieved indirectly.
Following separation of amplification products, a labeled, nucleic
acid probe is brought into contact with the amplified marker
sequence. The probe preferably is conjugated to a chromophore but
may be radiolabeled. In another embodiment, the probe is conjugated
to a binding partner, such as an antibody or biotin, and the other
member of the binding pair carries a detectable moiety.
[0155] In one embodiment, detection is by Southern blotting and
hybridization with a labeled probe. The techniques involved in
Southern blotting are well known to those of skill in the art and
can be found in many standard books on molecular protocols. See
Sambrook et al., 1989. Briefly, amplification products are
separated by gel electrophoresis. The gel is then contacted with a
membrane, such as nitrocellulose, permitting transfer of the
nucleic acid and non-covalent binding. Subsequently, the membrane
is incubated with a chromophore-conjugated probe that is capable of
hybridizing with a target amplification product. Detection is by
exposure of the membrane to x-ray film or ion-emitting detection
devices.
[0156] One example of the foregoing is described in U.S. Pat. No.
5,279,721 (specifically incorporated herein in its entirety by
express reference thereto), which discloses an apparatus and method
for the automated electrophoresis and transfer of nucleic acids.
The apparatus permits electrophoresis and blotting without external
manipulation of the gel and is ideally suited to carrying out
methods according to the present invention.
[0157] Methods of Nucleic Acid Delivery and DNA Transfection
[0158] In certain embodiments, it is contemplated that one or more
RNA, DNA, PNAs and/or substituted polynucleotide compositions
disclosed herein will be used to transfect an appropriate host
cell. Technology for introduction of PNAs, RNAs, and DNAs into
cells is well known to those of skill in the art.
[0159] Several non-viral methods for the transfer of expression
constructs into cultured mammalian cells also are contemplated by
the present invention. These include calcium phosphate
precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987;
Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation
(Wong and Neumann, 1982; Fromm et al., 1985; Tur-Kaspa et al.,
1986; Potter et al., 1984; Suzuki et al., 1998; Vanbever et al.,
1998), direct microinjection (Capecchi, 1980; Harland and
Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;
Fraley et al., 1979; Takakura, 1998) and lipofectamine-DNA
complexes, cell sonication (Fechheimer et al., 1987), gene
bombardment using high velocity microprojectiles (Yang et al.,
1990; Klein et al., 1992), and receptor-mediated transfection
(Curiel et al., 1991; Wagner et al., 1992; Wu and Wu, 1987; Wu and
Wu, 1988). Some of these techniques may be successfully adapted for
in vivo or ex vivo use.
[0160] Expression Vectors
[0161] The present invention contemplates a variety of AAV-based
expression systems, and vectors. In one embodiment the preferred
AAV expression vectors comprise at least a first nucleic acid
segment that encodes a therapeutic peptide, protein, or
polypeptide. In another embodiment, the preferred AAV expression
vectors disclosed herein comprise at least a first nucleic acid
segment that encodes an antisense molecule. In another embodiment,
a promoter is operatively linked to a sequence region that encodes
a functional mRNA, a tRNA, a ribozyme or an antisense RNA.
[0162] As used herein, the term "operatively linked" means that a
promoter is connected to a functional RNA in such a way that the
transcription of that functional RNA is controlled and regulated by
that promoter. Means for operatively linking a promoter to a
functional RNA are well known in the art.
[0163] The choice of which expression vector and ultimately to
which promoter a polypeptide coding region is operatively linked
depend directly on the functional properties desired, e.g., the
location and timing of protein expression, and the host cell to be
transformed. These are well known limitations inherent in the art
of constructing recombinant DNA molecules. However, a vector useful
in practicing the present invention is capable of directing the
expression of the functional RNA to which it is operatively
linked.
[0164] RNA polymerase transcribes a coding DNA sequence through a
site where polyadenylation occurs. Typically, DNA sequences located
a few hundred base pairs downstream of the polyadenylation site
serve to terminate transcription. Those DNA sequences are referred
to herein as transcription-termination regions. Those regions are
required for efficient polyadenylation of transcribed messenger RNA
(mRNA).
[0165] A variety of methods have been developed to operatively link
DNA to vectors via complementary cohesive termini or blunt ends.
For instance, complementary homopolymer tracts can be added to the
DNA segment to be inserted and to the vector DNA. The vector and
DNA segment are then joined by hydrogen bonding between the
complementary homopolymeric tails to form recombinant DNA
molecules.
[0166] Biological Functional Equivalents
[0167] Modification and changes to the structure of the
polynucleotides and polypeptides of wild-type rAAV vectors to
provide the improved rAAV virions as described in the present
invention to obtain functional viral vectors that possess desirable
characteristics, particularly with respect to improved delivery of
therapeutic gene constructs to selected mammalian cell, tissues,
and organs for the treatment, prevention, and prophylaxis of
various diseases and disorders, as well as means for the
amelioration of symptoms of such diseases, and to facilitate the
expression of exogenous therapeutic and/or prophylactic
polypeptides of interest via rAAV vector-mediated gene therapy. As
mentioned above, one of the key aspects of the present invention is
the creation of one or more mutations into specific polynucleotide
sequences that encode one or more of the therapeutic agents encoded
by the disclosed rAAV constructs. In certain circumstances, the
resulting polypeptide sequence is altered by these mutations, or in
other cases, the sequence of the polypeptide is unchanged by one or
more mutations in the encoding polynucleotide to produce modified
vectors with improved properties for effecting gene therapy in
mammalian systems.
[0168] When it is desirable to alter the amino acid sequence of a
polypeptide to create an equivalent, or even an improved,
second-generation molecule, the amino acid changes may be achieved
by changing one or more of the codons of the encoding DNA sequence,
according to Table 3.
[0169] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Since it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like
properties. It is thus contemplated by the inventors that various
changes may be made in the polynucleotide sequences disclosed
herein, without appreciable loss of their biological utility or
activity.
TABLE-US-00003 TABLE 3 Amino Acids Codons Alanine Ala A GCA GCC GCG
GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic
acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA
GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0170] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982,
incorporate herein by reference). It is accepted that the relative
hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like. Each amino acid has been assigned a hydropathic index on the
basis of their hydrophobicity and charge characteristics (Kyte and
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0171] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e. still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those that
are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred. It is also understood
in the art that the substitution of like amino acids can be made
effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101
(specifically incorporated herein in its entirety by express
reference thereto), states that the greatest local average
hydrophilicity of a protein, as governed by the hydrophilicity of
its adjacent amino acids, correlates with a biological property of
the protein.
[0172] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent protein. In such
changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those that are within .+-.1
are particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0173] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take several of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[0174] Therapeutic and Diagnostic Kits
[0175] The invention also encompasses one or more of the modified
rAAV vector compositions described herein together with one or more
pharmaceutically-acceptable excipients, carriers, diluents,
adjuvants, and/or other components, as may be employed in the
formulation of particular rAAV-polynucleotide delivery
formulations, and in the preparation of therapeutic agents for
administration to a mammal, and in particularly, to a human. In
particular, such kits may comprise one or more of the disclosed
rAAV compositions in combination with instructions for using the
viral vector in the treatment of such disorders in a mammal, and
may typically further include containers prepared for convenient
commercial packaging.
[0176] As such, preferred animals for administration of the
pharmaceutical compositions disclosed herein include mammals, and
particularly humans. Other preferred animals include murines,
bovines, equines, porcines, canines, and felines. The composition
may include partially or significantly purified rAAV compositions,
either alone, or in combination with one or more additional active
ingredients, which may be obtained from natural or recombinant
sources, or which may be obtainable naturally or either chemically
synthesized, or alternatively produced in vitro from recombinant
host cells expressing DNA segments encoding such additional active
ingredients.
[0177] Therapeutic kits may also be prepared that comprise at least
one of the compositions disclosed herein and instructions for using
the composition as a therapeutic agent. The container means for
such kits may typically comprise at least one vial, test tube,
flask, bottle, syringe or other container means, into which the
disclosed rAAV composition(s) may be placed, and preferably
suitably aliquoted. Where a second therapeutic polypeptide
composition is also provided, the kit may also contain a second
distinct container means into which this second composition may be
placed. Alternatively, the plurality of therapeutic biologically
active compositions may be prepared in a single pharmaceutical
composition, and may be packaged in a single container means, such
as a vial, flask, syringe, bottle, or other suitable single
container means. The kits of the present invention will also
typically include a means for containing the vial(s) in close
confinement for commercial sale, such as, e.g., injection or
blow-molded plastic containers into which the desired vial(s) are
retained.
[0178] Ribozymes
[0179] As mentioned above, one aspect of the invention concerns the
use of the modified capsid vectors to deliver catalytic RNA
molecules (ribozymes) to selected mammalian cells and tissues to
effect a reduction or elimination of expression of one or more
native DNA or mRNA molecules, so as to prevent or reduce the amount
of the translation product of such mRNAs. Ribozymes are biological
catalysts consisting of only RNA. They promote a variety of
reactions involving RNA and DNA molecules including site-specific
cleavage, ligation, polymerization, and phosphoryl exchange (Cech,
1989; Cech, 1990). Ribozymes fall into three broad classes: (1)
RNAse P, (2) self-splicing introns, and (3) self-cleaving viral
agents. Self-cleaving agents include hepatitis delta virus and
components of plant virus satellite RNAs that sever the RNA genome
as part of a rolling-circle mode of replication. Because of their
small size and great specificity, ribozymes have the greatest
potential for biotechnical applications. The ability of ribozymes
to cleave other RNA molecules at specific sites in a catalytic
manner has brought them into consideration as inhibitors of viral
replication or of cell proliferation and gives them potential
advantage over antisense RNA. Indeed, ribozymes have already been
used to cleave viral targets and oncogene products in living cells
(Koizumi et al., 1992; Kashani-Sabet et al., 1992; Taylor and
Rossi, 1991; von-Weizsacker et al., 1992; Ojwang et al., 1992;
Stephenson and Gibson, 1991; Yu et al., 1993; Xing and Whitton,
1993; Yu et al., 1995; Little and Lee, 1995).
[0180] Two kinds of ribozymes have been employed widely, hairpins
and hammerheads. Both catalyze sequence-specific cleavage resulting
in products with a 5N hydroxyl and a 2N,3N-cyclic phosphate.
Hammerhead ribozymes have been used more commonly, because they
impose few restrictions on the target site. Hairpin ribozymes are
more stable and, consequently, function better than hammerheads at
physiologic temperature and magnesium concentrations.
[0181] A number of patents have issued describing various ribozymes
and methods for designing ribozymes. See, for example, U.S. Pat.
Nos. 5,646,031; 5,646,020; 5,639,655; 5,093,246; 4,987,071;
5,116,742; and 5,037,746 (each of which is specifically
incorporated herein in its entirety by express reference thereto).
However, the ability of ribozymes to provide therapeutic benefit in
vivo has not yet been demonstrated.
[0182] Although proteins traditionally have been used for catalysis
of nucleic acids, another class of macromolecules has emerged as
useful in this endeavor. Ribozymes are RNA-protein complexes that
cleave nucleic acids in a site-specific fashion. Ribozymes have
specific catalytic domains that possess endonuclease activity (Kim
and Cech, 1987; Gerlach et al., 1987; Forster and Symons, 1987).
For example, a large number of ribozymes accelerate phosphoester
transfer reactions with a high degree of specificity, often
cleaving only one of several phosphoesters in an oligonucleotide
substrate (Cech et al., 1981; Michel and Westhof, 1990;
Reinhold-Hurek and Shub, 1992). This specificity has been
attributed to the requirement that the substrate bind via specific
base-pairing interactions to the internal guide sequence ("IGS") of
the ribozyme prior to chemical reaction.
[0183] Ribozyme catalysis has primarily been observed as part of
sequence-specific cleavage/ligation reactions involving nucleic
acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No.
5,354,855 (specifically incorporated herein in its entirety by
express reference thereto) reports that certain ribozymes can act
as endonucleases with a sequence-specificity greater than that of
known ribonucleases and approaching that of the DNA restriction
enzymes. Thus, sequence-specific ribozyme-mediated inhibition of
gene expression may be particularly suited to therapeutic
applications (Scanlon et al., 1991; Sarver et al., 1990). Recently,
it was reported that ribozymes elicited genetic changes in some
cells lines to which they were applied; the altered genes included
the oncogenes H-ras, c-fos and genes of HIV. Most of this work
involved the modification of a target mRNA, based on a specific
mutant codon that is cleaved by a specific ribozyme.
[0184] Six basic varieties of naturally occurring enzymatic RNAs
are known presently. Each can catalyze the hydrolysis of RNA
phosphodiester bonds in trans- (and thus can cleave other RNA
molecules) under physiological conditions. In general, enzymatic
nucleic acids act by first binding to a target RNA. Such binding
occurs through the target binding portion of an enzymatic nucleic
acid which is held in close proximity to an enzymatic portion of
the molecule that acts to cleave the target RNA. Thus, the
enzymatic nucleic acid first recognizes and then binds a target RNA
through complementary base pairing, and once bound to the correct
site, acts enzymatically to cut the target RNA. Strategic cleavage
of such a target RNA will destroy its ability to direct synthesis
of an encoded protein. After an enzymatic nucleic acid has bound
and cleaved its RNA target, it is released from that RNA to search
for another target and can repeatedly bind and cleave new
targets.
[0185] The enzymatic nature of a ribozyme is advantageous over many
technologies, such as antisense technology (where a nucleic acid
molecule simply binds to a nucleic acid target to block its
translation) since the concentration of ribozyme necessary to
affect a therapeutic treatment is lower than that of an antisense
oligonucleotide. This advantage reflects the ability of the
ribozyme to act enzymatically. Thus, a single ribozyme molecule is
able to cleave many molecules of target RNA. In addition, the
ribozyme is a highly specific inhibitor, with the specificity of
inhibition depending not only on the base pairing mechanism of
binding to the target RNA, but also on the mechanism of target RNA
cleavage. Single mismatches, or base-substitutions, near the site
of cleavage can completely eliminate catalytic activity of a
ribozyme. Similar mismatches in antisense molecules do not prevent
their action (Woolf et al., 1992). Thus, the specificity of action
of a ribozyme is greater than that of an antisense oligonucleotide
binding the same RNA site.
[0186] The enzymatic nucleic acid molecule may be formed in a
hammerhead, hairpin, a hepatitis .delta. virus, group I intron or
RNaseP RNA (in association with an RNA guide sequence) or
Neurospora VS RNA motif. Examples of hammerhead motifs are
described by Rossi et al. (1992). Examples of hairpin motifs are
described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),
Hampel and Tritz (1989), Hampel et al. (1990) and U.S. Pat. No.
5,631,359 (specifically incorporated herein in its entirety by
express reference thereto). An example of the hepatitis .delta.
virus motif is described by Perrotta and Been (1992); an example of
the RNaseP motif is described by Guerrier-Takada et al. (1983);
Neurospora VS RNA ribozyme motif is described by Collins (Saville
and Collins, 1990; Saville and Collins, 1991; Collins and Olive,
1993); and an example of the Group I intron is described in U.S.
Pat. No. 4,987,071 (specifically incorporated herein in its
entirety by express reference thereto). All that is important in an
enzymatic nucleic acid molecule of this invention is that it has a
specific substrate binding site which is complementary to one or
more of the target gene RNA regions, and that it have nucleotide
sequences within or surrounding that substrate binding site which
impart an RNA cleaving activity to the molecule. Thus the ribozyme
constructs need not be limited to specific motifs mentioned
herein.
[0187] In certain embodiments, it may be important to produce
enzymatic cleaving agents that exhibit a high degree of specificity
for the RNA of a desired target, such as one of the sequences
disclosed herein. The enzymatic nucleic acid molecule is preferably
targeted to a highly conserved sequence region of a target mRNA.
Such enzymatic nucleic acid molecules can be delivered exogenously
to specific cells as required, although in preferred embodiments
the ribozymes are expressed from DNA or RNA vectors that are
delivered to specific cells.
[0188] Small enzymatic nucleic acid motifs (e.g., of the hammerhead
or the hairpin structure) may also be used for exogenous delivery.
The simple structure of these molecules increases the ability of
the enzymatic nucleic acid to invade targeted regions of the mRNA
structure. Alternatively, catalytic RNA molecules can be expressed
within cells from eukaryotic promoters (e.g., Scanlon et al., 1991;
Kashani-Sabet et al., 1992; Dropulic et al., 1992; Weerasinghe et
al., 1991; Ojwang et al., 1992; Chen et al., 1992; Sarver et al.,
1990). Those skilled in the art realize that any ribozyme can be
expressed in eukaryotic cells from the appropriate DNA vector. The
activity of such ribozymes can be augmented by their release from
the primary transcript by a second ribozyme (PCT Intl. Pat. Appl.
Publ. No. WO 93/23569, and PCT Intl. Pat. Appl. Publ. No. WO
94/02595, each of which is specifically incorporated herein in its
entirety by express reference thereto; Ohkawa et al., 1992; Taira
et al., 1991; and Ventura et al., 1993).
[0189] Ribozymes may be added directly, or can be complexed with
cationic lipids, lipid complexes, packaged within liposomes, or
otherwise delivered to target cells. The RNA or RNA complexes can
be locally administered to relevant tissues ex vivo, or in vivo
through injection, aerosol inhalation, infusion pump or stent, with
or without their incorporation in biopolymers.
[0190] Ribozymes may be designed as described in PCT Intl. Pat.
Appl. Publ. No. WO 93/23569 and PCT Intl. Pat. Appl. Publ. No. WO
94/02595 (each of which is specifically incorporated herein in its
entirety by express reference thereto) and synthesized to be tested
in vitro and in vivo, as described. Such ribozymes can also be
optimized for delivery. While specific examples are provided, those
in the art will recognize that equivalent RNA targets in other
species can be utilized when necessary.
[0191] Hammerhead or hairpin ribozymes may be individually analyzed
by computer folding (Jaeger et al., 1989) to assess whether the
ribozyme sequences fold into the appropriate secondary structure,
as described herein. Those ribozymes with unfavorable
intramolecular interactions between the binding arms and the
catalytic core are eliminated from consideration. Varying binding
arm lengths can be chosen to optimize activity. Generally, at least
5 or so bases on each arm are able to bind to, or otherwise
interact with, the target RNA.
[0192] Ribozymes of the hammerhead or hairpin motif may be designed
to anneal to various sites in the mRNA message, and can be
chemically synthesized. The method of synthesis used follows the
procedure for normal RNA synthesis as described in Usman et al.
(1987) and in Scaringe et al. (1990) and makes use of common
nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
Average stepwise coupling yields are typically >98%. Hairpin
ribozymes may be synthesized in two parts and annealed to
reconstruct an active ribozyme (Chowrira and Burke, 1992).
Ribozymes may be modified extensively to enhance stability by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-fluoro, 2'-o-methyl, 2'-H (for a review see e.g.,
Usman and Cedergren, 1992). Ribozymes may be purified by gel
electrophoresis using general methods or by high-pressure liquid
chromatography and resuspended in water.
[0193] Ribozyme activity can be optimized by altering the length of
the ribozyme binding arms, or chemically synthesizing ribozymes
with modifications that prevent their degradation by serum
ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065;
Perrault et al, 1990; Pieken et al., 1991; Usman and Cedergren,
1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ.
No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat.
No. 5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688 (each of
which is specifically incorporated herein in its entirety by
express reference thereto), which describe various chemical
modifications that can be made to the sugar moieties of enzymatic
RNA molecules), modifications which enhance their efficacy in
cells, and removal of stem II bases to shorten RNA synthesis times
and reduce chemical requirements.
[0194] A preferred means of accumulating high concentrations of a
ribozyme(s) within cells is to incorporate the ribozyme-encoding
sequences into a DNA expression vector. Transcription of the
ribozyme sequences are driven from a promoter for eukaryotic RNA
polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase
III (pol III). Transcripts from pol II or pol III promoters will be
expressed at high levels in all cells; the levels of a given pol II
promoter in a given cell type will depend on the nature of the gene
regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic RNA polymerase promoters may also be used, providing
that the prokaryotic RNA polymerase enzyme is expressed in the
appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993;
Lieber et al., 1993; Zhou et al., 1990). Ribozymes expressed from
such promoters can function in mammalian cells (Kashani-Sabet et
al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yu et al., 1993;
L'Huillier et al., 1992; Lisziewicz et al., 1993). Although
incorporation of the present ribozyme constructs into
adeno-associated viral vectors is preferred, such transcription
units can be incorporated into a variety of vectors for
introduction into mammalian cells, including but not restricted to,
plasmid DNA vectors, other viral DNA vectors (such as adenovirus
vectors, etc.), or viral RNA vectors (such as retroviral, semliki
forest virus, sindbis virus vectors, etc.).
[0195] Sullivan et al. (PCT Intl. Pat. Appl. Publ. No. WO 94/02595;
specifically incorporated herein in its entirety by express
reference thereto) describes general methods for delivery of
enzymatic RNA molecules. Ribozymes may be administered to cells by
a variety of methods known to those familiar to the art, including,
but not restricted to, encapsulation in liposomes, by
iontophoresis, or by incorporation into other vehicles, such as
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres. For some indications, ribozymes may be
directly delivered ex vivo to cells or tissues with or without the
aforementioned vehicles. Alternatively, the RNA/vehicle combination
may be locally delivered by direct inhalation, by direct injection
or by use of a catheter, infusion pump or stent. Other routes of
delivery include, but are not limited to, intravascular,
intramuscular, subcutaneous or joint injection, aerosol inhalation,
oral (tablet or pill form), topical, systemic, ocular, intraocular,
retinal, subretinal, intraperitoneal, intracerebroventricular,
intrathecal delivery, and/or direct injection to one or more
tissues of the brain. More detailed descriptions of ribozyme and
rAAV vector delivery and administration are provided in PCT Intl.
Pat. Appl. Publ. No. WO 94/02595 and PCT Intl. Pat. Appl. Publ. No.
WO 93/23569, each of which is specifically incorporated herein in
its entirety by express reference thereto.
[0196] Ribozymes and the AAV vectored-constructs of the present
invention may be used to inhibit gene expression and define the
role (essentially) of specified gene products in the progression of
one or more neural diseases, dysfunctions, cancers, and/or
disorders. In this manner, other genetic targets may be defined as
important mediators of the disease. These studies lead to better
treatment of the disease progression by affording the possibility
of combination therapies (e.g., multiple ribozymes targeted to
different genes, ribozymes coupled with known small molecule
inhibitors, or intermittent treatment with combinations of
ribozymes and/or other chemical or biological molecules).
[0197] Antisense Oligonucleotides
[0198] In certain embodiments, the AAV constructs of the invention
will find utility in the delivery of antisense oligonucleotides and
polynucleotides for inhibiting the expression of a selected
mammalian mRNA in neural cells.
[0199] In the art the letters, A, G, C, T, and U respectively
indicate nucleotides in which the nucleoside is Adenosine (Ade),
Guanosine (Gua), Cytidine (Cyt), Thymidine (Thy), and Uridine
(Ura). As used in the specification and claims, compounds that are
"antisense" to a particular PNA, DNA or mRNA "sense" strand are
nucleotide compounds that have a nucleoside sequence that is
complementary to the sense strand. It will be understood by those
skilled in the art that the present invention broadly includes
oligonucleotide compounds that are capable of binding to the
selected DNA or mRNA sense strand. It will also be understood that
mRNA includes not only the ribonucleotide sequences encoding a
protein, but also regions including the 5'-untranslated region, the
3'-untranslated region, the 5'-cap region and the intron/exon
junction regions.
[0200] The invention includes compounds which are not strictly
antisense; the compounds of the invention also include those
oligonucleotides that may have some bases that are not
complementary to bases in the sense strand provided such compounds
have sufficient binding affinity for the particular DNA or mRNA for
which an inhibition of expression is desired. In addition, base
modifications or the use of universal bases such as inosine in the
oligonucleotides of the invention are contemplated within the scope
of the subject invention.
[0201] The antisense compounds may have some or all of the
phosphates in the nucleotides replaced by phosphorothioates
(X.dbd.S) or methylphosphonates (X.dbd.CH.sub.3) or other C.sub.1-4
alkylphosphonates. The antisense compounds optionally may be
further differentiated from native DNA by replacing one or both of
the free hydroxy groups of the antisense molecule with C.sub.1-4
alkoxy groups (R.dbd.C.sub.1-4 alkoxy). As used herein, C.sub.1-4
alkyl means a branched or unbranched hydrocarbon having 1 to 4
carbon-atoms.
[0202] The disclosed antisense compounds also may be substituted at
the 3' and/or 5' ends by a substituted acridine derivative. As used
herein, "substituted acridine," means any acridine derivative
capable of intercalating nucleotide strands such as DNA. Preferred
substituted acridines are 2-methoxy-6-chloro-9-pentylaminoacridine,
N-(6-chloro-2-methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-3-ami-
nopropanol, and
N-(6-chloro2-methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-5-amin-
opentanol. Other suitable acridine derivatives are readily apparent
to persons skilled in the art. Additionally, as used herein
"P(O)(O)-substituted acridine" means a phosphate covalently linked
to a substitute acridine.
[0203] As used herein, the term "nucleotides" includes nucleotides
in which the phosphate moiety is replaced by phosphorothioate or
alkylphosphonate and the nucleotides may be substituted by
substituted acridines.
[0204] In one embodiment, the antisense compounds of the invention
differ from native DNA by the modification of the phosphodiester
backbone to extend the life of the antisense molecule. For example,
the phosphates can be replaced by phosphorothioates. The ends of
the molecule may also be optimally substituted by an acridine
derivative that intercalates nucleotide strands of DNA. PCT Intl.
Pat. Appl. Publ. No. WO 98/13526 and U.S. Pat. No. 5,849,902 (each
of which is specifically incorporated herein in its entirety by
express reference thereto) describe a method of preparing three
component chimeric antisense compositions, and discuss many of the
currently available methodologies for synthesis of substituted
oligonucleotides having improved antisense characteristics and/or
half-life.
[0205] The reaction scheme involves .sup.1H-tetrazole-catalyzed
coupling of phosphoramidites to give phosphate intermediates that
are subsequently reacted with sulfur in 2,6-lutidine to generate
phosphate compounds. Oligonucleotide compounds are prepared by
treating the phosphate compounds with thiophenoxide (1:2:2
thiophenol/triethylamine/tetrahydrofuran, room temperature, 1 hr).
The reaction sequence is repeated until an oligonucleotide compound
of the desired length has been prepared. The compounds are cleaved
from the support by treating with ammonium hydroxide at room
temperature for 1 hr and then are further deprotected by heating at
about 50.degree. C. overnight to yield preferred antisense
compounds.
[0206] Selection of antisense compositions specific for a given
gene sequence is based upon analysis of the chosen target sequence
and determination of secondary structure, T.sub.m, binding energy,
relative stability, and antisense compositions were selected based
upon their relative inability to form dimers, hairpins, or other
secondary structures that would reduce or prohibit specific binding
to the target mRNA in a host cell. Highly preferred target regions
of the mRNA, are those that are at or near the AUG translation
initiation codon, and those sequences that were substantially
complementary to 5' regions of the mRNA. These secondary structure
analyses and target site selection considerations were performed
using v.4 of the OLIGO primer analysis software (Rychlik, 1997) and
the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).
Exemplary Definitions
[0207] In accordance with the present invention, polynucleotides,
nucleic acid segments, nucleic acid sequences, and the like,
include, but are not limited to, DNAs (including and not limited to
genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs)
RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs),
nucleosides, and suitable nucleic acid segments either obtained
from native sources, chemically synthesized, modified, or otherwise
prepared in whole or in part by the hand of man.
[0208] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and compositions similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and compositions are
described herein. For purposes of the present invention, the
following terms are defined below:
[0209] A, an: In accordance with long standing patent law
convention, the words "a" and "an" when used in this application,
including the claims, denotes "one or more".
[0210] Expression: The combination of intracellular processes,
including transcription and translation undergone by a
polynucleotide such as a structural gene to synthesize the encoded
peptide or polypeptide.
[0211] Promoter: a term used to generally describe the region or
regions of a nucleic acid sequence that regulates
transcription.
[0212] Regulatory element: a term used to generally describe the
region or regions of a nucleic acid sequence that regulates
transcription.
[0213] Structural gene: A gene or sequence region that is expressed
to produce an encoded peptide or polypeptide.
[0214] Transformation: A process of introducing an exogenous
polynucleotide sequence (e.g., a vector, a recombinant DNA or RNA
molecule) into a host cell or protoplast in which that exogenous
nucleic acid segment is incorporated into at least a first
chromosome or is capable of autonomous replication within the
transformed host cell. Transfection, electroporation, and naked
nucleic acid uptake all represent examples of techniques used to
transform a host cell with one or more polynucleotides.
[0215] Transformed cell: A host cell whose nucleic acid complement
has been altered by the introduction of one or more exogenous
polynucleotides into that cell.
[0216] Transgenic cell: Any cell derived or regenerated from a
transformed cell or derived from a transgenic cell, or from the
progeny or offspring of any generation of such a transformed host
cell.
[0217] Vector: A nucleic acid molecule (typically comprised of DNA)
capable of replication in a host cell and/or to which another
nucleic acid segment can be operatively linked so as to bring about
replication of the attached segment. A plasmid, cosmid, or a virus
is an exemplary vector.
[0218] The terms "substantially corresponds to", "substantially
homologous", or "substantial identity" as used herein denotes a
characteristic of a nucleic acid or an amino acid sequence, wherein
a selected nucleic acid or amino acid sequence has at least about
70 or about 75 percent sequence identity as compared to a selected
reference nucleic acid or amino acid sequence. More typically, the
selected sequence and the reference sequence will have at least
about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent
sequence identity, and more preferably at least about 86, 87, 88,
89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More
preferably still, highly homologous sequences often share greater
than at least about 96, 97, 98, or 99 percent sequence identity
between the selected sequence and the reference sequence to which
it was compared. The percentage of sequence identity may be
calculated over the entire length of the sequences to be compared,
or may be calculated by excluding small deletions or additions
which total less than about 25 percent or so of the chosen
reference sequence. The reference sequence may be a subset of a
larger sequence, such as a portion of a gene or flanking sequence,
or a repetitive portion of a chromosome. However, in the case of
sequence homology of two or more polynucleotide sequences, the
reference sequence will typically comprise at least about 18-25
nucleotides, more typically at least about 26 to 35 nucleotides,
and even more typically at least about 40, 50, 60, 70, 80, 90, or
even 100 or so nucleotides. Desirably, which highly homologous
fragments are desired, the extent of percent identity between the
two sequences will be at least about 80%, preferably at least about
85%, and more preferably about 90% or 95% or higher, as readily
determined by one or more of the sequence comparison algorithms
well-known to those of skill in the art, such as e.g., the FASTA
program analysis described by Pearson and Lipman (1988).
[0219] The term "naturally occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by the hand of man in a laboratory is naturally-occurring.
As used herein, laboratory strains of rodents that may have been
selectively bred according to classical genetics are considered
naturally occurring animals.
[0220] As used herein, a "heterologous" is defined in relation to a
predetermined referenced gene sequence. For example, with respect
to a structural gene sequence, a heterologous promoter is defined
as a promoter which does not naturally occur adjacent to the
referenced structural gene, but which is positioned by laboratory
manipulation. Likewise, a heterologous gene or nucleic acid segment
is defined as a gene or segment that does not naturally occur
adjacent to the referenced promoter and/or enhancer elements.
[0221] "Transcriptional regulatory element" refers to a
polynucleotide sequence that activates transcription alone or in
combination with one or more other nucleic acid sequences. A
transcriptional regulatory element can, for example, comprise one
or more promoters, one or more response elements, one or more
negative regulatory elements, and/or one or more enhancers.
[0222] As used herein, a "transcription factor recognition site"
and a "transcription factor binding site" refer to a polynucleotide
sequence(s) or sequence motif(s) which are identified as being
sites for the sequence-specific interaction of one or more
transcription factors, frequently taking the form of direct
protein-DNA binding. Typically, transcription factor binding sites
can be identified by DNA footprinting, gel mobility shift assays,
and the like, and/or can be predicted on the basis of known
consensus sequence motifs, or by other methods known to those of
skill in the art.
[0223] As used herein, the term "operably linked" refers to a
linkage of two or more polynucleotides or two or more nucleic acid
sequences in a functional relationship. A nucleic acid is "operably
linked" when it is placed into a functional relationship with
another nucleic acid sequence. For instance, a promoter or enhancer
is operably linked to a coding sequence if it affects the
transcription of the coding sequence. Operably linked means that
the DNA sequences being linked are typically contiguous and, where
necessary to join two protein coding regions, contiguous and in
reading frame. However, since enhancers generally function when
separated from the promoter by several kilobases and intronic
sequences may be of variable lengths, some polynucleotide elements
may be operably linked but not contiguous.
[0224] "Transcriptional unit" refers to a polynucleotide sequence
that comprises at least a first structural gene operably linked to
at least a first cis-acting promoter sequence and optionally linked
operably to one or more other cis-acting nucleic acid sequences
necessary for efficient transcription of the structural gene
sequences, and at least a first distal regulatory element as may be
required for the appropriate tissue-specific and developmental
transcription of the structural gene sequence operably positioned
under the control of the promoter and/or enhancer elements, as well
as any additional cis sequences that are necessary for efficient
transcription and translation (e.g., polyadenylation site(s), mRNA
stability controlling sequence(s), etc.
[0225] The term "substantially complementary," when used to define
either amino acid or nucleic acid sequences, means that a
particular subject sequence, for example, an oligonucleotide
sequence, is substantially complementary to all or a portion of the
selected sequence, and thus will specifically bind to a portion of
an mRNA encoding the selected sequence. As such, typically the
sequences will be highly complementary to the mRNA "target"
sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 base mismatches throughout the complementary portion of the
sequence. In many instances, it may be desirable for the sequences
to be exact matches, i.e. be completely complementary to the
sequence to which the oligonucleotide specifically binds, and
therefore have zero mismatches along the complementary stretch. As
such, highly complementary sequences will typically bind quite
specifically to the target sequence region of the mRNA and will
therefore be highly efficient in reducing, and/or even inhibiting
the translation of the target mRNA sequence into polypeptide
product.
[0226] Substantially complementary oligonucleotide sequences will
be greater than about 80 percent complementary (or `% exact-match`)
to the corresponding mRNA target sequence to which the
oligonucleotide specifically binds, and will, more preferably be
greater than about 85 percent complementary to the corresponding
mRNA target sequence to which the oligonucleotide specifically
binds. In certain aspects, as described above, it will be desirable
to have even more substantially complementary oligonucleotide
sequences for use in the practice of the invention, and in such
instances, the oligonucleotide sequences will be greater than about
90 percent complementary to the corresponding mRNA target sequence
to which the oligonucleotide specifically binds, and may in certain
embodiments be greater than about 95 percent complementary to the
corresponding mRNA target sequence to which the oligonucleotide
specifically binds, and even up to and including 96%, 97%, 98%,
99%, and even 100% exact match complementary to all or a portion of
the target mRNA to which the designed oligonucleotide specifically
binds.
[0227] Percent similarity or percent complementary of any of the
disclosed sequences may be determined, for example, by comparing
sequence information using the GAP computer program, version 6.0,
available from the University of Wisconsin Genetics Computer Group
(UWGCG). The GAP program utilizes the alignment method of Needleman
and Wunsch (1970). Briefly, the GAP program defines similarity as
the number of aligned symbols (i.e., nucleotides or amino acids)
that are similar, divided by the total number of symbols in the
shorter of the two sequences. The preferred default parameters for
the GAP program include: (1) a unary comparison matrix (containing
a value of 1 for identities and 0 for non-identities) for
nucleotides, and the weighted comparison matrix of Gribskov and
Burgess (1986), (2) a penalty of 3.0 for each gap and an additional
0.10 penalty for each symbol in each gap; and (3) no penalty for
end gaps.
EXAMPLES
[0228] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Improved rAAV Vectors Having Genetic Modifications in Specific
Capsid Proteins
[0229] Given advances in purification methods for rAAV2, the
requirements of the individual capsid protein species in rAAV2
particle formation were reexamined in the context of designing a
novel rAAV2 production system that would allow for the modification
of a specific capsid protein in regions of capsid sequence overlap.
Currently, highly purified and concentrated preparations of rAAV2
particles are possible from two plasmid-based production systems.
These systems differ in that one system supplies the necessary
adenovirus helper functions and AAV rep and cap genes from one
plasmid (pDG), while the other uses two plasmids to supply these
proteins (pIM45 and pXX6). These constructs are transfected into an
appropriate cell type along with a construct containing a transgene
expression cassette flanked by the AAV terminal repeats (e.g.,
pTR-UF5). This example describes an rAAV2 production system based
on modifications of the triple plasmid transfection method. In this
system, the expression of a specific capsid protein is restricted
to one pIM45 plasmid and complemented in trans with the remaining
two capsid proteins expressed from a second pIM45 plasmid. This
approach maintains expression of the capsid proteins in their
genomic context while providing a platform for the genetic
modification of a specific capsid protein or two of the capsid
proteins across their entire coding sequence. Missense mutation of
the capsid proteins' start codons generated pIM45 plasmids that
express a single capsid protein: pIM45-VP1, pIM45-VP2 (ACG or ATG
start codon), and pIM45-VP3. Such plasmids can be complemented with
plasmids expressing the remaining 2 capsid proteins (pIM45-VP2,3,
pIM45-VP1,3, and pIM45-VP1,2 (ACG or ATG start codon),
respectively) in order to produce viable rAAV2 vectors. Using the
system's plasmid components individually, a reevaluation of capsid
protein requirements for the production of rAAV2 particles revealed
that viable rAAV2-like particles are produced as long as the VP3
protein is present (VP1+2+3, VP1+3, VP2+3, and VP3 only). Focusing
on large peptide insertions in the VP1 and VP2 proteins without
altering the critical VP3 protein, the utility of this system is
demonstrated through the production of viable rAAV2 particles
containing 8-, 15-, and 29-kDa proteins inserted immediately
following amino acid 138 in both VP1 and VP2 proteins or in VP2
protein alone. Finally, rAAV2-like particles can be produced with
altered capsid protein stoichiometry if VP2 is significantly over
expressed.
[0230] Construction of rAAV2 Capsid Mutant Plasmids that Express 2
Capsid Proteins
[0231] To isolate the expression of a specific capsid protein to
one pIM45 plasmid and the remaining two capsid proteins to a second
pIM45 plasmid, missense mutation of the AAV2 cap ORF start codons
was employed as previously described. Using site-directed
mutagenesis of a pIM45 template, the VP1 start codon was mutated to
leucine to generate the construct, pIM45-VP2,3, the VP2 start codon
to alanine to generate the construct, pIM45-VP1,3, and the VP3
start codon to leucine to generate the construct, pIM45-VP1,2 (FIG.
1A-1 and FIG. 1A-2). Western blotting analysis of capsid protein
expression in whole cell lysates 48 hrs post transfection of 293
cells with these plasmids in the presence of Ad5 (MOI=10) was
carried out using the B1 antibody which recognizes all three capsid
proteins (FIG. 1A-1 and FIG. 1A-2). As previously reported, the
expression of VP1 and VP2 could be eliminated by missense mutation
of their start codons (FIG. 1A-1, lanes 2 and 3), and, in contrast,
mutation of the VP3 start codon resulted in expression of a smaller
VP3-like fragment (VP3a) (FIG. 1A-1, lane 4). Since this construct
did not eliminate all VP3-like proteins it was renamed,
pIM45-M203L. In the baculovirus study of AAV particle assembly, it
was suggested that mutation of the VP3 start codon allows
translational initiation to occur downstream at the next available
ATG codon with correct Kozak sequences. While no additional ATG
codons are found between the VP1 start codon and the start of VP3,
an examination of the VP3 capsid revealed that nine additional ATG
codons are present (amino acid positions 211, 235, 371, 402, 434,
523, 558, 604, and 634). Of these methionines, only those at amino
acid position 211, 235, 523, 558, and 604 are in a context that is
predicted favorable by Kozak. Since the VP3a fragment is slightly
smaller than wildtype VP3, the contribution of continued read
through translational initiation to the appearance of the VP3a
fragment was examined by mutating the next two available ATG codons
(M211 and M235) on a pIM45-M203L template yielding the plasmids,
pIM45-M203L, pIM45-M203,211L and pIM45-M203,211,235L (FIG. 2A).
Western blotting analysis of capsid protein expression in whole
cell lysates 48 hrs post transfection of 293 cells in the presence
of Ad5 (MOI=10) revealed that translational initiation could occur
at both these ATG codons. FIG. 1B-1 and FIG. 1B-2 (lane 2) again
demonstrates the formation of VP3a following the mutation M203L.
Combined mutation of M203 and M211 allowed less robust expression
of a second still shorter VP3-like fragment (VP3b, FIG. 1B-1 and
FIG. 1B-2, lane 3). Subsequent mutation of M235 in the
pIM45-M203,211L background led to disappearance of this VP3b
fragment generating pIM45-VP1,2 (FIG. 1B-1, lane 4). Collectively,
while missense mutagenesis of the VP1 start codon does not alter
the sequence of the VP2 and VP3 protein expressed (pIM45-VP2,3,
M1L), mutation of the VP2 start codon results in one point mutation
in the expressed VP1 protein (pIM45-VP1,3, T138A), and elimination
of all VP3-like proteins results in three mutations in the
remaining VP1 and VP2 proteins (pIM45-VP1,2, M203,211,235L).
[0232] An alternative method has been reported for eliminating VP3
expression that limits mutation of remaining capsid sequences to
one point mutation in the VP2 start codon. Changing the VP2 start
codon from ACG to ATG results in loss of VP3 expression
(pIM45-VP1,2A) with one point mutation in both the VP1 and VP2
proteins (T138M). Presumably, this stronger VP2 start codon
prevents efficient translational initiation at the downstream VP3
start codon. The VP2 start codon was mutated to ATG on a pIM45
template [pIM45-VP1,2A (FIG. 1C-1 and FIG. 1C-2)] as an alternative
means of eliminating VP3 protein (while maximizing VP2 expression).
As expected, Western blotting analysis of capsid protein expression
in whole cell lysates 48 hrs post transfection of 293 cells in the
presence of Ad5 (MOI=10) with pIM45-VP1,2A showed normal levels of
VP1 protein produced, with significantly increased expression of
VP2 protein (FIG. 1C-2, lane 2).
[0233] Construction of rAAV2 Capsid Plasmid Mutants that Express a
Single Capsid Protein
[0234] To complete the complementary pIM45 capsid groups, pIM45
plasmids that express a single capsid protein were generated next.
Employing the same missense mutations described above on templates
that now only express two capsid proteins, the plasmids, pIM45-VP1,
pIM45-VP2, pIM45-VP2A, and pIM45-VP3 (FIG. 2A and FIG. 2B) were
also generated. pIM45-VP1 has the VP2 start codon mutated to
alanine and M203, M211, and M235 mutated to L in the expressed VP1
protein. pIM45-VP2 has the VP1 start codon mutated to leucine and
M203, M211, and M235 mutated to L. The expressed VP2 protein
contains only M203, M211, and M235 mutations. pIM45-VP3 has the VP1
start codon mutated to leucine and the VP2 start codon mutated to
alanine. Like all VP3 protein in these complementary groups, the
VP3 coding sequence is not mutated. Finally, pIM45-VP2A has the VP1
start codon mutated to leucine and the VP2 start codon mutated to
methionine resulting in the single T138M modification of the VP2
protein being expressed. Western blotting analysis of capsid
protein expression in whole cell lysates 48 hr post transfection of
293 cells with pIM45-VP1, pIM45-VP2, pIM45-VP2A, and pIM45-VP3 in
the presence of Ad5 (MOI=10) demonstrated that a single capsid
protein could be expressed from the pIM45 cap ORF (FIG. 2A and FIG.
2B) and completed the catalogue of plasmids required of a system
for further genetic manipulation of a specific capsid protein
across its entire coding sequence.
[0235] VP3 N-Terminal M203 and M211 are Critical for AAV Particle
Formation
[0236] As control experiments for the production of AAV particles
from the complementary groups of single and double capsid
expressing pIM45 plasmids, particle production was examined from
the individual plasmids described. Since VP3 protein makes up the
bulk of the particle, and mutagenesis studies have indicated that
the N-terminal region of VP3 is important for AAV particle
formation, the effects of the three mutations required to eliminate
VP3 expression (M203,211,235L) were investigated on the recovery of
rAAV particles following standard production and purification
protocols. The plasmids pIM45-M203L, pIM45-M211L, pIM45-M235L, and
pIM45M-203,211,235L were cotransfected separately with pTR-UF5 and
pXX6 in a 1:1:8 molar ratio in 293 cells and 72 hrs later the cells
were harvested and particles were purified as previously reported.
Western blotting of capsid protein expression and dot blot analysis
of genome containing particles was carried out on the mutant virus
preparations (FIG. 3A-1 and FIG. 3A-2). No particles were recovered
from pIM45-M203L (lane 2) indicating that the combination of VP1,
VP2, and VP3a does not able form a stable AAV particle. Equally
important in the formation of the particle is M211 (lane 3), as
this mutation also prevented particle recovery. Whether it is the
M211L in VP1, VP2, or VP3 that leads to this defective phenotype is
unclear. This issue is addressed infra when pIM45-VP1,2 is
complemented with pIM45-VP3 to produce AAV particles (FIG. 4, #5).
Finally, particles were obtained from pIM45-M235L (FIG. 3A-1 and
FIG. 3A-2, lane 4) that package DNA efficiently.
[0237] AAV-Like Particles can be Produced that Lack VP10R VP2
Protein
[0238] While the effect of mutating the individual capsid start
codons on the formation of infectious AAV particles has been
reported, given the improvements in AAV2 production and
purification methods, control experiments were performed to
reexamine the role of each capsid protein in the formation of the
AAV2 particle capable of binding heparin. First examined were the
effects of the elimination of one capsid protein on AAV2 particle
recovery. pIM45-VP2,3; pIM45-VP1,3, pIM45-VP1,2; and pIM45-VP1,2A;
were transfected separately into 293 cells with pTR-UF5 and pXX6 in
a 1:1:8 molar ratio and 72 hrs later the cells were harvested and
particles were purified as previously reported. Western blotting,
A20 ELISA, and dot blot analysis of these virus preparations were
carried out (FIG. 3B-1 and FIG. 3B-2) and, in agreement with
previous reports, the elimination of the VP1 protein (pIM45-VP2,3)
resulted in the production of an AAV-like particle that packaged
genomes efficiently (lane 4). Surprisingly, in contrast with the
initial report mapping the capsid start codons, transfection of the
pIM45-VP1,3 plasmid resulted in the purification of an AAV-like
particle capable of packaging genomes efficiently composed of only
VP1 and VP3 (lane 3) that had only a modest decrease in infectivity
compared to particles containing all three capsid proteins
(two-fold decrease). Finally, regardless if VP2 is overexpressed,
particles composed of only VP1 and VP2 were not recovered (lane
2).
[0239] AAV-Like Particles can be Produced Composed Only of VP3
Capsid Proteins
[0240] As with the pIM45 plasmids that express two capsid proteins,
the ability of a single capsid protein to form an AAV-like particle
was tested. pIM45-VP1, pIM45-VP2, pIM45-VP2A, and pIM45-VP3 were
transfected separately into 293 cells with pTR-UF5 and pXX6 in a
1:1:8 molar ratio and harvested cells 72 hrs later and purified
particles as previously described. Western blotting of capsid
proteins, A20 ELISA, and dot blot analysis of virus preparations
were carried out with no detectable AAV-like particles obtained
from pIM45-VP1, pIM45-VP2, or pIM45-VP2A (FIG. 3C-1 and FIG. 3C-2,
lanes 2 and 3). Interestingly, like a recent insertional
mutagenesis study of the cap ORF, an AAV-like particle composed
exclusively of VP3 protein was purified (lane 3). Like the VP2,3
AAV-like particle, this particle had a significantly lower
infectious phenotype.
[0241] rAAV Particles with all Three Capsid Proteins can be
Produced from Capsid Complementation Groups
[0242] Given the results of the control experiments, the ability to
recover rAAV2 particles containing all three capsid proteins
following transfection of two complementary pIM45 plasmids was
tested (FIG. 4). To control for twice the Rep expression resulting
from two pIM45 plasmids, an additional plasmid was constructed,
pIM45-VP0, that expresses no capsid proteins as a result of 5 point
mutations (M1L, T138A, M203,211,235L). Complementary group VP0
(FIG. 4, #1) includes pIM45 and pIM45-VP0, group VP1 includes
pIM45-VP1 and pIM45-VP2,3 (FIG. 4, #2), group VP2 includes
pIM45-VP2 and pIM45-VP1,3 (FIG. 4, #3), group VP2A includes
pIM45-VP2A and pIM45-VP1,3 (FIG. 4, #4), and group VP3 includes
pIM45-VP3 and pIM45-VP1,2 (FIG. 4, #5). Western blotting of capsid
proteins, A20 ELISA, and dot blot analysis of virus preparations
were carried out following transfection of the individual groups
into 293 cells with pTR-UF5 and pXX6 in a 1:1:8 molar ratio. 72 hrs
post transfection the cells were harvested and particles were
purified as previously described. Infectious rAAV particles
containing all three capsid proteins with similar yields were
recovered (FIG. 5A-1 and FIG. 5A-2). Interestingly, the VP2A group
resulted in the formation of particles with an apparent alteration
of capsid protein stoichiometry and lower infectivity compared to
other groups (lane 4). The characteristics of this group suggest
that this preparation may contain a single unique particle that is
defective per se or alternatively two particles may be assembled
containing all three capsid proteins at normal levels and a
defective interfering particle composed of VP2 and VP3 proteins
with altered stoichiometry. Cotransfection of the pIM45-VP2A and
pIM45-VP3 plasmid should yield a particle with an altered VP2:VP3
ratio if such a defective interfering particle contributes to the
low titer of this group. Indeed, an AAV-like particle with an
overrepresentation of VP2 protein was purified that resembled the
VP2,3 and VP3 only particles with respect to infectivity (FIG. 5B-1
and FIG. 5B-2, lane 4).
[0243] Production of AAV Particles with Insertions in the VP1/VP2
Overlap Region
[0244] Since the VP1/VP2 overlap region has been shown to be on the
surface of the particle and flexible in the acceptance of targeting
epitopes, the ability of this region to accept larger insertions
was examined. Presumably, large insertions in the VP3 protein would
decrease ones success in obtaining a particle due to steric
hindrances in assembling the 60 modified capsid subunits. Sixty
ligands were considered excessive when inserting large molecules
into the AAV particle, so the strategy employed was to focus larger
insertions to VP1 and/or VP2 proteins. Large insertions in both VP1
and VP2 protein immediately after amino acid 138 may have less
steric constraints but may produce particles with defective
trafficking due to the juxtaposition of a large insertion to the
putative phospholipase motif in VP1 protein. Also, since VP1,
essentially an N-terminal fusion of 137 amino acids to VP2, and a
CD 34 sc antibody VP2 protein fusion are readily incorporated into
an AAV particle, insertion of large epitopes only at the N-terminus
of VP2 may have advantages. Notably, genetic modification of the
VP2 protein exclusively has not been accomplished from within a
pIM45 based AAV production scheme. To address the ability to insert
large peptide sequences in the VP1/VP2 overlap region of the cap
ORF, directional cloning sites were generated immediately after
amino acid 138 in plasmids that express VP1 and VP2 or VP2 only
(FIG. 6A). The choice of EagI/MluI restriction sites resulted in
two amino acids insertions on either side of further inserted
sequence (RP/RT). pIM45-VP1,2A and pIM45-VP2A were chosen as
templates for engineering EagI/MluI restriction sites immediately
after amino acid 138 in VP1 and VP2 or in VP2 only
(pIM45VP1,2AEM138, pIM45-VP2AEM138, FIG. 6A). Templates with VP2
protein over expression were used to help ensure that
genetically-modified VP2 would be present at sufficient levels for
assembly. The pIM45-VP1,2AEM138 plasmid was complemented with
pIM45-VP3, while the pIM45-VP2AEM138 plasmid was complemented with
pIM45-VP1,3 for the production of rAAV particles carrying insertion
sequences. Insertion of the coding sequence for leptin and GFP was
in these plasmids generated pIM45-VP1,2Alep, pIM45-VP2Alep,
pIM45-VP1,2AGFP, and pIM45-VP2AGFP. These plasmids and their
complements were transfected with in 293 cells in the presence of
pTR-UF5 (leptin-insertions) or pTR-dsRFP (GFP) and pXX6 in a 1:1:8
molar ratio. 72 hrs post transfection the cells were harvested and
virus particles were purified as previously described. Western
blotting, A20 ELISA, and dot blot analysis were carried out on the
virus preparations (FIG. 6B-1, FIG. 6B-2, FIG. 6C-1 and FIG. 6C-2).
For the leptin-inserted virus preparations successful insertion in
both VP1 and VP2 or VP2 only was possible in the purified particle,
but GFP-insertion in the purified particle was only possible in the
VP1 protein (VP2-GFP was excluded in both cases).
[0245] Discussion
[0246] This example increases the flexibility in probing the
surface of the particle by isolating the expression of a given
capsid protein to a separate plasmid. Such an approach allows for
manipulation of this capsid protein only within the produced
particle and allows for retesting regions of capsid overlap for the
acceptance of sequence modification. Alternatively, the system also
allows for the modification of only two of the capsid proteins
while leaving the third protein unmodified. Using the missense
mutation of capsid start codons to generate all required plasmids,
characterization of the catalogue of plasmids required for this
system yielded interesting results concerning the role of each
capsid protein in the assembly of AAV-like particles.
[0247] Elimination of VP3-like fragments illustrates importance of
VP3 N terminus, as particles with these mutations in the VP1 and
VP2 proteins were recovered following complementation of
pIM45-VP1,2 with pIM45-VP3. Evidence that ability to modify
individual capsid proteins in regions of overlap may allow
production of particles that were defective for production when
mutations are in all three proteins. Increases the flexibility in
manipulation of the particle for targeting purposes. Recently,
isolation of the expression of a C-terminal modified VP3 separately
allowed for modification of the c-terminus of VP3 with his tag and
production of viable recombinant virus follow nickel
chromatography. Like the study involving the VP3-6.times.His tag
where the modified capsid protein was isolated and VP1 and VP2 did
not carry the insertion, lethal mutations in the overlapping
N-terminus region of VP3 (M203,211) resulted in particles from
complementation group 3 when these mutations were only in VP1 and
VP2 with normal VP3.
[0248] The present system allows for complementation and recovery
of rAAV2 particles with all capsid proteins present. Since it
allows for the genetic modification of only one or two of the
capsid proteins, it can also be used for studies of previously
reported lethal mutations in overlapping capsid sequences to see if
mutations at the same positions in fewer capsid proteins rescue the
position for particle manipulation. Important genetic modification
would include insertion of genetic sequence for retargeting the
virus, purification of the virus, monitoring of the virus particle
following infection, or presentation of immunogenic epitopes on the
surface of the virus particle.
[0249] Insertions of large peptides (leptin and GFP) into the
overlapping region of VP1 and VP2 resulted in the purification of
virus like particles carrying these insertions. This required
preliminary isolation of the expression of VP1 and VP2
(pIM45-VP1,2A) or VP2 only (pIM45-VP2A) to a separate plasmid
followed by insertion of peptide sequences after amino acid 138
allowed for the production of peptide inserted AAV-like particles
following complementation with pIM45-VP3 or pIM45-VP1,3. This
example is the first report of the purification of an AAV-like
particle containing a mutation in the VP2 protein exclusively.
Estimated similar stoichiometry of capsid proteins in particle.
Retain ability to package genomes, bind A20, and are infectious as
they retain native tropism due to intact heparin binding motif. VP2
overexpression may have ensured the inclusion of modified VP2
protein large insertions with VP2 acg start codon produced
significantly less modified VP2 proteins.
Example 2
Heparin Sulfate Binding Motif in AAV2 Capsid Proteins Required for
Native Tropism
[0250] In this example, charged-to-alanine substitution mutants
were made to analyze the effects of single and combinatorial
mutations in the capsid gene. New point mutants that result in
assembly, packaging, and receptor binding deficiencies have been
discovered. Importantly, five amino acids, arginines 484, 487, 585,
and 588, and one lysine at position 532 have been identified that
appear to mediate the natural affinity of AAV for HSPG. Those
observations contribute to the current map of the AAV capsid and
provide a reagent for the discovery of novel, heparin independent
targeting ligands.
[0251] Materials and Methods
[0252] Plasmids.
[0253] Plasmid pIM45 (previously called pIM29-45) contains the Rep
and Cap coding sequences from AAV with expression controlled by
their natural promoters (McCarty et al., 1991). It was used as the
parent template for construction of all the AAV2 mutant
vectors.
[0254] Plasmid pXX6 supplies the adenovirus helper gene products in
trans to allow rAAV production in an adenovirus free environment
(Xiao et al., 1998). Plasmid pTR2-UF5 supplies the recombinant AAV
DNA to be packaged. It contains a cytomegalovirus promoter driving
expression of a green fluorescent protein (GFP) reporter gene
flanked by AAV2 terminal repeats (Klein et al., 1998). Plasmid
pTR5-UF11 was constructed using an expression cassette consisting
of a strong constitutive CBA promoter (Xu et al., 2001), GFP
reporter gene (Zolotukhin et al., 1996), woodchuck hepatitis virus
posttranscriptional regulatory element WPRE (Donello et al., 1998)
and bovine growth hormone gene polyadenylation signal. The cassette
was assembled using standard molecular biology techniques and
substituted for the lacZ cassette in the plasmid backbone
pAAV5RnlacZ containing AAV5 terminal repeats (Chiorini et al.,
1999). Plasmids pXYZ1, pXYZ5 contain the AAV1 and AAV5 Cap coding
sequences, respectively, in addition to AAV2 Rep coding sequence
with an ACG start codon under control of the AAV2 p5 promoter
(Zolotukhin et al., 2002). Plasmid pAAV5-2 contains the AAV5
nucleotides 260 to 4448 without terminal repeats (Chiorini et al.,
1999).
[0255] Construction of Mutant Capsid Plasmids.
[0256] Quickchange site directed mutagenesis (Stratagene) was
performed on plasmid pIM45 as per the manufacturer's instructions.
For each AAV2 mutant, two complementary PCR primers that contained
alanine or lysine substitutions in addition to a silent change for
restriction endonuclease screening purposes were used to introduce
changes into pIM45. For construction of AAV5-HS, pAAV5-2 was used
as the parental template. Sequences for the oligonucleotides used
are available upon request. PCR products were digested with DpnI to
remove methylated template DNA, phenol:cholorform:isoamyl alcohol
(25:24:1) extracted, ethanol precipitated, and transformed into
electrocompetent JM109 cells. Miniprep DNA was extracted from
overnight LB/amp cultures and screened with the appropriate
restriction enzyme. All mutants were sequenced prior to use.
Transfection quality plasmid DNA was produced by standard alkaline
lysis method of a 1-liter TB culture followed by PEG precipitation
and cesium chloride gradient purification.
[0257] Cell Culture
[0258] Human embryonic kidney 293's and cervical carcinoma HeLa
C12's, a gift from Dr. Phil Johnson (Clark et al., 1996) were grown
in Dulbecco Modified Eagle Medium (Gibco-BRL) supplemented with 100
U/mL penicillin, 100 U/mL streptomycin, 10% bovine calf serum,
sodium pyruvate and L-glutamine. Cells were incubated at 37.degree.
C. in a 5% CO.sub.2 atmosphere.
[0259] Production of rAAV2 Particles
[0260] To produce AAV2 virions, low passage 293's were seeded so
that they were approximately 75% confluent at transfection time. A
triple plasmid transfection protocol (Xiao et al., 1998) was
followed that included pIM45 to supply Rep and mutated capsid
genes, pTR2-UF5 (Klein et al., 1998) to supply recombinant DNA with
AAV2 terminal repeats and a CMV driven GFP reporter gene, and pXX6
(Xiao et al., 1998) to supply the adenovirus helper functions in
trans. A total of 60 .mu.g of plasmid DNA in a 1:1:1 molar ratio
was transfected by lipofectamine (Invitrogen).
[0261] To produce pseudotyped rAAV1 and rAAV5 particles, a total of
60 .mu.g of pXYZ1 or pXYZ5 (Zolotukhin et al., 2002) was
co-transfected with pTR2-UF5 plasmid DNA in a 1:1 molar ratio as
above. To produce rAAV5 and rAAV5-HS virions, a total of 60 .mu.g
of pAAV5 or pAAV5-HS was co-transfected with pTR5-UF 11.
[0262] Purification of rAAV has been described previously
(Zolotukhin et al., 1999; Zolotukhin et al., 2002). Briefly, 72 hrs
after transfection, cells were harvested and the pellets were
resuspended in lysis buffer (0.15 M NaCl, 50 mM Tris-Cl pH=8.5).
Virus was released by three cycles of freezing and thawing.
Benzonase (Sigma) was added to the cell lysate to a final
concentration of 140 U/mL and incubated at 37.degree. C. for 30
min. Cell debris was pelleted by centrifugation at 3,700.times.g
for 30 min and the supernatant was loaded onto a 15%-25%-40%-60%
iodixanol
(5,5'[2-hydroxy-1,3-propanediyl)bis(acetyl-amino)]bis[N,N'-bis(2,3dihydro-
xypropyl-2,4,6-triiodo-1,3-benzenecarboxamide] step gradient
(Nycomed). The 40% fraction was collected after centrifugation at
69,000.times.g for 1 hr and stored at -80.degree. C. until further
use.
[0263] Virus Titer Determination
[0264] To determine the concentration of intact capsid particles,
the A20 ELIZA (American Research Bioproducts) was used. The A20
antibody detects intact, fully assembled particles, both full and
empty (Wistuba et al., 1995). Iodixinal purified stocks were
serially diluted and processed by the manufacturer's recommended
protocol. Only readings within the linear range of the kit standard
were used.
[0265] To determine the concentration of DNA-containing particles,
real-time (RT)-PCR.TM. was performed using a Perkin Elmer-Applied
Biosystems (Foster City, Calif.) Prism 7700 sequence detector
system. Equal volumes of iodixanol purified virus stocks were
treated with 600 U/mL benzonase in 50 mM Tris-CL pH=7.5, 10 mM
MgCl.sub.2, 10 mM CaCl.sub.2 at 37.degree. C. for 30 mM. 280 U/mL
Proteinase K was added to reactions adjusted to 10 mM EDTA and 5%
SDS, and then incubated at 37.degree. C. for 30 mM. Reactions were
extracted with phenol/chloroform/isoamyl-alcohol (25:24:1) and
undigested DNA was precipitated overnight with ethanol and glycogen
carrier. Precipitated DNA pellets were resuspended in 100 .mu.l of
water. Five .mu.l was used for RT-PCR.TM. analysis in a reaction
mixture that included 900 nM each of GFP forward
(5'-TTCAAAGATGACGGGAACTACAA-3') (SEQ ID NO:3) and reverse
(5'-TCAATGCCCTTCAGCTCGAT-3') (SEQ ID NO:4) primers, 250 nM
Taqman.RTM. probe (5'-6-FAM-CCCGCGCTGAAGTCAAGTTCGAAG-TAMRA-3') (SEQ
ID NO:5), 1.times. Taqman.RTM. universal PCR master mix in a total
volume of 50 pt. Cycling parameters were 1 cycle each of 50.degree.
C., 5 min, and 95.degree. C., 10 min, followed by 40 cycles of
95.degree. C., 15 sec and 60.degree. C., 1 min. Only values within
the linear portion of a standard curve having a coefficient of
linearity greater than 0.98 were accepted. The average RT-PCR.TM.
titer was calculated from virus preparations assayed three
times.
[0266] To determine the infectious titer of the wt and mutant virus
stocks, a green cell assay (GCA) was performed essentially as
previously described (Zolotukhin et al., 1999). Briefly, HeLa C12
cells were seeded in a 96-well plate so that they were
approximately 75% confluent at infection time. Cells were infected
with 10-fold serial dilutions of iodixanol purified mutant viruses
and Ad5 at a constant multiplicity of infection (MOI)=10. Cells
were incubated at 37.degree. C. in a 5% CO.sub.2 atmosphere for 24
hrs and examined by fluorescence microscopy. The average GCA titer
was calculated by averaging the number of green cells counted in
individual wells from two or three virus preparations assayed three
times. Particle to infectivity ratios were calculated by dividing
the average RT-PCR.TM. titer by the average GCA titer. In some
figures, this number was expressed as a log.sub.10 value with rAAV2
arbitrarily set to one.
[0267] In Vitro Heparin Binding Assay
[0268] Bio-Rad microspin columns were treated with silicon dioxide
to minimize non-specific binding of the virus to the column wall. A
500 .mu.L heparin-agarose (Sigma H-6508) gravity column was
prepared by washing with 3 column volumes each of 1.times.TD (137
nM NaCl, 15 mM KCl, 10 mM Na.sub.2PO.sub.4, 5 mM MgCl.sub.2, 2 mM
KH.sub.2PO.sub.4, pH=7.4), 1.times.TD+2 M NaCl and 1.times.TD.
Approximately equal numbers of virus particles were added to
1.times.TD to a final volume of 600 .mu.L and loaded onto the
column. The column was washed with 7 column volumes of 1.times.TD.
Bound virus was eluted with 1.times.TD+2 M NaCl. The entire volume
of the flow through, wash, and eluate fractions were pooled
separately, denatured by boiling in SDS, and slot blotted onto
nitrocellulose for immunoblot analysis. The membrane (Osmonics) was
blocked in PBS/0.05% Tween-20+5% dry milk, and incubated with B1
antibody (Wistuba et al., 1997) at a 1:3000 dilution for 18 hrs at
4.degree. C. Anti-mouse IgG-horse radish peroxidase was used to
detect bands by enhanced chemiluminesence (Amersham-Pharmacia).
[0269] Fluorescence Activated Cell Sorting (FACS)
[0270] HeLa C12 cells were seeded in 6 well plates so that they
were approximately 75% confluent at infection time. Cells were
infected with an rAAV MOI=500 based on the genomic titer as
determined by DNA dot blot assay (Zolotukhin et al., 1999).
Adenovirus type-5 was used at an MOI=10 plaque forming units (pfu).
Twenty-four hrs postinfection, cells were washed, trypsinized, and
fixed in 2% paraformaldyhede. FACS analysis for GFP expression was
done in the ICBR Flow Cytometry lab of the University of Florida on
a Becton-Dickinson FACScan.
[0271] Cell Attachment Assay
[0272] 10.sup.6 Hela C12 cells were infected with rAAV2 at a genome
containing particle MOI=100 or R585A/R588A at an MOI=1000, as
determined by RT-PCR.TM.. Cells were incubated at 37.degree. C. in
a 5% CO.sub.2 atmosphere until harvesting. At indicated time
points, the infection media was removed and saved and the cells
were washed four times with PBS before being scraped.
Low-molecular-weight DNA from the infection media and the cell
pellet was extracted (Hirt, 1967). DNA pellets were resuspended in
0.2 M NaOH, incubated at 37.degree. C. for 20 min, and slot blotted
onto nitrocellulose. DNA was UV cross-linked to the nitrocellulose
and probed at 65.degree. C. for 18 hrs with [.alpha.-.sup.32P]-dATP
labeled GFP probe in hybridization buffer (7% SDS, 10 mM EDTA and
0.5 M Na.sub.2HPO.sub.4). Membranes were washed twice in
2.times.SSC/0.1% SDS, 0.2.times.SSC/0.1% SDS, 0.1.times.SSC/0.1%
SDS, and rinsed with water. The membranes were then exposed to film
and quantitated using a BAS-1000 phosphor imager (Fuji).
[0273] Results
[0274] Selection and Generation of AAV Mutants
[0275] A considerable body of information regarding the
determinants of HS-protein interactions suggests that their
association is driven mainly by electrostatic attraction between
acidic sulfate groups on the polysaccharide and basic R-groups on
amino acids in the target protein (Hermens et al., 1999; Hileman et
al., 1998). It was hypothesized that similar electrostatic
interactions would govern HSPG-AAV2 association. In order to
evaluate the role of particular amino acids in receptor binding, a
panel of mutants was generated by site directed mutagenesis of
selected residues. The selection was confined primarily to basic
amino acids (His, Lys, Arg) in VP3 as AAV-like particles composed
only of VP3 proteins have been purified by heparin affinity
chromatography. Any basic amino acid substitution mutant that
previously had demonstrated capsid instability or efficient
purification by heparin affinity chromatography (Wu et al., 2000)
was excluded from the pool of mutants.
[0276] Seven AAV serotypes have been reported (Bantel Schaal and
zur Hausen, 1984; Gao et al., 2002; Hoggan et al., 1996; Parks et
al., 1967; Rutledge et al., 1998). Several groups have shown that
rAAV2 and rAAV3 bind efficiently to heparin sulfate (Rabinowitz et
al., 2002; Shi et al., 2001; Wu et al., 2000). A single report
concerning rAAV1 suggests that it binds with low affinity, if at
all, to heparin (Rabinowitz et al., 2002). In contrast, rAAV4 and
rAAV5 do not bind heparin and instead recognize 2,3 O-linked and
2,6 N-linked sialic acid moieties (Kaludov et al., 2001). Indeed,
this may account for their different cellular tropisms. It was
reasoned that residues conserved among all five serotypes were
probably not participating directly in receptor discrimination and
binding and were excluded from further consideration. Additionally,
a number of charge-to-alanine substitution mutants in the AAV
capsid had been identified, and these had been characterized for
their ability to bind heparin sulfate columns (Wu et al., 2000) and
amino acid positions that did not affect heparin binding or had
been shown to be assembly mutants were excluded from further study.
Using a Clustal W algorithm, a sequence alignment of capsid
proteins from serotypes 1-5 was generated, and 9 basic residues in
AAV2 that were conserved in AAV3 and/or AAV1 but were uncharged or
acidic in AAV4 and AAV5 were identified that had not previously
been tested for heparin-agarose binding (Table 4). In addition to
these 9 amino acids, Wu et al. (2000) described a virus deficient
for heparin binding with alanine substitution mutations at
positions 585, 587, and 588. Finally, during the course of these
studies, the atomic structure of AAV2 was solved (Xie et al., 2002)
and suggested that residues 484, 513, and 532 might participate in
a heparin-binding pocket as they were located close to residues
585, 587, and 588. These six extra residues were also included to
complete the mutant panel (Table 4).
TABLE-US-00004 TABLE 4 RESIDUES CHOSEN FOR MUTAGENESIS AAV
Serotype.sup.b VP Residue.sup.a 2 3 1 4 5 358 H H H Q T 447 R R R S
S 459 R R D T G 484 R R R K R 487 R R R G G 509 H H H T E 513 R R R
R A 526 H H H A N 527 K K K G N 532 K K K K N 544 K K K P S 566 R R
K A Q 585 R S S S S 587 N N S S T 588 R T T N T .sup.aResidues
selected for mutagenesis were generated by a sequence alignment of
the VP1 capsid protein from each serotype using the Clustal W
algorithm (Vector NTi 5.2, Informax). .sup.bAmino acids are
represented by their one letter abbreviation. Blue letters
represent positively charged, basic amino acids. Red letters
represent any other amino acid.
[0277] Mutant Virus Production and Physical Characterization
[0278] A series of single and combinatorial capsid mutants were
generated from the pool of candidate residues in the AAV2 capsid
gene (Table 4). To designate the mutant viruses, the number of the
mutated amino acid based on its position in VP1 was used. Iodixanol
purified virus stocks were checked by western blot using the
monoclonal antibody B1. The B1 antibody recognizes a linear epitope
in the extreme carboxyl terminus of all three VP proteins from AAV
serotypes 1, 2, 3 and 5 (Rabinowitz et al., 2002; Wobus et al.,
2000). With the exception of H358A, capsid proteins were detected
in all virus stocks (FIG. 7). To confirm that assembled capsids,
rather than subunits or assembly intermediates, had been purified,
the particle concentration was measured with an A20 antibody ELISA
(Table 5). The A20 antibody recognizes a structural epitope that is
found only on assembled capsids with or without packaged DNA (Grimm
et al., 1998). Although there was some variability between stocks
due to different transfection efficiencies and purification
recoveries, only the H358A mutant was negative by A20 ELISA assay.
Excluding H358A, a particle concentration range was determined that
spanned 1.5 logs and correlated reasonably well with the B1
antibody results (FIG. 7; Table 5). Several possibilities may
account for this range of particle titers, including that capsid
subunits containing these mutations (i) form intact particles
inefficiently, (ii) are unstable during purification or (iii)
formed a particle with a partially disrupted A20 epitope. Since
none of these mutations fell within the antigenic regions that have
been mapped for A20 (Wobus et al., 2000), these results suggested
that the A20 epitope had probably not been modified but rather the
stability or assembly of some of the mutants was altered so that
fewer particles were recovered after iodixanol centrifugation (FIG.
7; Table 5).
TABLE-US-00005 TABLE 5 TITERS AND HEPARIN BINDING PROPERTIES OF
MUTANTS Infectious Particle Titer.sup.b Titer.sup.c Particle-to
Heparin Empty/ Mutant Virus.sup.a A20/mL Genome/mL (IU/mL)
infectivity Binding.sup.e Full.sup.g rAAV2 (WT) 1.5 .times.
10.sup.12 4.6 .times. 10.sup.11 .sup. 1.8 .times. 10.sup.10 25 +
3.4 H3558A <1.0 .times. 10.sup.8 <1.0 .times. 10.sup.6
<1.0 .times. 10.sup.4 N/D.sup.f N/D N/D R447A 1.2 .times.
10.sup.12 3.4 .times. 10.sup.10 1.3 .times. 10.sup.9 25 + 35.9
R459A 9.1 .times. 10.sup.10 7.2 .times. 10.sup.8 <1.0 .times.
10.sup.4 >72500 + 126.3 R484A 1.5 .times. 10.sup.11 3.0 .times.
10.sup.10 <1.0 .times. 10.sup.4 >2976667 +/- 5.1 R487A 5.4
.times. 10.sup.11 2.2 .times. 10.sup.11 2.3 .times. 10.sup.8 954
+/- 2.5 H509A 4.6 .times. 10.sup.10 2.3 .times. 10.sup.9 6.9
.times. 10.sup.5 3285 + 20.3 R513A 2.9 .times. 10.sup.11 1.7
.times. 10.sup.10 1.6 .times. 10.sup.8 106 + 17.9 K532A 1.1 .times.
10.sup.11 3.6 .times. 10.sup.10 <1.0 .times. 10.sup.4
>3633333 +/- 3.0 K544A 2.0 .times. 10.sup.11 1.7 .times.
10.sup.10 8.3 .times. 10.sup.8 20 + 11.9 R566A 5.1 .times.
10.sup.11 1.6 .times. 10.sup.10 7.4 .times. 10.sup.8 21 + 32.6
R585A 5.0 .times. 10.sup.11 4.8 .times. 10.sup.10 1.7 .times.
10.sup.7 2812 - 1.4 R587A 4.4 .times. 10.sup.11 1.3 .times.
10.sup.10 1.7 .times. 10.sup.7 165 + 34.7 R588A 2.4 .times.
10.sup.11 5.6 .times. 10.sup.10 3.0 .times. 10.sup.6 18521 - 4.2
H526A, K527A 1.4 .times. 10.sup.11 8.2 .times. 10.sup.10 5.5
.times. 10.sup.7 1489 + 1.8 R585A, R588A 1.2 .times. 10.sup.12 9.2
.times. 10.sup.11 1.9 .times. 10.sup.7 48421 - 1.2 R585K 1.3
.times. 10.sup.12 3.7 .times. 10.sup.10 4.0 .times. 10.sup.8 92 +
35.4 R585K, R588K 1.4 .times. 10.sup.12 3.9 .times. 10.sup.10 8.9
.times. 10.sup.7 436 + 34.9 AAV1 N/D 3.7 .times. 10.sup.10 1.1
.times. 10.sup.9 37 +/- N/D AAV5 N/D 3.4 .times. 10.sup.10 3.2
.times. 10.sup.6 10692 - N/D AAV5-HS N/D 8.0 .times. 10.sup.8
<1.0 .times. 10.sup.4 >80000 + N/D .sup.aTwo letters flanking
a number designate each mutant. The first letter is the one letter
abbreviation for the wild type amino acid followed by its numerical
position in VP1 followed by the one letter abbreviation for the
amino acid to which it was mutated. .sup.bA20 particle titers were
determined as described using the A20 ELISA assay. Genomic titers
were determined by RT-PCR .TM.. .sup.cInfectious titers were
determined by green cell assay as described by counting GFP
fluorescent cells. .sup.dParticle-to-infectivity ratio was
calculated by dividing the average genomic titer as determined by
RT-PCR .TM. by the average green cell assay titer. .sup.eDetermined
by heparin-agarose binding assay. +, >95% virus recovered in the
eluate; +/-, >50 recovered in the eluate; -, <5% of virus
recovered in the eluate. .sup.fN/D, not determined.
.sup.gEmpty-to-full ratio was determined by dividing the A20
particle titer by the average genomic titer.
[0279] To determine whether any mutations affected DNA packaging,
the titer of DNA containing virions was determined by real-time
(RT) PCR.TM. (Clark et al., 1999; Veldwijk et al., 2002) (Table 5)
and confirmed by DNA dot blot hybridization. Although there was
variation between preparations, the majority of the capsid mutants
were able to package detectable DNA (Table 5). As expected, H358A
was negative for DNA packaging, as it did not produce virus
particles. It was concluded that none of the capsids in the mutant
panel that made A20 positive particles were completely defective
for DNA packaging. However, by comparing the A20 ELISA and PCR
titers, it was noted that stocks of mutant R459A contained 40-fold
more empty particles than wild type rAAV2. Thus, R459 could have a
role in DNA packaging. Although less dramatic, mutants R447A,
R566A, R587A, R585K, and R585K/R588K had approximately 10-fold more
empty particles than rAAV2. The remainder of the virus preparations
packaged DNA at levels comparable to wild type AAV2 (Table 5).
[0280] In Vitro Heparin Binding of Capsid Mutants
[0281] To assess the ability of mutant capsids to bind heparin
sulfate, a modification of an assay previously described by Wu et
al. (2002) was used. Virus preparations that had been purified by
iodixanol step gradients were loaded on heparin agarose columns and
the entire volume of the flow through, wash, and eluate fractions
were pooled separately, denatured, and slot blotted onto
nitrocellulose for immunoblot analysis with B1 antibody. A
representative Western analysis for each mutant is shown in FIG. 8.
As expected, wild type AAV2 was not observed in the flow through or
wash fractions and most of the virus bound to the column was
recovered at the elution step. Eight other mutants, R447A, R459A,
H5009A, R513A, K544A, K566A, N587A, and H526A/K527A, had a
heparin-agarose binding phenotype indistinguishable from wild type.
The results with R513A confirmed a previous report (Wu et al.,
2000) in which a double mutant at positions 513 and 514 was
positive for heparin binding. In marked contrast, it was observed
that any capsid harboring a non-conservative mutation at position
585 or 588 was detected only in the flow-through and in the wash.
Intermediate heparin-agarose binding phenotypes in mutants R484A,
R487A and K532A were also detected with approximately equal levels
of signal detected in the flow through, wash, and eluate. The
results with K532A were inconsistent with previous results in which
a mutant containing alanine substitutions at positions 527 to 532
was found to be positive for heparin binding (Wu et al., 2000).
These data suggested that at least five amino acids had the
potential to contribute to the electrostatic attraction between AAV
and heparin sulfate. These included predominantly R585 and R588,
and to a lesser but detectable extent, R484, R487, and K532.
[0282] To confirm that the charge at R585 and R588 was primarily
responsible for heparin interaction, two viruses were generated
with conservative mutations, R585K and R585K/R588K, and tested them
in the in vitro heparin binding assay. Both lysine and arginine
residues are positively charged, however, lysine is slightly larger
due to an additional methyl residue in the side group. Both of
these capsids bound to heparin-agarose almost as well as wild type
virus (FIG. 8). In each case, most of the virus was recovered in
the eluate; however, the flow through and wash fractions also
contained minor amounts of virus. This result suggested that both
localized negative surface charge and the relative position of the
changes in this region of the capsid were responsible for mediating
the interaction with heparin-agarose.
[0283] Finally, as a control and to validate the heparin binding
assay, the ability of wild type rAAV2, rAAV1, and rAAV5 to bind to
heparin-agarose was compared. For this purpose, recombinant viruses
were produced and purified by using a pseudotyping protocol
developed to package AAV2 terminal repeat containing genomes into
alternative serotype capsids (FIG. 9A) (Rabinowitz et al., 2002;
Zolotukhin et al., 2002). Approximately equal amounts of input
virus as determined by Western blot signal intensity were applied
to a heparin-agarose column, and fractions from the column were
slot blotted onto nitrocellulose for immunodetection using the B1
antibody (FIG. 9B). As expected, rAAV2 was efficiently retained by
heparin-agarose under low ionic conditions but the majority of
rAAV1 and all of rAAV5 was seen in the flow through and wash. A low
amount of AAV1 was detected in the eluate. These data were
consistent with previous reports (Rabinowitz et al., 2002).
[0284] Multiple Mutations in the Aav2 Capsid Effect Viral
Transduction
[0285] To determine how the heparin-agarose binding phenotypes
correlated to infectivity, iodixanol stocks were tested for their
ability to transduce HeLa C12 cells by performing a green cell
assay (GCA). Cells in a 96-well plate were co-infected with Ad5 at
a constant MOI=10 pfu/cell and mutant AAV virus stocks in a 10-fold
dilution series. Twenty-four hrs post-infection (hpi), the number
of GFP expressing cells in individual wells were counted and a GCA
titer was calculated (Table 5). The detection limit of this assay
was approximately 10.sup.4 transducing units/mL. The GCA titers
were then normalized to genome containing physical particles by
calculating a particle-to-infectivity (P/I) ratio. This ratio is
equivalent to the number of genomes required to transduce one cell
(Table 5). To get a measure of the relative impact of a particular
mutation on viral infectivity, the P/I ratio of each mutant was
divided by the wild type capsid P/I ratio and the log.sub.in of
this value was plotted in FIG. 10. This provided a simple
comparison of how many genome-containing particles of each mutant
were required to achieve the same number of transduced cells as the
wild type virus.
[0286] Several phenotypes emerged from this analysis. Mutants
R477A, K544A, and K566A were virtually identical to wild type, and
mutants R513A, N587A, R585K, and R585K/R588K were only slightly
defective (approximately 1 log). These seven mutants were found
previously to bind heparin sulfate to the same extent as wild type
rAAV2 (FIG. 8).
[0287] Three of the mutants R459A, R484A, and K532A produced virus
that was essentially non-infectious with P/I ratio between
7.2.times.10.sup.4 and 3.6.times.10.sup.6 compared to the wild type
ratio of 25 (Table 5, FIG. 10). The P/I ratios for these mutants
were minimum estimates based on the GCA assay sensitivity of
1.times.10.sup.4 IU/mL. In fact, no transduction events were seen
with any of these mutants. R459A was the most severe example of
three mutants (R459A, H509A, and H526A/K527A) that were essentially
wild type for heparin binding but defective for transduction (FIG.
10). These mutants were presumably defective in some late stage of
viral infection.
[0288] Finally, all five of the mutants that were defective or
partially defective for heparin binding (R484A, R487A, K532A,
R585A, and R588A) were defective for transduction. However, the
loss of infectivity did not correlate completely with the loss of
heparin binding (compare FIG. 8 and FIG. 10). Two of these mutants
(R484A and K532A) were only partially defective for heparin binding
but severely defective (>5 logs) for transduction, suggesting
that some other step in viral infection was defective in these
mutants in addition to heparin binding. The remaining heparin
binding mutants (R487A, R585A, and R588A) had defects in
transduction that approximately correlated with their ability to
bind heparin.
[0289] Evaluation of R585A/R588A Cell Attachment in Vivo
[0290] As mentioned earlier, alanine substitutions at either
position 585 or 588 were the only mutations that completely
abolished binding to HS (FIG. 8), suggesting that these two
arginines were primarily responsible for heparin binding. Moreover,
the extent to which mutation of either or both of these residues
inhibited transduction (FIG. 10, 1.5-3 logs) was approximately the
same when soluble heparin sulfate is used to inhibit wild type
rAAV2 infection (Handa et al., 2000). Those mutants were,
therefore, examined in more detail.
[0291] To see if the defect in transduction of R585 and R588
mutants could be overcome by using higher input MOI's, cells were
co-infected with rAAV2 or the mutant viruses at an MOI=500 genome
containing particles/cell. Twenty-four hrs post-infection cells
were examined by fluorescence microscopy and counted by FACS. The
data from three independent experiments and representative
histograms are shown in FIG. 11. As expected, the defects in
transduction of the single mutants, R585A and R588A, could be
overcome by higher MOI's (56% and 25% transduction for R585A, and
R588A, respectively). Predictably, the level of recovery of the
double mutant, R585A/R588A, was lower (10% transduction). However,
it was clear that the fluorescence intensity profile for the
heparin binding mutants was quite different from wild type,
suggesting a significant delay in the onset of GFP expression by 24
hrs. In contrast, the level of transduction of the conservative
double mutant, R585K/R588K, and the heparin positive mutant, N587A,
was indistinguishable from wild type.
[0292] As a more direct assay for cell attachment, Hela C12 cells
were transfected and the location of viral DNA tracked. Cells were
infected with rAAV2 at an MOI=100 or R585A/R588A at an MOI=1000
genome containing particles as determined by RT-PCRTM. At 1, 4, and
20 hpi, the infection media was removed and saved, and the cells
were washed extensively to remove any residual unbound virus. The
cells were then harvested and low molecular weight DNA was
extracted from both the infection media (unbound) and the cell
pellet (bound or internalized) by the Hirt procedure and
transferred to nitrocellulose for Southern hybridization with an
[.alpha.-.sup.32P]-dATP labeled GFP probe (FIG. 12A and FIG.
12B).
[0293] At all time points rAAV2 DNA was detectable both
bound/internalized and in the infection media. In contrast, cells
infected with 10-fold more genomic copies of R585A/R588A showed the
vast majority of the signal only in the unbound fraction (FIG.
12A). Phosphor imager analysis determined that at each time point
approximately one third of the total rAAV2 DNA was attached or
internalized compared to only 1% of R585A/R588A (FIG. 12B). As
these infections were performed at 37.degree. C., the process of
internalization should not have been prevented. This result
demonstrated that the block in infection for mutant R585A/R588A
occurred at the cell attachment stage or internalization stage, and
correlated to heparin sulfate binding in vitro.
[0294] Loop Swapping Confers Heparin Binding to AAV5
[0295] Although the primary amino acid sequences are moderately
divergent, the architectural position of .beta.-sheets and loops is
predicted to be very similar among AAV serotypes (Rabinowitz and
Samulski, 2000). It was hypothesized that if R585 and R588 were the
critical residues involved in HSPG binding, then it should be
possible to substitute that region of AAV2 into AAV5 to create a
hybrid virus capable of interacting with heparin-agarose. To
achieve this, a recombinant virus, designated rAAV5-HS, was
generated by replacing a short loop containing residues 585 through
590 from AAV2 into a region predicted to be structurally equivalent
in AAV5 (FIG. 13A). Loop substitution rather than point mutagenesis
was done to account for the possibility of additional Van der Waals
interactions or hydrophobic contributions from nearby amino
acids.
[0296] Production and purification of rAAV5-HS was unaffected by
the six amino acid substitution (FIG. 13B; Table 5). When rAAV5-HS
was tested in the in vitro heparin-agarose binding assay, it was
indistinguishable from wild type rAAV2 (FIG. 13C). These data
suggested that this region of AAV5 was surface accessible, and that
heparin-agarose binding could be artificially conferred by the six
amino acids containing R585 and R588.
[0297] To compare the infectivity of rAAV5 and rAAV5-HS, packaged
viruses were generated that contained a recombinant AAV5 genome in
which the GFP reporter gene was flanked by AAV5 terminal repeats.
The infectivity of these viruses was compared to rAAV2 in a GCA
assay and particle-to-infectivity ratios were calculated as before
(FIG. 13D). rAAV5 was able to transduce Hela C12 cells at a low
efficiency, approximately 2.5 logs lower than AAV2. However, no
transduction was seen with AAV5-HS (<1.times.10.sup.4 IU/mL)
(Table 5; FIG. 13D). Given the minimum sensitivity of the GCA assay
this meant that the P/I ratio of AAV5-HS was at least 3.5 logs
higher than rAAV2 and at least 1 log higher than wild type rAAV5.
It was concluded that, although substitution of these five
heterologous amino acids into the AAV5 capsid restored heparin
binding to the level of AAV2 capsids, it was not sufficient to
produce AAV2 levels of infectivity in a cell line normally
permissive for AAV2.
[0298] Discussion
[0299] This example describes the identification of amino acids in
the capsid of AAV2 that mediate binding to heparin sulfate
proteoglycans. Several lines of evidence suggest that HSPG serves
as the primary receptor for AAV2 Inhibition of AAV2 infection can
be demonstrated by competition with soluble analogs, GAG
desulfation by sodium chlorate treatment, antibody competition,
enzymatic removal of heparin, and use of mutant cell lines that
express varying levels of HSPG (Handa et al., 2000; Qiu et al.,
2000; Summerford and Samulski, 1998; Wu et al., 2000). Binding to
heparin sulfate is usually the result of electrostatic charge
interactions between basic amino acids (R, K, or H) and negatively
charged sulfate residues (Hileman et al., 1998; Mulloy and
Linhardt, 2001). During the course of previous mutagenesis studies,
many of the basic amino acids in the AAV2 capsid that could
potentially contribute to heparin sulfate binding were eliminated
(Wu et al., 2000). In this example, the remaining basic residues
were examined by looking at their conservation in AAV serotypes 1
to 5. Those that were present in all five serotypes were not likely
to contribute significantly to heparin binding. Those that were
conserved in the heparin binding serotypes, AAV1 to 3, but not in
the remaining serotypes were targeted for mutagenesis. Finally, by
taking advantage of the fact that R585 and R588 had been previously
identified as potential heparin binding amino acids (Wu et al.,
2000) and that these amino acids were located in a cluster of basic
residues at the three fold axis of symmetry (Xie et al., 2002), all
of the basic amino acids in this cluster were also targeted for
mutagenesis. This approach yielded a total of 15 amino acids that
could have been involved in heparin binding and alanine mutations
were characterized at all of these positions. This approach, of
course, does not necessarily identify all possible heparin binding
amino acids. For example, R484, which is basic in all five
serotypes was tested because of its proximity to R585 and R588 and
subsequently proved to be involved in heparin binding.
[0300] Heparin Binding and Infectivity
[0301] These studies indicated that capsids with mutations at
residue 484, 487, 532, 585 or 588, were partially or completely
defective for heparin-agarose binding. The most severe defect was
found with mutations in R585 and R588. No binding to heparin
sulfate columns could be detected with either mutant (FIG. 8), and
both mutations reduced the particle-to-infectivity ratio by two to
three logs (Table 5). Mutants that contained substitutions at both
positions had even lower infectivity.
[0302] The phenotypes of R487A, R585A, and R588A, were probably due
largely to defective heparin binding. For example, the double
mutant R585A/R588A was approximately 500 fold more defective in
cell binding and internalization than wild type (FIG. 12B) when
corrected for the MOI, and approximately 2000 fold less infectious
(Table 5), as judged by the change in particle-to-infectivity
ratio. Another indication that heparin binding was primarily
responsible for the defects in R585 and R588 was the fact that
conservative mutations at these two positions (R585K and
R585K/R588K) produced virus particles with properties similar to
wild type (FIG. 8; FIG. 10; FIG. 11; Table 5). Results from the
conservative lysine substitutions at R585 and R588 are reasonably
consistent with electrostatic attraction being the primary mediator
for AAV-heparin interaction. R585K, the least defective heparin
binding mutant (FIG. 8), had transduction levels nearly equal to
rAAV2 (FIGS. 10), and R585K/R588K was only slightly more defective
for heparin binding (FIG. 8) and transduction (FIG. 10), and within
one log of wild type. Furthermore, when cells were infected at a
high MOI, robust transduction was observed for both mutants (FIG.
11). Finally, substitution of a six amino acid sequence containing
R585 and R588 imparted heparin binding to AAV5 that was comparable
to that seen with AAV2 (FIG. 13). Although similar studies were not
performed with the R487 position, it was clear that mutation of
R487 produced virus with a more modest defect in heparin binding
(FIG. 8) and in infectivity (FIG. 10).
[0303] In addition to R487A, R585, and R588, two other mutants were
found that were defective for heparin binding, R484A and K532A.
R484A and K532A, like R487A, had more modest effects on binding to
heparin sulfate, but unlike the other heparin binding mutants,
these two mutations had a dramatic effect on transduction
efficiency. Both R484A and R532A were more than 5 logs less
infectious than wild type capsids (Table 5; FIG. 10). This severe
defect is presumably due to a different block in the infection
process that is unrelated to heparin binding, but as yet it has not
been identified. The result from K532A is consistent with earlier
studies that identified a mutant (mut37) that contained six amino
acid substitutions that included K532A (Wu et al., 2000). Mut37 had
a phenotype identical to K532A in that it produced full virus
particles that were non-infectious and more recently has been shown
to have a modest defect (approximately 5-fold) in heparin binding
and internalization. This potentially maps this defect to a single
amino acid.
[0304] Computer Modeling
[0305] Using the recently published atomic structure of AAV2 (PDB
ID code: ILP3) (Xie et al., 2002), the positions of the heparin
binding mutations were examined. Symmetry transformation operations
from the original PDB file were applied to generate a VP3 trimer
arrangement in the context of an icosahedron. When viewed in ribbon
format looking directly down a three-fold axis, residues R484,
R487, R532, R585 and R588, represented as balls-and-sticks, were
located in a linear formation lining one side of each three-fold
related spike. When viewed across the top surface of the trimer,
residues R585 and R588, which are contributed by one of the
peptides in the trimer, were positioned above a linear arrangement
of R484, R487 and K532, which are contributed by a second peptide
in the trimer. Thus, it appears that a heparin binding motif is
formed from some combination of these five amino acids using amino
acids from two different polypeptides. An electrostatic potential
surface map of a VP3 trimer was also generated, in which areas of
positive and negative charge are represented. When viewed
perpendicular to the three-fold axis, the five amino acids mapped
by this example appear to contribute collectively to a basic patch
on one side of each three-fold-related spike. The charge,
clustering, and surface presentation of these residues were all
consistent with a model of electrostatic attraction. Two other
basic residues, H526 and K527, contribute to the basic cluster at
the three fold spike but these residues do not appear to be
involved in heparin binding (FIG. 8).
[0306] The five mutations that affected heparin binding were
located in the large loop IV region, which among AAV serotypes has
low overall sequence conservation and includes all of the
previously identified insertion and substitution mutations that
affect heparin binding. Interestingly, with the exception of N587,
the stretch of amino acids encompassing 585 to 590 is unique to
AAV2 and is not present in AAV3, which is the other AAV serotype
that has been shown to bind efficiently to heparin sulfate.
Mutation of N587 had no effect on heparin-agarose binding and only
minor effects on transduction. Conceivably, residues R484, R487,
and K532 could be the dominant residues involved in heparin sulfate
binding for AAV3.
[0307] The apparent dissociation constant (K.sub.d) of AAV2 and
heparin sulfate was determined by competition analysis to be
2.times.10.sup.9 M (Qiu et al., 2000). Although this is higher than
some heparin-protein interactions, it is sufficiently strong to
suggest cooperative binding by one HS glycosaminoglycan chain to
multiple attachment points. This example does not address whether
heparin sulfate could form a bridge between basic residues in one
of the threefold spikes to those in another. However, as the
average chain length of heparin glycosaminoglycans varies between
50 and 200 disaccharide repeats that adopt a helical conformation
40-160 nm in length, it is conceivable that a heparin sulfate chain
could wrap around the exterior of the capsid through cooperative
binding of multiple spikes at the threefold axis of symmetry.
Although a rigorous computational docking analysis was not
undertaken, a heparin molecule (PDB ID code 1NTP) was manually
superimposed in several orientations that placed multiple reactive
sulfate and amine groups within accepted electrostatic attraction
distances on pairs of residues spanning the spikes.
[0308] Mutants that Bind Heparin but are Still Defective
[0309] Several new mutants were found that bound heparin sulfate as
well as wild type but still produced defective particles. H538A was
defective for particle assembly. There are a number of reported
examples of mutations that disrupt AAV2 particle formation, several
of which are located in the conserved .beta.-strand regions
(Rabinowitz et al., 1999; Shi et al., 2001; Wu et al., 2000). Since
H358 is neither surface accessible nor in a conserved
.beta.-strand, it is possible that it acts internally to stabilize
the monomer subunit structure.
[0310] Mutants R459A, H509A, and H526A/K527 bound heparin-agarose
efficiently but had particle-to-infectivity ratios that were two to
more than three logs higher than wild type. Like K532A and R484A,
these mutants are presumably defective in some stage of the
infectious entry pathway between secondary receptor binding and
uncoating. Ongoing studies in the lab are examining the block in
infectivity for these mutants.
[0311] DNA Packaging
[0312] The process of DNA packaging is thought to occur by an
active process requiring NTP consumption coupled to the helicase
activity of the small Rep proteins (King et al., 2001). Although
none of the mutations that assembled an A20-positive particle were
completely deficient for DNA packaging, mutant R459A produced a
40-fold excess of empty capsid particles compared to rAAV2. Other
studies have reported that short insertions at positions 323, 339,
466, 520, 540, 595, and 597 that did not interfere with capsid
formation still reduced DNA packaging to levels detectable only by
PCR.TM. amplification (Shi et al., 2001). In addition, a point
mutant, R432A, prevented DNA packaging (Wu et al., 2000). Although
the relationship between these mutations and their mechanism of
action is unclear, it is possible that they disrupt protein-capsid
or DNA-capsid interactions.
Summary of Exemplary Production System
[0313] An exemplary rAAV production system has been described to
produce modified rAAV vectors that comprise one or more altered
capsid proteins. FIG. 14 shows the results of an immunoslotblot of
total capsid protein following standard purification procedures of
a representative expression system of the invention. FIG. 15 shows
a dot blot autoradiograph of DNA extracted from pTR-UF5 and the
system plasmid combinations. FIG. 16A, FIG. 16B, FIG. 16C, and FIG.
16D show the in vivo transduction ability of recombinant AAV
vectors produced using various system components. FIG. 17A and FIG.
17B show an Immunoblot and dot blot autoradiograph of virions
produced from pTR-UF5; pIM45-VP1,2; pIM45-VP1,3; and pIM45-VP2,3
plasmids following standard purification protocols. FIG. 18A, FIG.
18B, FIG. 18C, and FIG. 18D show the in vivo transduction ability
of recombinant AAV vectors containing only two capsid proteins,
while FIG. 19 depicts an immunoblot of protein fractions collected
from iodixinol purified passed over a heparin-agarose column. Using
an anti-VP1,2,3 monoclonal antibody, FIG. 20 shows a dot blot
autoradiograph of DNA extracted from pTR-UF5 and rAAV R585A, R588A,
while FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D summarize an
exemplary system that demonstrates the in vivo transduction ability
of pTR-UF5 and R585A, R588A.
[0314] FIG. 22 shows a slot blot autoradiograph of an in vivo DNA
tracking time course experiment of pTR-UF5, rAAV R585A, R588A,
while FIG. 23 shows a schematic diagram of the pIM45 vector showing
the rep and cap sequences.
REFERENCES
[0315] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein in their
entirety by express reference thereto: [0316] U.S. Pat. No.
4,216,209. [0317] U.S. Pat. No. 4,237,244. [0318] U.S. Pat. No.
4,554,101. [0319] U.S. Pat. No. 4,683,195. [0320] U.S. Pat. No.
4,683,195. [0321] U.S. Pat. No. 4,683,202. [0322] U.S. Pat. No.
4,683,202. [0323] U.S. Pat. No. 4,800,159. [0324] U.S. Pat. No.
4,800,159. [0325] U.S. Pat. No. 4,883,750. [0326] U.S. Pat. No.
4,883,750. [0327] U.S. Pat. No. 4,987,071. [0328] U.S. Pat. No.
4,987,071. [0329] U.S. Pat. No. 5,037,746. [0330] U.S. Pat. No.
5,093,246. [0331] U.S. Pat. No. 5,098,887. [0332] U.S. Pat. No.
5,116,742. [0333] U.S. Pat. No. 5,145,684. [0334] U.S. Pat. No.
5,145,684. [0335] U.S. Pat. No. 5,219,727. [0336] U.S. Pat. No.
5,219,727. [0337] U.S. Pat. No. 5,238,921. [0338] U.S. Pat. No.
5,297,721. [0339] U.S. Pat. No. 5,334,711. [0340] U.S. Pat. No.
5,334,711. [0341] U.S. Pat. No. 5,348,978. [0342] U.S. Pat. No.
5,354,855. [0343] U.S. Pat. No. 5,354,855. [0344] U.S. Pat. No.
5,399,346. [0345] U.S. Pat. No. 5,399,363. [0346] U.S. Pat. No.
5,399,363. [0347] U.S. Pat. No. 5,449,661. [0348] U.S. Pat. No.
5,455,166. [0349] U.S. Pat. No. 5,466,468. [0350] U.S. Pat. No.
5,466,468. [0351] U.S. Pat. No. 5,543,158. [0352] U.S. Pat. No.
5,543,158. [0353] U.S. Pat. No. 5,552,157 [0354] U.S. Pat. No.
5,552,157. [0355] U.S. Pat. No. 5,552,397. [0356] U.S. Pat. No.
5,565,213. [0357] U.S. Pat. No. 5,565,213. [0358] U.S. Pat. No.
5,567,434. [0359] U.S. Pat. No. 5,567,434. [0360] U.S. Pat. No.
5,580,579 [0361] U.S. Pat. No. 5,602,306. [0362] U.S. Pat. No.
5,631,359. [0363] U.S. Pat. No. 5,631,359. [0364] U.S. Pat. No.
5,639,655. [0365] U.S. Pat. No. 5,639,940 [0366] U.S. Pat. No.
5,641,515 [0367] U.S. Pat. No. 5,641,515. [0368] U.S. Pat. No.
5,646,020. [0369] U.S. Pat. No. 5,646,031. [0370] U.S. Pat. No.
5,648,211. [0371] U.S. Pat. No. 5,656,016. [0372] U.S. Pat. No.
5,697,899. [0373] U.S. Pat. No. 5,712,124. [0374] U.S. Pat. No.
5,720,936. [0375] U.S. Pat. No. 5,725,871. [0376] U.S. Pat. No.
5,738,868. [0377] U.S. Pat. No. 5,738,868. [0378] U.S. Pat. No.
5,741,516. [0379] U.S. Pat. No. 5,741,516. [0380] U.S. Pat. No.
5,744,311. [0381] U.S. Pat. No. 5,756,353. [0382] U.S. Pat. No.
5,770,219. [0383] U.S. Pat. No. 5,779,708 [0384] U.S. Pat. No.
5,780,045 [0385] U.S. Pat. No. 5,783,208 [0386] U.S. Pat. No.
5,789,655. [0387] U.S. Pat. No. 5,792,451. [0388] U.S. Pat. No.
5,795,587. [0389] U.S. Pat. No. 5,795,587. [0390] U.S. Pat. No.
5,797,898. [0391] U.S. Pat. No. 5,804,212. [0392] U.S. Pat. No.
5,863,736. [0393] U.S. Pat. No. 5,863,736. [0394] Eur. Pat. Appl.
Publ. No. EP0273085. [0395] Eur. Pat. Appl. Publ. No. EP0329822.
[0396] Eur. Pat. Appl. Publ. No. EP0360257. [0397] Eur. Pat. Appl.
Publ. No. EP320308. [0398] Eur. Pat. Appl. Publ. No. EP92110298.4.
[0399] Great Britain Pat. Appl. No. GB2202328. [0400] PCT Int'l.
Pat. Appl. No. PCT/US87/00880. [0401] PCT Int'l. Pat. Appl. No.
PCT/US87/00880. [0402] PCT Int'l. Pat. Appl. No. PCT/US88/10315.
[0403] PCT Int'l. Pat. Appl. No. PCT/US88/10315. [0404] PCT Int'l.
Pat. Appl. No. PCT/US89/01025. [0405] PCT Int'l. Pat. Appl. No.
PCT/US89/01025. [0406] PCT Int'l. Pat. Appl. Publ. No. WO89/06700.
[0407] PCT Int'l. Pat. Appl. Publ. No. WO89/06700. [0408] PCT
Int'l. Pat. Appl. Publ. No. WO90/07641. [0409] PCT Int'l. Pat.
Appl. Publ. No. WO91/03162. [0410] PCT Int'l. Pat. Appl. Publ. No.
WO91/03162. [0411] PCT Int'l. Pat. Appl. Publ. No. WO92/07065.
[0412] PCT Int'l. Pat. Appl. Publ. No. WO92/07065. [0413] PCT
Int'l. Pat. Appl. Publ. No. WO93/15187. [0414] PCT Int'l. Pat.
Appl. Publ. No. WO93/15187. [0415] PCT Int'l. Pat. Appl. Publ. No.
WO93/23569. [0416] PCT Int'l. Pat. Appl. Publ. No. WO93/23569.
[0417] PCT Int'l. Pat. Appl. Publ. No. WO94/02595. [0418] PCT
Int'l. Pat. Appl. Publ. No. WO94/02595. [0419] PCT Int'l. Pat.
Appl. Publ. No. WO94/13688. [0420] PCT Int'l. Pat. Appl. Publ. No.
WO94/13688. [0421] Acton et al., "Expression cloning of SR-BI, a
CD36-related class B scavenger receptor," J. Biol. Chem.,
269:21003-09, 1994. [0422] Adair et al., "Vascular development in
chick embryos: a possible role for adenosine," Am. J. Physiol.,
256:H240-46, 1989. [0423] Afione et al., "In vivo model of
adeno-associated virus vector persistence and rescue," J. Virol.,
70:3235-41, 1996. [0424] Afione et al., "Delayed expression of
adeno-associated virus vector DNA," Intervirology, 42:213-20, 1999.
[0425] Agarwal et al., "Linoleyl hydroperoxide transcriptionally
upregulates heme oxygenase-1 gene expression in human renal
epithelial and aortic endothelial cells," J. Am. Soc. Nephrol.,
9:1990-97, 1998. [0426] Agbandje et al., "The structure of human
parvovirus B19 at 8 .ANG. resolution," Virol., 203:106-15, 1994.
[0427] Agbandje et al., "Structure determination of feline
panleuopenia virus empty particles," Proteins, 16:155-71, 1993.
[0428] Agbandje-McKenna et al., "Functional implications of the
structure of the murine parvovirus, minute virus of mice,"
Structure, 6:1369-81, 1998.
[0429] Aiello et al., "Vascular endothelial growth factor in ocular
fluid of patients with diabetic retinopathy and other retinal
disorders," N. Engl. J. Med., 331:1480-87, 1994. [0430] Aiello et
al., "Vascular endothelial growth factor-induced retinal
permeability is mediated by protein kinase C in vivo and suppressed
by an orally effective beta-isoform-selective inhibitor," Diabetes,
46:1473-80, 1997. [0431] Aird et al., "Vascular bed-specific
expression of an endothelial cell gene is programmed by the tissue
microenvironment," J. Cell Biol., 138:1117-24, 1997. [0432] Akagi
et al., "Transcriptional activation of a hybrid promoter composed
of cytomegalovirus enhancer and .beta.-actin/.beta.-globin gene in
glomerular epithelial cells in vivo," Kidney Int., 51:1265-69,
1997. [0433] Alejandro et al., "Long-term function (6 years) of
islet allografts in type 1 diabetes," Diabetes, 46:1983-89, 1997.
[0434] Allen and Choun, "Large unilamellar liposomes with low
uptake into the reticuloendothelial system," FEBS Lett., 223:42-46,
1987. [0435] Altschuler et al., "A method for generating
transcripts with defined 5' and '3 termini by autolytic
processing," Gene, 122:85-90, 1992. [0436] Ardekani et al., "Two
repressor elements inhibit expression of the von Willebrand factor
gene promoter in vitro," Thromb. Haemost., 80:488-94, 1998. [0437]
Arreaza et al., "Neonatal activation of CD28 signaling overcomes T
cell energy and prevents autoimmune diabetes by an IL-4-dependent
mechanism," J. Clin. Invest., 100:2243-53, 1997. [0438] Asahara et
al., "Isolation of putative progenitor endothelial cells for
angiogenesis," Science, 275:964-67, 1997. [0439] Atchison et al.,
"Adenovirus-associated defective virus particles," Science
149(3685):754-56, 1965. [0440] Atchison et al., "Electron
microscopy of adenovirus-associated virus (AAV) in cell cultures,"
Virology, 29:353-57, 1966. [0441] Atkinson and Eisenbarth, "Type 1
diabetes: new perspectives on disease pathogenesis and treatment,"
Lancet, 358:221-29, 2001. [0442] Atkinson and Leiter, "The NOD
mouse model of type 1 Type I diabetes: as good as it gets?" Nat.
Med., 5:601-04, 1999. [0443] Atkinson and Maclaren, "The
pathogenesis of insulin-dependent diabetes mellitus," N. Engl. J.
Med., 331:1428-36, 1994. [0444] Auricchio et al., "A single-step
affinity column for purification of serotype-5 based
adeno-associated viral vectors,"Mol. Ther., 4:372-74, 2001. [0445]
Bach, "Insulin dependent diabetes mellitus as a beta-cell targeted
disease of immunoregulation," J. Autoimmun., 8:439-463, 1995.
[0446] Bach, "Insulin-dependent diabetes mellitus as an autoimmune
disease," Endocr. Rev., 15:516-42, 1994. [0447] Bach and Chatenoud,
"Tolerance to islet autoantigens in Type 1 diabetes," Annu. Rev.
Immunol., 19:131-61, 2001. [0448] Balasa and Sarvetnick, "The
paradoxical effects of interleukin 10 in the immunoregulation of
autoimmune diabetes," J. Autoimmun., 9:283-86, 1996. [0449] Balasa
et al., "A mechanism for IL-10-mediated diabetes in the nonobese
diabetic (NOD) mouse: ICAM-1 deficiency blocks accelerated
diabetes," J. Immunol., 165:7330-37, 2000a. [0450] Balasa et al.,
"Islet-specific expression of IL-10 promotes diabetes in nonobese
diabetic mice independent of Fas, perforin, TNF receptor-1, and TNF
receptor-2 molecules," J. Immunol., 165:2841-47, 2000b. [0451]
Balazsovits et al., "Analysis of the effect of liposome
encapsulation on the vesicant properties, acute and cardiac
toxicities, and antitumor efficacy of doxorubicin," Cancer
Chemother. Pharmacol., 23:81-86, 1989. [0452] Bantel et al.,
"Characterization of the DNA of a defective human parvovirus
isolated from a genital site," Virology, 134:52-63, 1984. [0453]
Barbis et al., "Mutations adjacent to the dimple of the canine
parvovirus capsid structure affect sialic acid binding," Virology,
191:301-08, 1992. [0454] Barcz et al., "The influence of
theobromine on angiogenic activity and proangiogenic cytokines
production of human ovarian cancer cells," Oncol. Rep., 5:517-20,
1998. [0455] Barrijal et al., "Nucleolin forms a specific complex
with a fragment of the viral (minus) strand of minute virus of mice
DNA," Nucleic Acids Res., 20:5053-60, 1992. [0456] Bartlett and
Samulski, "Fluorescent viral vectors: a new technique for the
pharmacological analysis of gene therapy,"Nat. Med., 4:635-7, 1998.
[0457] Bartlett et al., "Long-term expression of a fluorescent
reporter gene via direct injection of plasmid vector into mouse
skeletal muscle: Comparison of human creatine kinase and CMV
promoter expression levels in vivo," Cell Transplant.,
5(3):411-419, 1996. [0458] Bartlett et al., "Targeted
adeno-associated virus vector transduction of nonpermissive cells
mediated by a bispecific F(ab'.gamma.)2 antibody," Nat.
Biotechnol., 17:181-86, 1999. [0459] Baskar et al., "Developmental
analysis of the cytomegalovirus enhancer in transgenic animals," J.
Virol., 70:3215-26, 1996. [0460] Baskar et al., "The enhancer
domain of the human cytomegalovirus major immediate-early promoter
determines cell type-specific expression in transgenic mice," J.
Virol., 70:3207-14, 1996. [0461] Becerra et al., "Synthesis of
adeno-associated virus structural proteins requires both
alternative mRNA splicing and alternative initiations from a single
transcript," J. Virol., 62:2745-54, 1988. [0462] Becerra et al.,
"Direct mapping of adeno-associated virus capsid proteins B and C:
a possible ACG initiation codon," Proc. Nat'l. Acad. Sci. USA,
82:7919-23, 1985. [0463] Beck et al., "Repeated delivery of
adeno-associated virus vectors to the rabbit airway," J. Virol.,
73:9446-55, 1999. [0464] Beck et al., "Igf1 gene disruption results
in reduced brain size, CNS hypomyelination, and loss of hippocampal
granule and striatal parvalbumin-containing neurons," Neuron,
14:717-30, 1995. [0465] Bendelac et al., "Syngeneic transfer of
autoimmune diabetes from diabetic NOD mice to healthy neonates.
Requirement for both L3T4+ and Lyt-2+ T cells," J. Exp. Med.,
166:823-32, 1987. [0466] Benhamou et al., "Decreased alloreactivity
to human islets secreting recombinant viral interleukin 10,"
Transplantation, 62:1306-12, 1996. [0467] Bennett et al.,
"Adenovirus-mediated delivery of rhodopsin-promoted bcl-2 results
in a delay in photoreceptor cell death in the rd/rd mouse," Gene
Ther., 5(9):1156-1164, 1998. [0468] Bennett et al., "Real-time,
noninvasive in vivo assessment of adeno-associated virus-mediated
retinal transduction," Invest. Ophthalmol. Vis. Sci., 38:2857-2863,
1997. [0469] Bennett et al., "Stable transgene expression in rod
photoreceptors after recombinant adeno-associated virus-mediated
gene transfer to monkey retina," Proc. Nat'l Acad. Sci. USA,
96:9920-25, 1999. [0470] Berns and Bohenzky, "Adeno-associated
viruses: an update," Adv. Virus Res., 32:243-306, 1987. [0471]
Berns and Giraud, "Adenovirus and adeno-associated virus as vectors
for gene therapy," Ann. N.Y. Acad. Sci., 772:95-104, 1995. [0472]
Berns and Giraud, "Biology of adeno-associated virus," Curr. Top.
Microbiol. Immunol., 218:1-23, 1996. [0473] Berns and Linden, "The
cryptic life style of adeno-associated virus," Bioessays,
17:237-45, 1995. [0474] Berns et al., In VIRUS PERSISTENCE, Mehay
et al. (ed.), Cambridge Univ. Press, pp. 249-265, 1982. [0475]
Berns, In FIELDS VIROLOGY, Fields, (ed.), Raven Press,
Philadelphia, Pa., pp. 2173-97, 1996. [0476] Berns, THE
PARVOVIRUSES, Plenum Press, New York, 1984. [0477] Berns et al.,
"Regulation of adeno-associated virus DNA replication," Biochim.
Biophys. Acta, 951:425-29, 1988. [0478] Berns et al., "Detection of
adeno-associated virus (AAV)-specific nucleotide sequences in DNA
isolated from latently infected Detroit 6 cells," Virology,
68:556-60, 1975. [0479] Bikfalvi and Han, "Angiogenic factors are
hematopoietic growth factors and vice versa," Leukemia, 8:523-29,
1994. [0480] Binley et al., "An adenoviral vector regulated by
hypoxia for the treatment of ischaemic disease and cancer," Gene
Ther., 6:1721-27, 1999. [0481] Birikh et al., "The structure,
function and application of the hammerhead ribozyme," Eur. J.
Biochem., 245:1-16, 1997. [0482] Blacklow, "Adeno-associated
viruses of humans, p. 165-174," in PARVOVIRUSES AND HUMAN DISEASE,
Pattison (ed.), CRC Press, Boca Raton, Fla., 1988. [0483] Blacklow
et al., "Studies of the enhancement of an adenovirus-associated
virus by herpes simplex virus," J. Gen. Virol., 10:29-36, 1971.
[0484] Blacklow et al., "Isolation of adenovirus-associated viruses
from man," Proc. Nat'l. Acad. Sci. USA, 58:1410-15, 1967. Blacklow
et al., "Serologic evidence for human infection with
adenovirus-associated viruses," J. Nat'l. Cancer Inst., 40:319-27,
1968a. [0485] Blacklow et al., "Epidemiology of
adenovirus-associated virus infection in a nursery population," Am.
J. Epidemiol., 88:368-78, 1968b. [0486] Blacklow et al., "A
seroepidemiologic study of adenovirus-associated virus infection in
infants and children," Am. J. Epidemiol., 94:359-66, 1971. [0487]
Boast et al., "Characterization of physiologically regulated
vectors for the treatment of ischemic disease," Hum. Gene Ther.,
10:2197-208, 1999. [0488] Borriello and Krauter, "Multiple murine
alpha 1-protease inhibitor genes show unusual evolutionary
divergence," Proc. Nat'l. Acad. Sci. USA, 88:9417-21, 1991. [0489]
Boskovic and Twining, "Local control of .alpha.1-proteinase
inhibitor levels: regulation of .alpha.1-proteinase inhibitor in
the human cornea by growth factors and cytokines," Biochim.
Biophys. Acta, 1403:37-46, 1998. [0490] Bottino et al.,
"Transplantation of allogenic islets of Langerhans in the rat
liver: effects of macrophage depletion on graft survival and
microenvironment activation," Diabetes, 47:316-23, 1998. [0491]
Bourlais et al., "Ophthalmic drug delivery systems--recent
advances," Prog. Retin Eye Res., 17(1):33-58, 1998. [0492] Bowman
et al., "Immunological and metabolic effects of prophylactic
insulin therapy in the NOD-scid/scid adoptive transfer model of
IDDM," Diabetes, 45:205-08, 1996. [0493] Brantly et al., "Use of a
highly purified alpha 1-antitrypsin standard to establish ranges
for the common normal and deficient alpha 1-antitrypsin
phenotypes," Chest, 100: 703-08, 1991. [0494] Brass et al.,
"Evaluation of University of Wisconsin cold-storage solution in
warm hypoxic perfusion of rat liver: the addition of fructose
reduces injury," Gastroenterology, 105:1455-63, 1993. [0495]
Breakefield et al., TREATMENT OF GENETIC DISEASES, Churchill
Livingstone, Inc., 1991. [0496] Briggs et al., "Purification and
biochemical characterization of the promoter-specific transcription
factor, Sp1," Science, 234:47-52, 1986. [0497] Brown and Jampol,
"New concepts of regulation of retinal vessel tone," Arch.
Ophthalmol., 114:199-204, 1996. [0498] Brown et al., "Critical
evaluation of ECV304 as a human endothelial cell model defined by
genetic analysis and functional responses: a comparison with the
human bladder cancer derived epithelial cell line T24/83," Lab.
Invest., 80:37-45, 2000. [0499] Brown et al., "Isolation and
characterization of LRP6, a novel member of the low density
lipoprotein receptor gene family," Biochem. Biophys. Res. Commun.,
248:879-88, 1998. [0500] Bruijn et al., "Aggregation and motor
neuron toxicity of an ALS-linked SOD1 mutant independent from
wild-type SOD1," Science, 281:1851-1854, 1998. [0501] Buller,
"Herpes simplex virus types 1 and 2 completely help
adenovirus-associated virus replication," J. Virol., 40:241-47,
1981. [0502] Buller and Rose, "Characterization of
adenovirus-associated virus-induced polypeptides in KB cells," J.
Virol., 25:331-38, 1978. [0503] Burcin et al., "Adenovirus-mediated
regulable target gene expression in vivo," Proc. Nat'l. Acad. Sci.
USA, 96:355-60, 1999. [0504] Caldovic and Hackett, "Development of
position-independent expression vectors and their transfer into
transgenic fish,"Mol. Mar. Biol. Biotechnol., 4(1):51-61, 1995.
[0505] Cameron et al., "IL-4 prevents insulitis and
insulin-dependent diabetes mellitus in nonobese diabetic mice by
potentiation of regulatory T helper-2 cell function," J. Immunol.,
159:4686-92, 1997. [0506] Cameron et al., "Biolistic-mediated
interleukin 4 gene transfer prevents the onset of type 1 Type I
diabetes," Hum. Gene Ther., 11:1647-56, 2000. [0507] Cao et al.,
"Developmental and hormonal regulation of murine scavenger
receptor, class B, type 1," Mol. Endocrinol., 13:1460-73, 1999.
[0508] Capecchi, "High efficiency transformation by direct
microinjection of DNA into cultured mammalian cells," Cell,
22:479-88, 1980. [0509] Carrell et al., "Structure and variation of
human alpha 1-antitrypsin," Nature, 298:329-34, 1982. [0510]
Carroll et al., "Long-term (>3-year) insulin independence in a
patient with pancreatic islet cell transplantation following upper
abdominal exenteration and liver replacement for fibrolamellar
hepatocellular carcinoma," Transplantation, 59:875-79, 1995. [0511]
Carter and Flotte, "Development of adeno-associated virus vectors
for gene therapy of cystic fibrosis," Curr. Top. Microbiol.
Immunol., 218:119-44, 1996. [0512] Carter et al., In THE
PARVOVIRUSES, Berns (ed.), Plenum, N.Y., pp. 153-207, 1983. [0513]
Carter, "The growth of adeno-associated virus," In HANDBOOK OF
PARVOVIRUSES, Tijssen (ed.), CRC Press, Boca Raton, pp. 155-68,
1990. [0514] Carter et al., "Physical map and strand polarity of
specific fragments of adenovirus-associated virus DNA produced by
endonuclease R-EcoRI," J. Virol., 16:559-68, 1975. [0515] Carter et
al., "Properties of an adenovirus type 2 mutant, Ad2d1807, having a
deletion near the right-hand genome terminus: failure to help AAV
replication," Virology, 126:505-16, 1983. [0516] Carter et al., In
HANDBOOK OF PARVOVIRUSES, CRC Press, Boca Raton, pp. 169-226, 1990.
[0517] Carver et al., "Transgenic livestock as bioreactors: stable
expression of human alpha-1-antitrypsin by a flock of sheep,"
Biotechnology (New York), 11(11):1263-1270, 1993. [0518] Casto et
al., "Studies on the relationship between adeno-associated virus
type 1 (AAV-1) and adenoviruses. II. Inhibition of adenovirus
plaques by AAV; its nature and specificity," Virology, 33:452-58,
1967. [0519] Cech, "Self-splicing of group I introns," Annu. Rev.
Biochem., 59:543-69, 1990. [0520] Cech, "RNA as an enzyme,"
Biochem. Int., 18:7-14, 1989. [0521] Cech et al., "In vitro
splicing of the ribosomal RNA precursor of Tetrahymena: involvement
of a guanosine nucleotide in the excision of the intervening
sequence," Cell, 27(3 Pt 2):487-496, 1981. [0522] Chakravarthy et
al., "Nitric oxide synthase activity and expression in retinal
capillary endothelial cells and pericytes,"Curr. Eye Res.,
14:285-94, 1995. [0523] Challberg, "A method for identifying the
viral genes required for herpesvirus DNA replication," Proc. Nat'l.
Acad. Sci. USA, 83:9094-103, 1986. [0524] Chandran et al., "Recent
trends in drug delivery systems: liposomal drug delivery
system--preparation and characterization," Indian J. Exp.
Biol.,
35(8):801-809, 1997. [0525] Chang and Prud'homme, "Intramuscular
administration of expression plasmids encoding interferon-gamma
receptor/IgG1 or IL-4/IgG1 chimeric proteins protects from
autoimmunity," J. Gene Med., 1:415-23, 1999. [0526] Chao et al.,
"Several log increase in therapeutic transgene delivery by distinct
adeno-associated viral serotype vectors,"Mol. Ther., 2:619-23,
2000. [0527] Chapman and Rossman, "Structure, sequence, and
function correlations among parvoviruses," Virol., 194:491-508,
1993. [0528] Chejanovsky and Carter, "Mutagenesis of an AUG codon
in the adeno-associated virus rep gene: effects on viral DNA
replication," Virology, 173:120-28, 1989. [0529] Chen and Okayama,
"High-efficiency transformation of mammalian cells by plasmid DNA,"
Mol. Cell. Biol., 7:2745-52, 1987. [0530] Chen et al.,
"Multitarget-ribozyme directed to cleave at up to nine highly
conserved HIV-1 env RNA regions inhibits HIV-1
replication--potential effectiveness against most presently
sequenced HIV-1 isolates," Nucl. Acids Res., 20:4581-4589, 1992.
[0531] Cheung et al., "Integration of the adeno-associated virus
genome into cellular DNA in latently infected human Detroit 6
cells," J. Virol., 33:739-48, 1980. [0532] Chiocca et al.,
"Transfer and expression of the lacZ gene in rat brain neurons
mediated by herpes simplex virus mutants," The New Biologist,
2:739-46, 1990. [0533] Chiorini et al., "Cloning and
characterization of adeno-associated virus type 5," J. Virol.,
73:1309-19, 1999. [0534] Chiorini et al., "High-efficiency transfer
of the T cell co-stimulatory molecule B7-2 to lymphoid cells using
high-titer recombinant adeno-associated virus vectors," Hum. Gene
Ther., 6:1531-41, 1995. [0535] Chiorini et al., "Cloning of
adeno-associated virus type 4 (AAV4) and generation of recombinant
AAV4 particles," J. Virol., 71:6823-33, 1997. [0536] Chowrira and
Burke, "Extensive phosphorothioate substitution yields highly
active and nuclease-resistant hairpin ribozymes," Nucl. Acids Res.,
20:2835-2840, 1992. [0537] Churg et al., ".alpha.-1-antitrypsin and
a broad spectrum metalloprotease inhibitor, RS113456, have similar
acute anti-inflammatory effects," Lab. Invest., 81:1119-31, 2001.
[0538] Cipolla et al., "High glucose concentrations dilate cerebral
arteries and diminish myogenic tone through an endothelial
mechanism," Stroke, 28:405-11, 1997. [0539] Clark et al., "Highly
purified recombinant adeno-associated virus vectors are
biologically active and free of detectable helper and wild-type
viruses," Hum. Gene Ther. 10:1031-39, 1999. [0540] Clark et al.,
"Recombinant adeno-associated viral vectors mediate long-term
transgene expression in muscle," Hum. Gene Ther., 8:659-69, 1997.
[0541] Clark et al., "A stable cell line carrying
adenovirus-inducible rep and cap genes allows for infectivity
titration of adeno-associated virus vectors," Gene Ther.,
3:1124-32, 1996. [0542] Clark et al., "Cell lines for the
production of recombinant adeno-associated virus," Hum. Gene Ther.,
6:1329-41, 1995. [0543] Clemmons, "IGF binding proteins: regulation
of cellular actions," Growth Regul., 2:80-87, 1992. [0544]
Cleveland, "From Charcot to SOD1: mechanisms of selective motor
neuron death in ALS," Neuron, 23:515-520, 1999. [0545] Collins and
Olive, "Reaction conditions and kinetics of self-cleavage of a
ribozyme derived from Neurospora VS RNA," Biochem.,
32(11):2795-2799, 1993. [0546] Conrad et al., "Safety of
single-dose administration of an adeno-associated virus (AAV)-CFTR
vector in the primate lung," Gene Ther., 3:658-68, 1996. [0547]
Cook and McCormick, "Inhibition by cAMP of Ras-dependent activation
of Raf," Science, 262:1069-72, 1993. [0548] Cosentino et al., "High
glucose increases nitric oxide synthase expression and superoxide
anion generation in human aortic endothelial cells," Circulation,
96:25-28, 1997. [0549] Coune, "Liposomes as drug delivery system in
the treatment of infectious diseases: potential applications and
clinical experience," Infection, 16:141-47, 1988. [0550] Couvreur,
"Polyalkyleyanoacrylates as colloidal drug carriers," Crit. Rev.
Ther. Drug Carrier Syst., 5:1-20, 1988. [0551] Couvreur et al.,
"Tissue distribution of antitumor drugs associated with
polyalkylcyanoacrylate nanoparticles," J. Pharm. Sci., 69:199-202,
1980. [0552] Couvreur et al., "Nanocapsules, a new lysosomotropic
carrier," FEBS Lett., 84:323-26, 1977. [0553] Cowan et al.,
"Elafin, a serine elastase inhibitor, attenuates post-cardiac
transplant coronary arteriopathy and reduces myocardial necrosis in
rabbits after heterotopic cardiac transplantation," J. Clin.
Invest., 97:2452-68, 1996. [0554] Cozzi et al., "Characterization
of pigs transgenic for human decay-accelerating factor,"
Transplantation, 64(10):1383-1392, 1997. [0555] Cretin et al.,
"Human islet allotransplantation: world experience and current
status," Dig. Surg., 15:656-62, 1998. [0556] Crute et al., "Herpes
simplex virus 1 helicase-primase: a complex of three herpes-encoded
gene products," Proc. Nat'l. Acad. Sci. USA, 86:2186-94, 1989.
[0557] Cukor et al., In THE PARVOVIRUSES, Berns (ed.), Plenum
Press, NY, pp. 33-66, 1983. [0558] Cunningham and Wells, "High
resolution epitope mapping of hGH-receptor interactions by
alanine-scanning mutagenesis," Science, 244:1081-85, 1989. [0559]
Curiel et al., "Adenovirus enhancement of
transferrin-polylysine-mediated gene delivery," Proc. Nat'l. Acad.
Sci. USA, 88:8850-54, 1991. [0560] Cusi and DeFronzo, "Treatment of
NIDDM, IDDM and other insulin-resistant states with IGF-I:
physiological and clinical considerations," Diabetes Rev.,
3:206-36, 1995. [0561] D'Angelo et al., "cAMP-dependent protein
kinase inhibits the mitogenic action of vascular endothelial growth
factor and fibriblast growth factor in capillary endothelial cells
by blocking Raf activation," J. Cell Biochem., 67:353-366, 1997.
[0562] Daiger et al., "Data services and software for identifying
genes and mutations causing retinal degeneration," Invest.
Ophthalmol.Vis. Sci., 39:S295, 1998. [0563] Daiger et al.,
"Correlation of phenotype with genotype in inherited retinal
degeneration,m" Behavioral Brain Sci., 18:452-67, 1995. [0564] Daly
et al., "Neonatal gene transfer leads to widespread correction of
pathology in a murine model of lysosomal storage disease," Proc.
Nat'l Acad. Sci. USA, 96:2296-300, 1999. [0565] Damert et al.,
"Activator-protein-1 binding potentiates the hypoxia-inducible
factor-1-mediated hypoxia-induced transcriptional activation of
vascular-endothelial growth factor expression in C6 glioma cells,"
Biochem. J., 327:419-23, 1997. [0566] Datta et al., "The receptor
binding domain of apolipoprotein E, linked to a model class A
amphipathic helix, enhances internalization and degradation of LDL
by fibroblasts," Biochemistry, 39:213-220, 2000. [0567] Davidson et
al., "Recombinant adeno-associated virus type 2, 4, and 5 vectors:
transduction of variant cell types and regions in the mammalian
central nervous system," Proc. Nat'l. Acad. Sci. USA, 97:3428-43,
2000. [0568] Davies et al., "Interleukin-4 secretion by the
allograft fails to affect the allograft-specific interleukin-4
response in vitro," Transplantation, 67:1583-89, 1999. [0569] Davis
et al., "In vivo activation and in situ BDNF-stimulated nuclear
translocation of mitogen-activated/extracellular signal-regulated
protein kinase is inhibited by ethanol in the developing rat
hippocampus," Neurosci. Lett., 272:95-98, 1999. [0570] Delovitch
and Singh, "The nonobese diabetic mouse as a model of autoimmune
diabetes: immune dysregulation gets the NOD," Immunity, 7:727-38,
1997. [0571] DeLuca and Schaffer, "Activities of herpes simplex
virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides,"
Nucl. Acids Res., 15:4491-511, 1987. [0572] DeLuca et al.,
"Isolation and characterization of deletion mutants of herpes
simplex virus type 1 in the gene encoding immediate-early
regulatory protein ICP4," J. Virol., 56:558-70, 1985. [0573] Deng
et al., "IL-10 and TGF-.beta. gene transfer to rodent islets:
effect on xenogeneic islet graft survival in naive and
B-cell-deficient mice," Trans. Proc., 29:2207-08, 1997. [0574]
Deshpande et al., "The human transcription enhancer factor-1,
TEF-1, can substitute for Drosophila scalloped during wingblade
development," J. Biol. Chem., 272:10664-68, 1997. [0575] DesJardin
and Hauswirth, "Developmentally important DNA elements within the
bovine opsin upstream region," Inv. Ophth. Vis. Sci., 37:154-65,
1996. [0576] Dhami et al., "Acute cigarette smoke-induced
connective tissue breakdown is mediated by neutrophils and
prevented by .alpha.1-antitrypsin," Am. J. Respir. Cell Mol. Biol.,
22:244-52, 2000. [0577] Dills et al., "Association of elevated
IGF-I levels with increased retinopathy in late-onset diabetes,"
Diabetes, 40:1725-30, 1991. [0578] Ding et al., "A single amino
acid determines the immunostimulatory activity of interleukin 10,"
J. Exp. Med., 191:213-23, 2000. [0579] Donello et al., "Woodchuck
hepatitis virus contains a tripartite posttranscriptional
regulatory element," J. Virol., 72:5085-92, 1998. [0580] Dong et
al., "Quantitative analysis of the packaging capacity of
recombinant adeno-associated virus," Hum. Gene Ther., 7:2101-12,
1996. [0581] Douglas et al., "Nanoparticles in drug delivery,"
Crit. Rev. Ther. Drug Carrier Syst., 3:233-61, 1987. [0582] Drenser
et al., "Ribozyme-targeted destruction of RNAs associated with
ADRP," Inv. Ophth. Vis. Sci., 39:681-689, 1998. [0583] Dropulic et
al., "Functional characterization of a U5 ribozyme: intracellular
suppression of human immunodeficiency virus type 1 expression," J.
Virol., 66(3):1432-41, 1992. [0584] Dryja and Berso, "Retinitis
pigmentosa and allied diseases. Implications of genetic
heterogeneity," Invest. Ophthalmol. Vis. Sci., 36:1197-1200, 1995.
[0585] Duan et al., "Dynamin is required for recombinant
adeno-associated virus type 2 infection," J. Virol., 73:10371-76,
1999. [0586] Duan et al., "A new dual-vector approach to enhance
recombinant adeno-associated virus-mediated gene expression through
intermolecular cis activation," Nat. Med., 6:595-98, 2000. [0587]
Dunn, "Problems related to immunosuppression. Infection and
malignancy occurring after solid organ transplantation,"Crit. Care
Clin., 6:955-77, 1990. [0588] Dunn et al., "Insulin-like growth
factor 1 (IGF-1) alters drug sensitivity of HBL100 human breast
cancer cells by inhibition of apoptosis induced by diverse
anticancer drugs," Cancer Res., 57:2687-93, 1997. [0589] During et
al., "Peroral gene therapy of lactose intolerance using an
adeno-associated virus vector," Nature Med., 4:1131-1135, 1998.
[0590] Dusseau and Hutchins, "Hypoxia-induced angiogenesis in chick
chorioallantoic membranes: a role for adenosine," Respir. Physiol.,
17:33-44, 1988. [0591] Dusseau et al., "Stimulation of angiogenesis
by adenosine on the chick chorioallantoic membrane," Circ. Res.,
59:163-70, 1986. [0592] Ebert and Bunn, "Regulation of
transcription by hypoxia requires a multiprotein complex that
includes hypoxia-inducible factor 1, an adjacent transcription
factor, and p300/CREB binding protein," Mol. Cell Biol.,
18:4089-96, 1998. [0593] Ebert et al., "Transgenic production of a
variant of human tissue-type plasminogen activator in goat milk:
generation of transgenic goats and analysis of expression,"
Biotechnology (New York), 9(9):835-838, 1991. [0594] Eglitis and
Anderson, "Retroviral vectors for introduction of genes into
mammalian cells," Biotechniques, 6(7):608-614, 1988. [0595] Eglitis
et al., "Retroviral-mediated gene transfer into hemopoietic cells,"
Avd. Exp. Med. Biol., 241:19-27, 1988. [0596] Eisen and Brown, "DNA
arrays for analysis of gene expression," Methods Enzymol.,
303:179-205, 1999. [0597] Ellis et al., Increased H.sub.2O.sub.2,
vascular endothelial growth factor and receptors in the retina of
the BBZ/Wor diabetic rat." Free Radic. Biol. Med., 28:91-101, 2000.
[0598] Elroy-Stein and Moss, "Cytoplasmic expression system based
on constitutive synthesis of bacteriophage T7 RNA polymerase in
mammalian cells," Proc. Nat'l. Acad. Sci. USA, 87:6743-7, 1990.
[0599] Ethier et al., "Adenosine stimulates proliferation of human
endothelial cells in culture," Am. J. Physiol., 265:H131-38, 1993.
[0600] Faktorovich et al., "Photoreceptor degeneration in inherited
retinal dystrophy delayed by basic fibroblast growth factor,"
Nature, 347:83-86, 1990. [0601] Faller and Baltimore, "Liposome
encapsulation of retrovirus allows efficient super infection of
resistant cell lines," J. Virol., 49:269-72, 1984. [0602]
Fechheimer et al., "Transfection of mammalian cells with plasmid
DNA by scrape loading and sonication loading," Proc. Nat'l. Acad.
Sci. USA, 84:8463-67, 1987. [0603] Fedor and Uhlenbeck, "Substrate
sequence effects on `hammerhead` RNA catalytic efficiency," Proc.
Nat'l Acad. Sci. USA, 87:1668-1672, 1990. [0604] Fellowes et al.,
"Amelioration of established collagen induced arthritis by systemic
IL-10 gene delivery," Gene Ther., 7:967-77, 2000. [0605] Ferrari et
al., "Second strand synthesis is a rate-limiting step for efficient
transduction by recombinant adeno-associated virus vectors,"J.
Virol., 70:3227-34, 1996. [0606] Ferrari et al., "New developments
in the generation of Ad-free, high-titer rAAV gene therapy
vectors," Nature Med., 3:1295-97, 1997. [0607] Ferreira et al.,
"The role of the 5'-flanking region in the cell-specific
transcription of the human von Willebrand factor gene," Biochem.
J., 293:641-48, 1993. [0608] Fife et al., "Endothelial cell
transfection with cationic liposomes and herpes simplex-thymidine
kinase mediated killing," Gene Ther., 5:614-20, 1998. [0609]
Finkenzeller et al., "Sp1 recognition sites in the proximal
promoter of the human vascular endothelial growth factor gene are
essential for platelet-derived growth factor-induced gene
expression," Oncogene, 15:669-76, 1997. [0610] Fischer et al.,
"Induction of alpha1-antitrypsin synthesis in human articular
chondrocytes by interleukin-6-type cytokines: evidence for a local
acute-phase response in the joint," Arthritis Rheum., 42:1936-45,
1999. [0611] Fisher et al., "Transduction with recombinant
adeno-associated virus for gene therapy is limited by
leading-strand synthesis," J. Virol., 70:520-32, 1996. [0612]
Fisher et al., "Recombinant adeno-associated virus for muscle
directed gene therapy," Nat. Med., 3:306-12, 1997. [0613]
Fisher-Adams et al., "Integration of adeno-associated virus vectors
in CD34' human hematopoietic progenitor cells after transduction,"
Blood, 88:492-504, 1996. [0614] Flamme and Risau, "Induction of
vasculogenesis and hematopoiesis in vitro," Development,
116:435-39, 1992. [0615] Flannery et al., "Efficient
photoreceptor-targeted gene expression in vivo by recombinant
adeno-associated virus," Proc. Nat'l. Acad. Sci. USA, 94:6916-21,
1997. [0616] Flotte, "Stable in vivo expression of the cystic
fibrosis transmembrane conductance regulator with an
adeno-associated virus vector," Proc. Nat'l. Acad. Sci. USA,
90:10613-10617, 1993. [0617] Flotte and Carter, "Adeno-associated
virus vectors for gene therapy," Gene Ther., 2:357-62, 1995. [0618]
Flotte and Carter, "Adeno-associated virus vectors for gene therapy
of cystic fibrosis," Methods Enzymol., 292:717-32, 1998. [0619]
Flotte and Ferkol, "Genetic therapy. Past, present, and future,"
Pediatr. Clin. North Am., 44:153-78, 1997. [0620] Flotte et al.,
"Adeno-associated virus vector gene expression occurs in
nondividing cells in the absence of vector DNA integration," Am. J.
Respir. Cell Mol. Biol., 11:517-21, 1994. [0621] Flotte et al.,
"Stable in vivo expression of the cystic fibrosis transmembrane
conductance regulator with an adeno-associated virus vector," Proc.
Nat'l. Acad. Sci. USA, 90:10613-17, 1993. [0622] Flotte et al.,
"Efficient ex vivo transduction of pancreatic islet cells with
recombinant adeno-associated virus vectors," Diabetes, 50:515-20,
2001. [0623] Flotte et al., "An improved system for packaging
recombinant adeno-associated virus vectors capable of in vivo
transduction," Gene Ther., 2:29-37, 1995. [0624] Flotte et al., "A
fluorescence video-endoscopy technique for detection of gene
transfer and expression," Gene Ther., 5:166-73, 1998. [0625] Flotte
et al., "A phase I study of an adeno-associated virus-CFTR gene
vector in adult CF patients with mild lung disease," Hum. Gene
Ther., 7:1145-59, 1996. [0626] Flotte et al., "Gene expression from
adeno-associated virus vectors in airway epithelial cells," Am. J.
Respir. Cell Mol. Biol., 7:349-56, 1992. [0627] Forster and Symons,
"Self-cleavage of plus and minus RNAs of a virusoid and a
structural model for the active sites," Cell, 49:211-220, 1987.
[0628] Forsythe et al., "Activation of vascular endothelial growth
factor gene transcription by hypoxia-inducible factor 1," Mol. Cell
Biol., 16:4604-13, 1996. [0629] Fraley et al., "Entrapment of a
bacterial plasmid in phospholipid vesicles: Potential for gene
transfer," Proc. Nat'l. Acad. Sci. USA, 76:3348-52, 1979. [0630]
Frank, "On the pathogenesis of diabetic retinopathy. A 1990
update," Ophthalmology, 98:586-93, 1991. [0631] Franz et al.,
"Transgenic animal models: new avenues in cardiovascular
physiology," J. Mol. Med., 75(2):115-119, 1997. [0632] Fredholm et
al., "Nomenclature and classification of purinoceptors," Pharmacol.
Rev., 46:143-56, 1994. [0633] Fresta and Puglisi, "Application of
liposomes as potential cutaneous drug delivery systems. In vitro
and in vivo investigation with radioactively labeled vesicles," J.
Drug Target, 4:95-101, 1996. [0634] Frohman, In PCR PROTOCOLS: A
GUIDE TO METHODS AND APPLICATIONS, Academic Press, New York, 1990.
[0635] Frohman et al., "Tissue distribution and molecular
heterogeneity of human growth hormone-releasing factor in the
transgenic mouse," Endocrinology, 127(5):2149-2156, 1990. [0636]
Fromm et al., "Expression of genes transferred into monocot and
dicot plant cells by electroporation," Proc. Nat'l. Acad. Sci. USA,
82:5824-28, 1985. [0637] Fry and Wood, "Gene therapy: potential
applications in clinical transplantation," Expert Rev. Mol. Med.,
1999:1-20, 1999. [0638] Fry et al., "The structure and function of
a foot-and-mouth disease virus-oligosaccharide receptor complex,"
EMBO J., 18:543-54, 1999. [0639] Fujita et al., "Lymphocytic
insulitis in a nonobese diabetic (NOD) strain of mice: an
immunohistochemical and electron microscope investigation," Biomed.
Res., 3:429, 1982. [0640] Fukuda et al., "A peptide mimic of
E-selectin ligand inhibits sialyl Lewis X-dependent lung
colonization of tumor cells," Cancer Res., 60:450-56, 2000. [0641]
Gabizon and Papahadjopoulos, "Liposomes formulations with prolonged
circulation time in blood and enhanced uptake by tumors," Proc.
Nat'l. Acad. Sci. USA, 85:6949-53, 1988. [0642] Gade et al.,
"Nitric oxide mediates hyperglycemia-induced defective migration in
cultured endothelial cells," J. Vasc. Surg., 26:319-26, 1997.
[0643] Gallichan et al., "Pancreatic IL-4 expression results in
islet-reactive Th2 cells that inhibit diabetogenic lymphocytes in
the nonobese diabetic mouse," J. Immunol., 1163:1696-703, 1999.
[0644] Gallichan et al., "Lentivirus-mediated transduction of islet
grafts with interleukin 4 results in sustained gene expression and
protection from insulitis," Hum. Gene Ther., 9:2717-26, 1998.
[0645] Gao and Huang, "Cytoplasmic expression of a reporter gene by
co-delivery of T7 RNA polymerase and T7 promoter sequence with
cationic liposomes," Nucl. Acids Res., 21:2867-2872, 1993. [0646]
Gao et al., "Novel adeno-associated viruses from rhesus monkeys as
vectors for human gene therapy," Proc. Nat'l. Acad. Sci. USA,
99:11854-59, 2002. [0647] Gao et al., "High-titer adeno-associated
viral vectors from a Rep/Cap cell line and hybrid shuttle virus,"
Hum. Gene Ther., 9:2353-62, 1998. [0648] Garver et al., "Clonal
gene therapy: transplanted mouse fibroblast clones express human
alpha 1-antitrypsin gene in vivo," Science, 237:762-64, 1987.
[0649] Geboes et al., "Morphological identification of
.alpha.-I-antitrypsin in the human small intestine,"
Histopathology, 6:55-60, 1982. [0650] Gerlach et al., "Construction
of a plant disease resistance gene from the satellite RNA of
tobacco rinspot virus," Nature (London), 328:802-805, 1987. [0651]
Giannoukakis et al., "Targeting autoimmune diabetes with gene
therapy, Diabetes, 48:2107-21, 1999. [0652] Gidday and Park,
"Adenosine-mediated autoregulation of retinal arteriolar tone in
the piglet," Invest. Ophthalmol. Vis. Sci., 34:2713-19, 1993.
[0653] Gidday et al., "KATP channels mediate adenosine-induced
hyperemia in retina," Invest. Ophthalmol. Vis. Sci., 37:2624-33,
1996. [0654] Gille et al., "Transforming growth
factor-alpha-induced transcriptional activation of the vascular
permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA
binding and transactivation," Embo. J., 16:750-59, 1997. [0655]
Gilman, In CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al.
(eds.), John Wiley & Sons, New York, pp. 4.7.1-4.7.8, 1987.
[0656] Giraud et al., "Recombinant junctions formed by
site-specific integration of adeno-associated virus into an
episome," J. Virol., 69:6917-24, 1995. [0657] Giraud et al.,
"Site-specific integration by adeno-associated virus is directed by
a cellular DNA sequence," Proc. Nat'l. Acad. Sci. USA, 91:10039-43,
1994. [0658] Girod et al., "Genetic capsid modifications allow
efficient re-targeting of adeno-associated virus type 2," Nat.
Med., 5:1052-56, 1999. [0659] Gnatenko et al., "Characterization of
recombinant adeno-associated virus-2 as a vehicle for gene delivery
and expression into vascular cells," J. Investig. Med., 45:87-98,
1997. [0660] Go et al., "Interleukin 10, a novel B cell stimulatory
factor: unresponsiveness of X chromosome-linked immunodeficiency B
cells," J. Exp. Med., 172:1625-31, 1990. [0661] Goldstein et al.,
"Nitric oxide: a review of its role in retinal function and
disease," Vision Res., 36:2979-74, 1996. [0662] Gopal, "Gene
transfer method for transient gene expression, stable transfection,
and cotransfection of suspension cell cultures," Mol. Cell Biol.,
5:1188-90, 1985. [0663] Goudy et al., "Elucidation of time and dose
dependencies using AAV-IL-10 gene therapy for prevention of type 1
diabetes in the NOD mouse," Mol. Ther., 5:S17 (Abstr. 46), 2002.
[0664] Goudy et al., "Adeno-associated virus vector-mediated IL-10
gene delivery prevents Type 1 Type I diabetes in NOD mice," Proc.
Nat'l. Acad. Sci. USA, 98:13913-18, 2001. [0665] Graham and van der
Eb, "Transformation of rat cells by DNA of human adenovirus 5,"
Virology, 4:536-39, 1973. [0666] Graham et al., "Characteristics of
a human cell line transformed by DNA from human adenovirus type 5,"
J. Gen. Virol., 36:59-74, 1977. [0667] Grant and King, "IGF-1 and
blood vessels," Diabetes Rev., 3:113-28, 1995. [0668] Grant et al.,
"Inhibition of IGF-1 and .beta.-FGF stimulated growth of human
retinal endothelial cells by the somatostatin analogue, octreotide:
a potential treatment for ocular neovascularization," Regul. Pept.,
48:267-78, 1993b. [0669] Grant et al., "Insulin-like growth
factor-I modulates endothelial cell chemotaxis," J. Clin.
Endocrinol. Metab., 65:370-71, 1987. [0670] Grant et al.,
"Insulin-like growth factor I as an angiogenic agent: in vivo and
in vitro studies," Ann. NY Acad. Sci., 692:230-42, 1993a. [0671]
Grant et al., "Insulin-like growth factors in vitreous: studies in
control and diabetic subjects with neovascularization," Diabetes,
35:416-20, 1986. [0672] Grant et al., "Adenosine receptor
activation induces vascular endothelial growth factor in human
retinal endothelial cells," Circ. Res., 85:699-706, 1999. [0673]
Grant et al., "Adenosine mediates growth factor expression through
A2B adenosine receptor (AdoR) in human retinal endothelial cells
(HREC)," Diabetes, 47(Suppl):A39, 1998. [0674] Graser et al.,
"Identification of a CD8 T cell that can independently mediate
autoimmune diabetes development in the complete absence of CD4 T
cell helper functions," J. Immunol, 164:3913-18, 2000. [0675]
Greelish et al., "Stable restoration of the sarcoglycan complex in
dystrophic muscle perfused with histamine and a recombinant
adeno-associated viral vector," Nat. Med., 5:439-43, 1999. [0676]
Green and Roeder, "Transcripts of the adeno-associated virus
genome: mapping of the major RNAs," J. Virol., 36:79-92, 1980.
[0677] Grifman et al., "Incorporation of tumor-targeting peptides
into recombinant adeno-associated virus capsids," Mol. Ther.,
3:964-75, 2001. [0678] Grimm et al., "Titration of AAV-2 particles
via a novel capsid ELISA: packaging of genomes can limit production
of recombinant AAV-2," Gene Ther., 6:1322-30, 1999. [0679] Grimm et
al., "Novel tools for production and purification of recombinant
adenoassociated virus vectors," Hum. Gene Ther., 9:2745-60, 1998.
[0680] Grupping et al., "Low density lipoprotein binding and uptake
by human and rat islet cells," Endocrinology, 138:4064-68, 1997.
[0681] Guan et al., "Characterization of the mouse von Willebrand
factor promoter," Blood, 94:3405-12, 1999. [0682] Guenette et al.,
"Cathepsin B, a cysteine protease implicated in metastatic
progression, is also expressed during regression of the rat
prostate and mammary glands," Eur. J. Biochem., 226:311-21, 1994.
[0683] Guerrier-Takada et al., "The RNA moiety of ribonuclease P is
the catalytic subunit of the enzyme," Cell, 35:849, 1983. [0684]
Guo et al., "In vivo effects of leflunomide on normal pancreatic
islet and syngeneic islet graft function," Transplantation,
63:716-21, 1997. [0685] Guy et al., "Adeno-associated
viral-mediated catalase expression suppresses optic neuritis in
experimental allergic encephalomyelitis," Proc. Nat'l Acad. Sci.
USA, 95:13847-13852, 1998. [0686] Guy et al., "Reporter expression
persists 1 year after adeno-associated virus-mediated gene transfer
to the optic nerve," Arch. Ophthalmol., 117:929-37, 1999. [0687]
Hahn et al., "Toxic effects of cyclosporine on the endocrine
pancreas of Wistar rats," Transplantation, 41:44-47, 1986. [0688]
Halbert et al., "Successful readministration of adeno-associated
virus vectors to the mouse lung requires transient
immunosuppression during the initial exposure," J. Virol.,
72:9795-805, 1998. [0689] Hampel and Tritz, "RNA catalytic
properties of the minimum (-)s TRSV sequence," Biochem., 28:4929,
1989. [0690] Hampel et al., "Hairpin` catalytic RNA model: evidence
for helices and sequence requirement for substrate RNA," Nucl.
Acids Res., 18:299, 1990. [0691] Handa et al., "Adeno-associated
virus (AAV)-3-based vectors transduce haematopoietic cells not
susceptible to transduction with AAV-2-based vectors," J. Gen.
Virol., 81:2077-84, 2000. [0692] Handa et al., "Establishment and
characterization of KB cell lines latently infected with
adeno-associated virus type 1," Virology, 82:84-92, 1977. [0693]
Hangai et al., "Inducible nitric oxide synthase in retinal
ischemia-reperfusion injury," Exp. Eye Res., 63:501-09, 1996.
[0694] Harland and Weintraub, "Translation of mammalian mRNA
injected into Xenopus oocytes is specifically inhibited by
antisense RNA," J. Cell Biol., 101:1094-99, 1985. [0695] Hashimoto
et al., "Adenosine as an endogenous mediator of hypoxia for
induction of vascular endothelial growth factor mRNA in U-937
cells," Biochem. Biophys. Res. Commun., 204:318-24, 1994. [0696]
Haskell and Bowen, "Efficient production of transgenic cattle by
retroviral infection of early embryos," Mol. Reprod. Dev.,
40(3):386-390, 1995. [0697] Haskins et al., "T-lymphocyte clone
specific for pancreatic islet antigen," Diabetes, 37:1444-48, 1988.
[0698] Hauswirth et al., "Production and purification of
recombinant adeno-associated virus," Methods Enzymol., 316:743-61,
2000. [0699] Hauswirth et al., "Production and purification of
recombinant AAV vectors," In: VERTEBRATE PHOTOTRANSDUCTION AND THE
VISUAL CYCLE. METHODS IN ENZYMOLOGY 316, Palczewski (ed.), New
York, Academic Press, in press, 2000. [0700] Heath and Martin, "The
development and application of protein-liposome conjugation
techniques," Chem. Phys. Lipids, 40:347-58, 1986. [0701] Heath et
al., "Liposome-mediated delivery of pteridine antifolates to cells:
in vitro potency of methotrexate and its alpha and gamma
substituents," Biochim. Biophys. Acta, 862:72-80, 1986. [0702]
Heilbronn et al., "The adeno-associated virus rep gene suppresses
herpes simplex virus-induced DNA amplification," J. Virol.,
64:3012-18, 1990. [0703] Hemsley et al., "A simple method for
site-directed mutagenesis using the polymerase chain reaction,"
Nucleic Acids Res., 17:6545-51, 1989. [0704] Henry-Michelland et
al., "Attachment of antibiotics to nanoparticles; Preparation,
drug-release and antimicrobial activity in vitro," Int. J. Pharm.,
35:121-27, 1987. [0705] Hering et al., "Clinical islet
transplantation--registry report, accomplishments in the past and
future research needs," Cell Transplant., 2:269-82, discussion
283-305, 1993. [0706] Hermens et al., "Purification of recombinant
adeno-associated virus by iodixanol gradient ultracentrifugation
allows rapid and reproducible preparation of vector stocks for gene
transfer in the nervous system," Hum. Gene Ther., 10:1885-91, 1999.
[0707] Hermonat and Muzyczka, "Use of adeno-associated virus as a
mammalian DNA cloning vector: transduction of neomycin resistance
into mammalian tissue culture cells," Proc. Nat'l. Acad. Sci. USA,
81:6466-70, 1984. [0708] Hermonat et al., "Genetics of
adeno-associated virus: isolation and preliminary characterization
of adeno-associated virus type 2 mutants," J. Virol., 51:329-39,
1984. [0709] Hernandez et al., "Latent adeno-associated virus
infection elicits humoral but not cell-mediated immune responses in
a nonhuman primate model," J. Virol., 73:8549-58, 1999. [0710]
Hertel et al., "A kinetic and thermodynamic framework for the
hammerhead ribozyme reaction," Biochemistry, 33:3374-3385, 1994.
[0711] Herzog et al., "Stable gene transfer and expression of human
blood coagulation factor IX after intramuscular injection of
recombinant adeno-associated virus," Proc. Nat'l. Acad. Sci. USA,
94:5804-09, 1997. [0712] Hey et al., "Cloning of a novel member of
the low-density lipoprotein receptor family," Gene, 216:103-11,
1998. [0713] Hileman et al., "Glycosaminoglycan-protein
interactions: definition of consensus sites in glycosaminoglycan
binding proteins," Bioessays, 20:156-67, 1998. [0714] Hirano et
al., "Morphological and functional changes of islets of Langerhans
in FK506-treated rats," Transplantation, 53:889-94, 1992. [0715]
Hirano et al., "Expression of human scavenger receptor class B type
I in cultured human monocyte-derived macrophages and
atherosclerotic lesions," Circ. Res., 85:108-16, 1999. [0716] Hirt,
"Selective extraction of polyoma DNA from infected mouse cell
cultures," J. Mol. Biol., 26:365-69, 1967. [0717] Hoggan, "Presence
of small virus-like particles in various adenovirus type 2, 5, 7,
and 12 preparations," Fed. Proc., 24:248, 1965. [0718] Hoggan et
al. In PROCEEDING OF THE FOURTH LEPETIT COLLOQUIUM, Cacoyac,
Mexico, North Holland, Amsterdam, pp. 243-249, 1972. [0719] Hoggan
et al., "Studies of small DNA viruses found in various adenovirus
preparations: physical, biological, and immunological
characteristics," Proc. Nat'l. Acad. Sci. USA, 55:1467-74, 1966.
[0720] Hoggan et al., "Helper-dependent infectious deoxyribonucleic
acid from adenovirus-associated virus," J. Virol., 2:850-51, 1968.
[0721] Holzknecht and Platt, "The fine cytokine line between graft
acceptance and rejection," Nat. Med., 6:497-98, 2000. [0722] Hoover
et al., Eds., In REMINGTON'S PHARMACEUTICAL SCIENCES, 16.sup.th
Edition, Mack Publishing Co., Easton, Pa., 1980.
[0723] Hogue et al., "Nuclear transport of the major capsid protein
is essential for adeno-associated virus capsid formation," J.
Virol., 73:7912-15, 1999. [0724] Hsu et al., "Expression of
interleukin-10 activity by Epstein-Barr virus protein BCRF1,"
Science, 250:830-32, 1990. [0725] Huang and Hearing, "Adenovirus
early region 4 encodes two gene products with redundant effects in
lytic infection," J. Virol., 63:2605-15, 1989. [0726] Hussain et
al., "The mammalian low-density lipoprotein receptor family," Annu.
Rev. Nutr., 19:141-72, 1999. [0727] Hwang et al., "Gastric
retentive drug-delivery systems," Crit. Rev. Ther. Drug Carrier
Syst., 15(3):243-284, 1998. [0728] Hyer et al., "A two-year
follow-up study of serum insulin-like growth factor-I in diabetics
with retinopathy," Metabolism, 38:586-89, 1989. [0729] Im and
Muzyczka, "The AAV origin binding protein Rep68 is an ATP-dependent
site-specific endonuclease with DNA helicase activity," Cell,
61:447-57, 1990. [0730] Imaizumi et al., "Liposome-entrapped
superoxide dismutase reduces cerebral infarction in cerebral
ischemia in rats," Stroke, 21:1312-17, 1990a. [0731] Imaizumi et
al., "Liposome-entrapped superoxide dismutase ameliorates infarct
volume in focal cerebral ischemia,"Acta. Neurochir. Suppl.,
51:236-238, 1990b. [0732] Inoue and Russell, "Packaging cells based
on inducible gene amplification for the production of
adeno-associated virus vectors," J. Virol., 72:7024-31, 1998.
[0733] Ishii et al., "cDNA cloning of a new low-density lipoprotein
receptor-related protein and mapping of its gene (LRP3) to
chromosome bands 19q12-q13.2," Genomics, 51:132-35, 1998. [0734]
Jacobsen et al., "Molecular characterization of a novel human
hybrid-type receptor that binds the alpha2-macroglobulin
receptor-associated protein," J. Biol. Chem., 271:31379-83, 1996.
[0735] Jacobson et al., "Retinal degenerations with truncation
mutations in the cone-rod homeobox (CRX) gene," Invest. Ophthalmol.
Vis. Sci., 39:2417-2426, 1998. [0736] Jaeger et al., "Improved
predictions of secondary structures for RNA," Proc. Natl. Acad.
Sci. USA, 86:7706-10, 1989. [0737] Jager et al., "Endothelial
cell-specific transcriptional targeting from a hybrid long terminal
repeat retrovirus vector containing human prepro-endothelin-1
promoter sequences," J. Virol., 73:9702-09, 1999. [0738] Jaggar et
al., "Endothelial cell-specific expression of tumor necrosis
factor-.alpha. from the KDR or E-selectin promoters following
retroviral delivery," Hum. Gene Ther., 8:2239-47, 1997. [0739]
Janciauskiene, "Conformational properties of serine proteinase
inhibitors (serpins) confer multiple pathophysiological roles,"
Biochim. Biophys. Acta, 1535:221-35, 2001. [0740] Jindal,
"Post-transplant diabetes mellitus--a review," Transplantation,
58:1289-98, 1994. [0741] Johansson et al., "Alpha-1-antitrypsin is
present in the specific granules of human eosinophilic
granulocytes," Clin. Exp. Allergy, 31:379-86, 2001. [0742] Johnson
and Curtis, "Preventive therapy for periodontal diseases," Adv.
Dent. Res., 8:337-48, 1994. [0743] Johnson et al., "Cytotoxicity of
a replication-defective mutant of herpes simplex virus type 1," J.
Virol., 66:2952-65, 1992a. [0744] Johnson et al., "Efficiency of
gene transfer for restoration of normal airway epithelial function
in cystic fibrosis," Nat. Genet., 2:21-25, 1992b. [0745] Johnson et
al., "Improved cell survival by the reduction of immediate-early
gene expression in the replication-defective mutants of herpes
simplex virus type 1 but not by mutation of the virion host shutoff
function," J. Virol., 68:6347-62, 1994. [0746] Johnston et al.,
"HSV/AAV hybrid amplicon vectors extend transgene expression in
human glioma cells," Hum. Gene Ther., 8:359-70, 1997. [0747] Jones
et al., "Improved methods for binding protein models in electron
density maps and the location of errors in these models," Acta.
Crystallograph. A, 47:110-19, 1991. [0748] Jooss et al.,
"Transduction of dendritic cells by DNA viral vectors directs the
immune response to transgene products in muscle fibers," J. Virol.,
727:4212-23, 1998. [0749] Joslin et al., "The SEC receptor
recognizes a pentapeptide neodomain of alpha 1-antitrypsin-protease
complexes," J. Biol. Chem., 266:11282-88, 1991. [0750] Joyce, "RNA
evolution and the origins of life," Nature, 338:217-244, 1989.
[0751] Kaludov et al., "Adeno-associated virus serotype 4 (AAV4)
and AAV5 both require sialic acid binding for hemagglutination and
efficient transduction but differ in sialic acid linkage
specificity," J. Virol., 75:6884-93, 2001. [0752] Kang, et al.,
"Up-regulation of luciferase gene expression with antisense
oligonucleotides: implications and applications in functional assay
development," Biochemistry, 37(18):6235-9, 1998. [0753] Kaplitt et
al., "Long-term gene expression and phenotypic correction using
adeno-associated virus vectors in the mammalian brain," Nat.
Genet., 8:148-54, 1994. [0754] Kapturczak et al., "Adeno-associated
virus (AAV) as a vehicle for therapeutic gene delivery:
improvements in vector design and viral production enhance
potential to prolong graft survival in pancreatic islet cell
transplantation for the reversal of type 1 diabetes," Curr. Mol.
Med., 1:245-58, 2001. [0755] Kashani-Sabet et al., "Reversal of the
malignant phenotype by an anti-ras ribozyme," Antisense Res. Dev.,
2:3-15, 1992. [0756] Kaufman et al., "Differential roles of
Mac-1+cells, and CD4+and CD8+T lymphocytes in primary nonfunction
and classic rejection of islet allografts," J. Exp. Med.,
172:291-302, 1990. [0757] Kay et al., "Evidence for gene transfer
and expression of factor IX in haemophilia B patients treated with
an AAV vector," Nat. Genet., 24:257-261, 2000. [0758] Kearns et
al., "Recombinant adeno-associated virus (AAV-CFTR) vectors do not
integrate in a site-specific fashion in an immortalized epithelial
cell line," Gene Ther., 3:748-55, 1996. [0759] Kenyon et al.,
"Islet cell transplantation: beyond the paradigms," Diabetes Metab.
Rev., 12:361-72, 1996. [0760] Kenyon et al., "Islet
transplantation: present and future perspectives," Diabetes Metab.
Rev., 14:303-13, 1998. [0761] Keppler et al., "Human colon
carcinoma cells synthesize and secrete a 1-proteinase inhibitor,"
Biol. Chem. Hoppe-Seyler, 377:301-11, 1996. [0762] Kessler et al.,
"Gene delivery to skeletal muscle results in sustained expression
and systemic delivery of a therapeutic protein," Proc. Nat'l. Acad.
Sci. USA, 93:14082-87, 1996. [0763] Khleif et al., "Inhibition of
cellular transformation by the adeno-associated virus rep gene,"
Virology, 181:738-41, 1991. [0764] Kief and Warner, "Coordinate
control of syntheses of ribosomal ribonucleic acid and ribosomal
proteins during nutritional shift-up in Saccharomyces cerevisiae,"
Mol. Cell Biol., 1:1007-1015, 1981. [0765] Kim and Cech,
"Three-dimensional model of the active site of the self-splicing
rRNA precursor of Tetrahymena," Proc. Nat'l. Acad. Sci. USA
84:8788-8792, 1987. [0766] Kimura et al., "Hypoxia response element
of the human vascular endothelial growth factor gene mediates
transcriptional regulation by nitric oxide: control of
hypoxia-inducible factor-1 activity by nitric oxide," Blood,
95:189-97, 2000. [0767] King et al., "DNA helicase-mediated
packaging of adeno-associated virus type 2 genomes into preformed
capsids," EMBO J., 20:3282-91, 2001. [0768] King et al., "Receptors
and growth-promoting effects of insulin and insulin-like growth
factors on cells from bovine retinal capillaries and aorta," J.
Clin. Invest., 75:1028-36, 1985. [0769] Klein et al.,
"Neuron-specific transduction in the rat septohippocampal or
nigrostriatal pathway by recombinant adeno-associated virus
vectors," Exper. Neurol. 150:183-94, 1998. [0770] Klein et al.,
"High-velocity microprojectiles for delivering nucleic acids into
living cells. 1987," Biotechnology, 24:384-386, 1992. [0771] Knipe,
"The role of viral and cellular nuclear proteins in herpes simplex
virus replication," Adv. Virus Res., 37:85-123, 1989. [0772] Knipe
et al., "Characterization of two conformational forms of the major
DNA-binding protein encoded by herpes simplex virus 1," J. Virol.,
44:736-41, 1982. [0773] Knoell et al., "Alpha 1-antitrypsin and
protease complexation is induced by lipopolysaccharide,
interleukin-10, and tumor necrosis factor-alpha in monocytes," Am.
J. Respir. Crit. Care Med., 157:246-55, 1998. [0774] Koeberl et
al., "Persistent expression of human clotting factor IX from mouse
liver after intravenous injection of adeno-associated virus
vectors," Proc. Nat'l Acad. Sci. USA, 94:1426-1431, 1997. [0775]
Kohner and Oakley, "Diabetic retinopathy," Metabolism, 24:1085-102,
1975. [0776] Koizumi et al., "Ribozymes designed to inhibit
transformation of NIH3T3 cells by the activated c-Ha-ras gene,"
Gene, 117:179-84, 1992. [0777] Kolaczynski and Caro, "Insulin-like
growth factor-1 therapy in diabetes: physiologic basis, clinical
benefits, and risks," Ann. Intern. Med., 120:47-55, 1994. [0778]
Korhonen et al., "Endothelial-specific gene expression directed by
the tie gene promoter in vivo," Blood, 86:1828-35, 1995. [0779]
Kotin, "Prospects for the use of adeno-associated virus as a vector
for human gene therapy," Hum. Gene Ther., 5:793-801, 1994. [0780]
Kotin and Berns, "Organization of adeno-associated virus DNA in
latently infected Detroit 6 cells," Virology, 170:460-67, 1989.
[0781] Kotin et al., "Characterization of a preferred site on human
chromosome 19q for integration of adeno-associated virus DNA by
non-homologous recombination," EMBO Journal, 11:5071-78, 1992.
[0782] Kotin et al., "Mapping and direct visualization of a
region-specific viral DNA integration site on chromosome
19q13-qter," Genomics, 10:831-34, 1991. [0783] Kotin et al.,
"Site-specific integration by adeno-associated virus," Proc. Nat'l.
Acad. Sci. USA, 87:2211-15, 1990. [0784] Kraulis, "MOLSCRIPT: a
program to produce both detailed and schematic plots of protein
structures," J. Appl. Cryst., 24:946-50, 1991. [0785] Kroemer et
al., "Differential involvement of Th1 and Th2 cytokines in
autoimmune diseases," Autoimmunity, 24:25-33, 1996. [0786]
Kronenberg et al., "Electron cryo-microscopy and image
reconstruction of adeno-associated virus type 2 empty capsids,"
EMBO Rep., 2:997-1002, 2001. [0787] Kuby, In IMMUNOLOGY, 2nd
Edition. W.H. Freeman & Company, New York, 1994. [0788]
Kvietikova et al., "The transcription factors ATF-1 and CREB-1 bind
constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA
recognition site," Nucleic Acids Res., 23:4542-50, 1995. [0789]
Kwoh et al., "Transcription-based amplification system and
detection of amplified human immunodeficiency virus type 1 with a
bead-based sandwich hybridization format," Proc. Natl. Acad. Sci.
USA, 86(4):1173-1177, 1989. [0790] Kyte and Doolittle, "A simple
method for displaying the hydropathic character of a protein," J.
Mol. Biol., 157:105-32, 1982. [0791] L'Huillier et al.,
"Cytoplasmic delivery of ribozymes leads to efficient reduction in
alpha-lactalbumin mRNA levels in C1271 mouse cells," EMBO J.,
11(12):4411-4418, 1992. [0792] LaFace and Peck, "Reciprocal
allogeneic bone marrow transplantation between NOD mice and
diabetes-nonsusceptible mice associated with transfer and
prevention of autoimmune diabetes," Diabetes, 38:894-901, 1989.
[0793] Lam and Tso, "Nitric oxide synthase (NOS) inhibitors
ameliorate retinal damage induced by ischemia in rats," Res.
Commun. Mol. Pathol. Pharmacol., 92:329-40, 1996. [0794] Langford
and Miell, "The insulin-like growth factor-I binding protein axis:
physiology, pathophysiology and therapeutic manipulation," Eur. J.
Clin. Invest., 23:503-16, 1993. [0795] Lasic, "Novel applications
of liposomes," Trends Biotechnol., 16:307-21, 1998. [0796] Laughlin
et al., "Defective-interfering particles of the human parvovirus
adeno-associated virus," Virology, 94:162-74, 1979. [0797] Laughlin
et al., "Cloning of infectious adeno-associated virus genomes in
bacterial plasmids," Gene, 23:65-73, 1983. [0798] Lem et al.,
"Tissue-specific and developmental regulation of rod opsin chimeric
genes in transgenic mice," Neuron, 6(2):201-10, 1991. [0799] Lem et
al., "Retinal degeneration is rescued in transgenic rd mice by
expression of the cGMP phosphodiesterase beta subunit," Proc. Nat'l
Acad. Sci. USA, 89:4422-4426, 1992. [0800] Lewin et al., "Ribozyme
rescue of photoreceptor cells in a transgenic rat model of
autosomal dominant retinitis pigmentosa," Nat. Med., 4:967-971,
1998. [0801] Li et al., "Synthetic muscle promoters: activities
exceeding naturally occurring regulatory sequences," Nat.
Biotechnol., 17:241-45, 1999. [0802] Li et al., "Role for highly
regulated rep gene expression in adeno-associated virus vector
production," J. Virol., 71:5236-43, 1997. [0803] Liblau et al.,
"Th1 and Th2CD4+T-cells in the pathogenesis of organ specific
autoimmune diseases," Immunology Today, 16:34-38, 1995. [0804]
Lieber et al., "Stable high-level gene expression in mammalian
cells by T7 phage RNA polymerase," Methods Enzymol., 217:47-66,
1993. [0805] Like and Rossini, "Streptozotocin-induced pancreatic
insulitis: new model of diabetes mellitus," Science, 193:415-17,
1976. [0806] Like et al., "Prevention of diabetes in
BioBreeding/Worcester rats with monoclonal antibodies that
recognize T lymphocytes or natural killer cells," J. Exp. Med.,
164:1145-59, 1986. [0807] Limb et al., "Distribution of TNF alpha
and its reactive vascular adhesion molecules in fibrovascular
membranes of proliferative diabetic retinopathy," Br. J.
Ophthalmol., 80:168-73, 1996. [0808] Linden and Woo, "AAVant-garde
gene therapy," Nat. Med., 5:21-22, 1999. [0809] Linden et al.,
"Site-specific integration by adeno-associated virus," Proc. Nat'l.
Acad. Sci. USA, 93:11288-94, 1996. [0810] Linetsky et al.,
"Improved human islet isolation using a new enzyme blend,
liberase," Diabetes, 46:1120-23, 1997. [0811] Linetsky et al.,
"Endotoxin contamination of reagents used during isolation and
purification of human pancreatic islets," Transplant Proc.,
30:345-46, 1998. [0812] Liptak et al., "Functional order of
assembly of herpes simplex virus DNA replication proteins into
prereplicative site structures," J. Virol., 70:1759-67, 1996.
[0813] Lisziewicz et al., "Inhibition of human immunodeficiency
virus type 1 replication by regulated expression of a polymeric Tat
activation response RNA decoy as a strategy for gene therapy in
AIDS," Proc. Nat'l. Acad. Sci. USA, 90:8000-8004, 1993. [0814]
Little and Lee, "Generation of a mammalian cell line deficient in
glucose-regulated protein stress induction through targeted
ribozyme driven by a stress-inducible promoter," J. Biol. Chem.,
270:9526-34, 1995. [0815] Liu and Thorp, "Cell surface heparan
sulfate and its roles in assisting viral infections," Med. Res.
Rev., 22:1-25, 2002. [0816] Loeb et al., "Enhanced expression of
transgenes from adeno-associated virus vectors with the woodchuck
hepatitis virus posttranscriptional regulatory element:
implications for gene therapy,
" Hum. Gene Ther., 10:2295-305, 1999. [0817] Lopez-Berestein et
al., "Liposomal amphotericin B for the treatment of systemic fungal
infections in patients with cancer: a preliminary study," J.
Infect. Dis., 2151:704, 1985a. [0818] Lopez-Berestein et al.,
"Protective effect of liposomal-amphotericin B against C. albicans
infection in mice," Cancer Drug Delivery, 2:183, 1985b. [0819] Lu
et al., "High efficiency retroviral mediated gene transducion into
single isolated immature and replatable CD34.sup.3+ hematopoietic
stem/progenitor cells from human umbilical cord blood," J. Exp.
Med., 178(6):2089-2096, 1993. [0820] Lukonis and Weller,
"Characterization of nuclear structures in cells infected with
herpes simplex virus type 1 in the absence of viral DNA
replication," J. Virol., 70:1751-58, 1996. [0821] Luo et al.,
"Noninflammatory expression of E-selectin is regulated by cell
growth," Blood, 93:3785-91, 1999. [0822] Lusby and Berns, "Mapping
of the 5' termini of two adeno-associated virus 2 RNAs in the left
half of the genome," J. Virol., 41:518-26, 1982. [0823] Lusby et
al., "Nucleotide sequence of the inverted terminal repetition in
adeno-associated virus DNA," J. Virol., 34:402-09, 1980. [0824]
Lutty et al., "Adenosine stimulates canine retinal microvascular
endothelial cell migration and tube formation," Curr. Eye Res.,
17:594-607, 1998. [0825] Lutty et al., "5' nucleotidase and
adenosine during retinal vasculogenesis and oxygen-induced
retinopathy," Invest. Ophthalmol. Vis. Sci., 41:218-29, 2000.
[0826] Lynch et al., "Adeno-associated virus vectors for vascular
gene delivery," Circ. Res., 80:497-505, 1997. [0827] Macen et al.,
"SERP1, a serine proteinase inhibitor encoded by myxoma virus, is a
secreted glycoprotein that interferes with inflammation," Virology,
195:348-63, 1993. [0828] MacNeil et al., "IL-10, a novel growth
cofactor for mature and immature T cells," J. Immunol.,
145:4167-73, 1990. [0829] Maloy et al., In MICROBIAL GENETICS, 2nd
Edition, Jones and Barlett Publishers, Boston, Mass., 1994. [0830]
Mandel et al., "Midbrain injection of recombinant adeno-associated
virus encoding rat glial cell line-derived neurotrophic factor
protects nigral neurons in a progressive 6-hydroxydopamine-induced
degeneration model of Parkinson's disease in rats," Proc. Nat'l.
Acad. Sci. USA, 94:14083-88, 1997. [0831] Maniatis et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, 1982. [0832] Marcus et al.,
"Adeno-associated virus RNA transcription in vivo," Eur. J.
Biochem., 121:147-54, 1981. [0833] Margalit, "Liposome-mediated
drug targeting in topical and regional therapies," Crit. Rev. Ther.
Drug Carrier Syst., 12(2-3):233-261, 1995. [0834] Massetti et al.,
"Current indications and limits of pancreatic islet transplantation
in diabetic nephropathy," J. Nephrol., 10:245-2521, 1997. [0835]
Mathiowitz et al., "Biologically erodable microspheres as potential
oral drug delivery systems," Nature, 386(6623):410-414, 1997.
[0836] Matsushita et al., "Adeno-associated virus vectors can be
efficiently produced without helper virus," Gene Ther., 5:938-45,
1998. [0837] McAuthor and Raulet, "CD28-induced costimulation of T
helper type 2 cells mediated by induction of responsiveness to
interleukin 4," J. Exp. Med., 178:1645, 1993. [0838] McCarthy et
al., "Herpes simplex virus type 1 ICP27 deletion mutants exhibit
altered patterns of transcription and are DNA deficient," J.
Virol., 63:18-27, 1989. [0839] McCarty et al., "Sequences required
for coordinate induction of adeno-associated virus p19 and p40
promoters by Rep protein," J. Virol., 65:2936-45, 1991. [0840]
McCown et al., "Differential and persistent expression patterns of
CNS gene transfer by an adeno-associated virus (AAV) vector," Brain
Res., 713:99-107, 1996. [0841] McKenna et al., "Three-dimensional
structure of Aleutian mink disease parvovirus: implications for
disease pathogenicity," J. Virol., 73:6882-91, 1999. [0842]
McLauchlan et al., "Herpes simplex virus 1E63 acts at the
posttranscriptional level to stimulate viral mRNA 3' processing,"
J. Virol., 66:6939-45, 1992. [0843] McLaughlin et al.,
"Adeno-associated virus general transduction vectors: analysis of
proviral structures," J. Virol., 62:1963-73, 1988. [0844] Merimee
et al., "Insulin-like growth factors: studies in diabetics with and
without retinopathy," N. Engl. J. Med., 309:527-30, 1983. [0845]
Merritt and Bacon, "Raster3D Photorealistic Molecular Graphics," p.
505-24, METHODS IN ENZYMOLOGY, Vol. 277, 1997. [0846]
Meyer-Schwickerath et al., "Vitreous levels of the insulin-like
growth factors I and II, and the insulin-like growth factor binding
proteins 2 and 3, increase in neovascular eye disease. Studies in
nondiabetic and diabetic subjects," J. Clin. Invest., 92:2620-25,
1993. [0847] Miao et al., "The kinetics of rAAV integration in the
liver [letter]," Nat. Genet., 19:13-15, 1998. [0848] Michel and
Westhof, "Modeling of the three-dimensional architecture of group I
catalytic introns based on comparative sequence analysis," J. Mol.
Biol., 216:585-610, 1990. [0849] Mietus-Snyder et al.,
"Transcriptional activation of scavenger receptor expression in
human smooth muscle cells requires AP-1/c-Jun and C/EB113: both
AP-1 binding and INK activation are induced by phorbol esters and
oxidative stress," Arterioscler. Thromb. Vasc. Biol., 18:1440-49,
1998. [0850] Miller et al., "Both the Lyt-.sup.2+ and L3T.sup.4+ T
cell subsets are required for the transfer of diabetes in nonobese
diabetic mice," J. Immunol., 140:52-58, 1988. [0851] Minet et al.,
"ERK activation upon hypoxia: involvement in HIF-1 activation,"
FEBS Lett., 468:53-58, 2000. [0852] Mishra and Rose,
"Adeno-associated virus DNA replication is induced by genes that
are essential for HSV-1 DNA synthesis," Virology, 179:632-39, 1990.
[0853] Mitchell and Tjian, "Transcriptional regulation in mammalian
cells by sequence-specific DNA binding proteins," Science,
245:371-78, 1989. [0854] Miyamoto et al., "Novel functions of human
a(1)-protease inhibitor after S-nitrosylation: inhibition of
cysteine protease and antibacterial activity," Biochem. Biophys.
Res. Commun., 267:918-23, 2000. [0855] Mizutani et al.,
"Accelerated death of retinal microvascular cells in human and
experimental diabetic retinopathy," J. Clin. Invest., 97:2883-90,
1996. [0856] Monahan et al., "Direct intramuscular injection with
recombinant AAV vectors results in sustained expression in a dog
model of hemophilia," Gene Ther., 5:40-49, 1998. [0857] Moore et
al., "Homology of cytokine synthesis inhibitory factor (IL-10) to
the Epstein-Barr virus gene BCRFI," Science, 248:1230-34, 1990.
[0858] Morabito et al., "Characterization of developmentally
regulated and retina-specific nuclear protein binding to a site in
the upstream region of the rat opsin gene," J. Biol. Chem.,
266:9667-72, 1991. [0859] Mori and Fukatsu, "Anticonvulsant effect
of DN-1417a derivative of thyrotropin-releasing hormone and
liposome-entrapped DN-1417 on amygdaloid-kindled rats," Epilepsia,
33:994-1000, 1992. [0860] Moritani et al., "Transgenic expression
of IL-10 in pancreatic islet A cells accelerates autoimmune
insulitis and diabetes in non-obese diabetic mice," Int. Immunol.,
6:1927-36, 1994. [0861] Morris, "Spatial localization does not
require the presence of local cues," Learn. Motiv., 12(2):239-260,
1981. [0862] Morris et al., "Ibotenate lesions of hippocampus
and/or subiculum: dissociating components of allocentric spatial
learning," Eur. J. Neurosci., 2:1016, 1990. [0863] Morwald et al.,
"A novel mosaic protein containing LDL receptor elements is highly
conserved in humans and chickens," Arterioscler. Thromb. Vasc.
Biol., 17:996-1002 (1997). [0864] Moskalenko et al., "Epitope
mapping of human anti-adeno-associated virus type 2 neutralizing
antibodies: implications for gene therapy and virus structure," J.
Virol., 74:1761-66, 2000. [0865] Mueller et al., "Pancreatic
expression of interleukin-4 abrogates insulitis and autoimmune
diabetes I nonobese diabetic (NOD) mice," J. Exp. Med.,
184:1093-99, 1996. [0866] Mukai et al., "G protein antagonists. A
novel hydrophobic peptide competes with receptor for G protein
binding," J. Biol. Chem., 267:16237-43, 1992. [0867] Muller et al.,
"Efficient transfection and expression of heterologous genes in
PC12 cells," Cell Biol., 9:221-29, 1990. [0868] Mulloy and
Linhardt, "Order out of complexity--protein structures that
interact with heparin," Curr. Opin. Struct. Biol., 11:623-28, 2001.
[0869] Muralidhar et al., "Site-directed mutagenesis of
adeno-associated virus type 2 structural protein initiation codons:
effects on regulation of synthesis and biological activity," J.
Virol., 68:170-76, 1994. [0870] Murphy et al., "Long-term
correction of obestity and diabetes in genetically obese mice by a
single intramuscular injection of recombinant adeno-associated
virus encoding mouse leptin," Proc. Nat'l. Acad. Sci. USA,
94:13921-26, 1997. [0871] Muzyczka and Berns, "Parvoviridae: The
viruses and their replication," p. 2327-2360, In FIELDS VIROLOGY,
Fourth ed., P. M. Howley (ed.), Lippincott Williams and Wilkins,
New York, 2001. [0872] Muzyczka and McLaughlin, "Use of
adeno-associated virus as a mamalian transduction vector," In
CURRENT COMMUNICATIONS IN MOLECULAR BIOLOGY: VIRAL VECTORS, Glzman
and Hughes (eds.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., pp. 39-44, 1988. [0873] Muzyczka, "Use of
adeno-associated virus as a general transduction vector for
mammalian cells," Curr. Top Microbiol. Immunol., 158:97-129, 1992.
[0874] Muzyczka et al., "The genetics of adeno-associated virus,"
Adv. Exp. Med. Biol., 179:151-61, 1984. [0875] Nakai et al.,
"Adeno-associated viral vector-mediated gene transfer of human
blood coagulation factor IX into mouse liver," Blood, 91:4600-4607,
1998. [0876] Nakai et al., "Isolation of recombinant
adeno-associated virus vector-cellular DNA junctions from mouse
liver," J. Virol., 73:5438-5447, 1999. [0877] Nathans et al.,
"Molecular genetics of human color vision: the genes encoding blue,
green, and red pigments," Science, 232:193-202, 1986. [0878] Nees
et al., "The coronary endothelium: a highly active metabolic
barrier for adenosine," Basic Res. Cardiol., 80:515-29, 1985.
[0879] Nettelbeck et al., "A strategy for enhancing the
transcriptional activity of weak cell type-specific promoters,"
Gene Ther., 5:1656-64, 1998. [0880] Nettelbeck, Jr. and Muller, "A
dual specificity promoter system combining cell cycle-regulated and
tissue-specific transcriptional control," Gene Ther., 6:1276-81,
1999. [0881] Ni et al., "In vitro replication of adeno-associated
virus DNA," J. Virol., 68:1128-38, 1994. [0882] Nicholls et al.,
"Protein folding and association: insights from the interfacial and
thermodynamic properties of hydrocarbons," PROTEINS, Structure,
Function and Genetics, 11:281-296, 1991. [0883] Nickerson et al.,
"Cytokines and the Th1/Th2 paradigm in transplantation," Curr.
Opin. Immunol., 6:757-64, 1994. [0884] Nicklin et al., "Efficient
and selective AAV2-mediated gene transfer directed to human
vascular endothelial cells," Mol. Ther., 4:174-81, 2001. [0885]
Nicolau and Gersonde, "Incorporation of inositol hexaphosphate into
intact red blood cells, I. fusion of effector-containing lipid
vesicles with erythrocytes," Naturwissenschaften (Germany),
66:563-66, 1979. [0886] Nicolau and Sene, "Liposome-mediated DNA
transfer in eukaryotic cells," Biochem. Biophys. Acta, 721:185-90,
1982. [0887] Niemann et al., "Binding of SPAAT, the 44-residue
C-terminal peptide of alpha 1-antitrypsin, to proteins of the
extracellular matrix," J. Cell Biochem., 66:346-57, 1997. [0888]
Nitta et al., "Systemic delivery of interleukin 10 by intramuscular
injection of expression plasmid DNA prevents autoimmune diabetes in
nonobese diabetic mice," Hum. Gene Ther., 9:1701-07, 1998. [0889]
Nussler et al., "Hepatic nitric oxide generation as a putative
mechanism for failure of intrahepatic islet cell grafts,"
Transplant Proc., 24:2997, 1992. [0890] O'Blenes et al., "Gene
transfer of the serine elastase inhibitor claim protects against
vein graft degeneration," Circulation, 102(19 Suppl. 3):III-289-95,
2000. [0891] Ohara et al., "One-sided polymerase chain reaction:
the amplification of cDNA," Proc. Nat'l. Acad. Sci. USA,
86(15):5673-5677, 1989. [0892] Ohkawa et al., "Activities of
HIV-RNA targeted ribozymes transcribed from a `shot-gun` type
ribozyme-trimming plasmid," Nucl. Acids Symp. Ser., 27:15-16, 1992.
[0893] Ojwang et al., "Inhibition of human immunodeficiency virus
type 1 expression by a hairpin ribozyme," Proc. Nat'l. Acad. Sci.
USA, 89(22):10802-10806, 1992. [0894] Oldstone, "Prevention of type
I diabetes in nonobese diabetic mice by virus infection," Science,
23:500, 1988. [0895] Olsen et al., "Alpha-1-antitrypsin content in
the serum, alveolar macrophages, and alveolar lavage fluid of
smoking and nonsmoking normal subjects," J. Clin. Invest.,
55:427-430, 1975. [0896] Ono et al., "Transgenic medaka fish
bearing the mouse tyrosinase gene: expression and transmission of
the transgene following electroporation of the orange-colored
variant," Pigment Cell Res., 10(3):168-175, 1997. [0897] Ostwald et
al., "Effect of nitric oxide synthase inhibition on blood flow
after retinal ischemia in cats," Invest. Ophthalmol. Vis. Sci.,
36:2396-403, 1995. [0898] Parish et al., "The effect of bone marrow
and thymus chimerism between non-obese diabetic (NOD) and NOD-E
transgenic mice, on the expression and prevention of diabetes,"
Eur. J. Immunol., 23:2667, 1993. [0899] Parks et al.,
"Physicochemical characterization of adeno-associated satellite
virus type 4 and its nucleic acid," J. Virol., 1:980-87, 1967.
[0900] Parks et al., "Physical assay and growth cycle studies of a
defective adeno-satellite virus," J. Virol., 1:171-80, 1967. [0901]
Paterna et al., Influence of promoter and WHV post-transcriptional
regulatory element on AAV-mediated transgene expression in the rat
brain," Gene Ther., 7:1304-11, 2000. [0902] Paterson et al., "The
regions of the herpes simplex virus type 1 immediate early protein
Vmw175 required for site specific DNA binding closely correspond to
those involved in transcriptional regulation," Nucleic Acids Res.,
16:11005-25, 1988a. [0903] Paterson et al., "Mutational dissection
of the HSV-1 immediate-early protein Vmw175 involved in
transcriptional transactivation and repression," Virology,
166:186-96, 1988b. [0904] Patterson et al., "Cloning and functional
analysis of the promoter for KDR/flk-1, a receptor for vascular
endothelial growth factor," J. Biol. Chem., 270:23111-18, 1995.
[0905] Peel et al., "Efficient transduction of green fluorescent
protein in spinal cord neurons using adeno-associated virus vectors
containing cell type-specific promoters," Gene Ther., 4:16-24,
1997. [0906] Peltier and Hansen, "Immunoregulatory activity,
biochemistry, and phylogeny of ovine uterine serpin," Am. J.
Reprod. Immunol.,
45:266-72, 2001. [0907] Penn, "Why do immunosuppressed patients
develop cancer?" Crit. Rev. Onogen., 1:27-52, 1989. [0908] Pennline
et al., "Recombinant human IL-10 prevents the onset of diabetes in
the nonobese diabetic mouse," Clin. Immunol. Immunopathol.,
71:169-75, 1994. [0909] Pereira and Muzyczka, "The cellular
transcription factor SP1 and an unknown cellular protein are
required to mediate Rep protein activation of the adeno-associated
virus p19 promoter," J. Virol., 71:1747-56, 1997. [0910] Pereira et
al., "The adeno-associated virus (AAV) Rep protein acts as both a
repressor and an activator to regulate AAV transcription during a
productive infection," J. Virol., 71:1079-88, 1997. [0911] Perlino
et al., "The human alpha 1-antitrypsin gene is transcribed from two
different promoters in macrophages and hepatocytes," Embo. J.,
6:2767-71, 1987. [0912] Perlmutter and Punsal, "Distinct and
additive effects of elastase and endotoxin on expression of
.alpha.1 proteinase inhibitor in mononuclear phagocytes," J. Biol.
Chem., 263:16499-503, 1988. [0913] Perlmutter et al., "Expression
of the alpha 1-proteinase inhibitor gene in human monocytes and
macrophages," Proc. Nat'l Acad. Sci. USA, 82:795-799, 1985. [0914]
Perlmutter et al., "Identification of a serpin-enzyme complex
receptor on human hepatoma cells and human monocytes," Proc. Nat'l
Acad. Sci. USA, 87:3753-57, 1990. [0915] Perlmutter et al.,
"Interferon beta 2/interleukin 6 modulates synthesis of alpha
1-antitrypsin in human mononuclear phagocytes and in human hepatoma
cells," J. Clin. Invest., 84:138-144, 1989. [0916] Perreault et
al., "Mixed deoxyribo- and ribo-oligonucleotides with catalytic
activity," Nature, 344(6266):565, 1990. [0917] Perrey et al., "The
LDL receptor is the major pathway for .beta.-VLDL uptake by mouse
peritoneal macrophages," Atherosclerosis, 154:51-60, 2001. [0918]
Perrotta and Been, "Cleavage of oligoribonucleotides by a ribozyme
derived from the hepatitis delta virus RNA sequence," Biochem.,
31(1):16, 1992. [0919] Peters et al., "Agonist-induced
desensitization of A2B adenosine receptors," Biochem. Pharmacol.,
55:873-82, 1998. [0920] Philip et al., "Efficient and sustained
gene expression in primary T lymphocytes and primary and cultured
tumor cells mediated by adeno-associated virus plasmid DNA
complexed to cationic liposomes," Mol. Cell Biol., 14(4):2411-2418,
1994. [0921] Phillips et al., "Cutting edge: interactions through
the IL-10 receptor regulate autoimmune diabetes," J. Immunol.,
167:6087-91, 2001. [0922] Picard and Schaffner, "A
Lymphocyte-specific enhancer in the mouse immunoglobulin kappa
gene," Nature, 307:83, 1984. [0923] Pieken et al., "Kinetic
characterization of ribonuclease-resistant 2'-modified hammerhead
ribozymes," Science, 253(5017):314, 1991. [0924] Pikul et al., "In
vitro killing of melanoma by liposome-delivered intracellular
irradiation," Arch. Surg., 122:1417-20, 1987. [0925] Pileggi et
al., "Heine oxygenase-1 induction in islet cells results in
protection from apoptosis and improved in vivo function after
transplantation," Diabetes, 50:1983-91, 2001. [0926]
Pinto-Alphandary et al., "A new method to isolate
polyalkylcyanoacrylate nanoparticle preparations," J. Drug Target,
3(2):167-169, 1995. [0927] Pinto-Sietsma and Paul, "Transgenic rats
as models for hypertension," J. Hum. Hypertens., 11(9):577-581,
1997. [0928] Pitas, "Expression of the acetyl low density
lipoprotein receptor by rabbit fibroblasts and smooth muscle cells.
Up-regulation by phorbol esters," J. Biol. Chem., 265:12722-27,
1990. [0929] Pitas et al., "Uptake of chemically modified low
density lipoproteins in vivo is mediated by specific endothelial
cells," J. Cell Biol., 100:103-17, 1985. [0930] Pitluk and Ward,
"Unusual Spl-GC box interaction in a parvovirus promoter," J.
Virol., 65:6661-70, 1991. [0931] Pober, "Immunobiology of human
vascular endothelium," Immunol. Res., 19:225-32, 1999. [0932]
Polans et al., "Turned on by Ca.sup.2+! The physiology and
pathology of Ca.sup.2+-binding proteins in the retina," Trends
Neurosci., 19:547-554, 1996. [0933] Ponnazhagan et al., "Lack of
site-specific integration of the recombinant adeno-associated virus
2 genomes in human cells," Hum. Gene Ther., 8:275-84, 1997. [0934]
Ponnazhagan et al., "Adeno-associated virus type 2-mediated
transduction in primary human bone marrow-derived CD34.sup.+
hematopoietic progenitor cells: donor variation and correlation of
transgene expression with cellular differentiation," J. Virol.,
71:8262-67, 1997. [0935] Portera-Cailliau et al., "Apoptotic
photoreceptor cell death in mouse models of retinitis pigmentosa,"
Proc. Nat'l. Acad. Sci. USA, 91:974-978, 1994. [0936] Potter et
al., "Enhancer-dependent expression of human K immunoglobulin genes
introduced into mouse pre-B lymphocytes by electroporation," Proc.
Nat'l. Acad. Sci. USA, 81:7161-65, 1984. [0937] Prasad et al.,
"Adeno-associated virus vector mediated gene transfer to pancreatic
.beta. cells," Gene Ther., 7:1553-61, 2000. [0938] Prokop and
Bajpai, "Recombinant DNA Technology I," Conference on Progress in
Recombinant DNA Technology Applications, Potosi, M I, Jun. 3-8,
1990, Ann. N.Y. Acad. Sci., 646:1-383, 1991. [0939] Punglia et al.,
"Regulation of vascular endothelial growth factor expression by
insulin-like growth factor I," Diabetes, 46:1619-26, 1997. [0940]
Qing et al., "Human fibroblast growth factor receptor 1 is a
co-receptor for infection by adeno-associated virus 2," Nat. Med.,
5:71-77, 1999. [0941] Qiu and Brown, "A 110-kDa nuclear shuttle
protein, nucleolin, specifically binds to adeno-associated virus
type 2 (AAV-2) capsid," Virology, 257:373-82, 1999. [0942] Qiu et
al., "The interaction of heparin sulfate and adeno-associated virus
2," Virology, 269:137-47, 2000. [0943] Quinlan et al., "The
intranuclear location of a herpes simplex virus DNA binding protein
is determined by the status of viral DNA replication," Cell,
36:857-68, 1984. [0944] Quintanar-Guerrero et al., "Preparation and
characterization of nanocapsules from preformed polymers by a new
process based on emulsification-diffusion techinque," Phamr. Res.,
15(7):1056-1062, 1998. [0945] Rabinovitch, "An update on cytokines
in the pathogenesis of insulin-dependent diabetes mellitus,"
Diabetes Metab. Rev., 14:129-51, 1998. [0946] Rabinovitch,
"Immunoregulatory and cytokine imbalances in the pathogenesis of
IDDM: therapeutic intervention by immunostimulation?" Diabetes,
44:613-621, 1994. [0947] Rabinovitch et al., "Combined therapy with
interleukin-4 and interleukin-10 inhibits autoimmune diabetes
recurrence in syngeneic islet-transplanted nonobese diabetic mice.
Analysis of cytokine mRNA expression in the graft,"
Transplantation, 60:368-74, 1995. [0948] Rabinowitz and Samulski,
"Adeno-associated virus expression systems for gene transfer,"
Curr. Opin. Biotechnol., 9:470-75, 1998. [0949] Rabinowitz and
Samulski, "Building a better vector: the manipulation of AAV
virions," Virology, 278:301-08, 2000. [0950] Rabinowitz et al.,
"Cross-packaging of a single adeno-associated virus (AAV) type 2
vector genome into multiple AAV serotypes enables transduction with
broad specificity," J. Virol., 76:791-801, 2002. [0951] Rabinowitz
et al., "Insertional mutagenesis of AAV2 capsid and the production
of recombinant virus," Virology, 265:274-85. 1999. [0952] Ranuncoli
et al., "Islet cell transplantation: in vivo and in vitro
functional assessment of nonhuman primate pancreatic islets," Cell
Transplant, 9:409-14, 2000. [0953] Rapoport et al., "Interleukin 4
reverses T cell proliferative unresponsiveness and prevents the
onset of diabetes in nonobese diabetic mice," J. Exp. Med.,
178:87-99, 1993. [0954] Rasband and Bright, "NIH Image," Microbeam
Anal. Soc. J., 4:137-49, 1995. [0955] Ray et al.,
".alpha.-1-antitrypsin immunoreactivity in islet cells of adult
human pancreas," Cell Tissue Res., 185:63-68, 1977. [0956] Rego et
al., "Oxidative stress, hypoxia, and ischemia-like conditions
increase the release of endogenous amino acids by distinct
mechanisms in cultured retinal cells," J. Neurochem., 66:2506-16,
1996. [0957] Reinhold-Hurek and Shub, "Self-splicing introns in
tRNA genes of widely divergent bacteria," Nature, 357:173-176,
1992. [0958] REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed., Mack
Publishing Company, 1975. [0959] Rendahl et al., "Regulation of
gene expression in vivo following transduction by two separate rAAV
vectors," Nature Biotech., 16:757-62, 1998. [0960] Renneisen et
al., "Inhibition of expression of human immunodeficiency virus-1 in
vitro by antibody-targeted liposomes containing antisense RNA to
the env region," J. Biol. Chem., 265:16337-42, 1990. [0961] Rice
and Knipe, "Genetic evidence for two distinct transactivation
functions of the herpes simplex virus alpha protein ICP27," J.
Virol., 64:1704-15, 1990. [0962] Richard et al., "p42/p44
mitogen-activated protein kinases phosphorylate hypoxia-inducible
factor 1.alpha. (HIF-1.alpha.) and enhance the transcriptional
activity of HIF-1," J. Biol. Chem., 274:32631-37, 1999. [0963]
Ricordi et al., "Automated method for isolation of human pancreatic
islets," Diabetes, 37:413-20, 1988. [0964] Ridgeway, "Mammalian
expression vectors," In: Vectors: A survey of molecular cloning
vectors and their uses, Rodriguez and Denhardt (ed.), Stoneham:
Butterworth, pp. 467-492, 1988. [0965] Ried et al.,
"Adeno-associated virus capsids displaying immunoglobulin-binding
domains permit antibody-mediated vector retargeting to specific
cell surface receptors," J. Virol., 76:4559-66, 2002. [0966] Rippe
et al., "DNA-mediated gene transfer into adult rat hepatocytes in
primary culture," Mol. Cell Biol., 10:689-95, 1990. [0967] Robbins
and Evans, "Prospects for treating autoimmune and inflammatory
diseases by gene therapy," Gene Therapy, 3:187-89, 1996. [0968]
Robertson, "Pancreatic islet cell transplantation: likely impact on
current therapeutics for Type 1 diabetes mellitus," Drugs,
61:2017-20, 2001. [0969] Robinson et al., "Oligodeoxynucleotides
inhibit retinal neovascularization in a murine model of
proliferative retinopathy," Proc. Nat'l Acad. Sci. USA, 93:4851-56,
1996. [0970] Roizman and Sears, In FIELDS VIROLOGY, Fields et al.
(eds.), Lippincott-Raven, Philadelphia, pp. 2231-95, 1996. [0971]
Rolling et al., "Adeno-associated virus-mediated gene transfer into
rat carotid arteries," Gene Therapy, 4:757-761, 1997. [0972]
Rolling et al., "Evaluation of adeno-associated virus-mediated gene
transfer into the rat retina by clinical fluorescence photography,"
Hum. Gene Ther., 10:641-48, 1999. [0973] Rose and Koczot,
"Adenovirus-associated virus multiplication. VII. Helper
requirement for viral deoxyribonucleic acid and ribonucleic acid
synthesis," J. Virol., 10:1-8, 1972. [0974] Rose et al., "Evidence
for a single-stranded adenovirus-associated virus genome: formation
of a DNA density hybrid on release of viral DNA," Proc. Nat'l.
Acad. Sci. USA, 64(3):863-869, 1969. [0975] Rosen et al.,
"Mutations in Cu/Zn superoxide dismutase gene are associated with
familial amyotrophic lateral sclerosis," Nature, 362:59-62, 1993.
[0976] Rosen et al., "Enhanced expression of inducible nitric oxide
synthase in murine macrophages and glomerular mesangial cells by
elevated glucose levels: possible mediation via protein kinase C,"
Biochem. Biophys. Res. Commun., 207:80-88, 1995. [0977] Rosenberg,
"Clinical islet cell transplantation. Are we there yet?" Int. J.
Pancreatol., 24:145-68, 1998. [0978] Rossi et al., "Ribozymes as
anti-HIV-1 therapeutic agents: principles, applications, and
problems," AIDS Res. Hum. [0979] Retrovir., 8(2):183, 1992. [0980]
Rossini et al., "Studies of streptozotocin-induced insulitis and
diabetes," Proc. Nat'l. Acad. Sci. USA, 74:2485-89, 1977. [0981]
Rossman, "The canyon hypothesis. Hiding the host cell receptor
attachment site on a viral surface from immune surveillance," J.
Biol. Chem., 264:14587-90, 1989. [0982] Ruffing et al., "Mutations
in the carboxy terminus of adeno-associated virus 2 capsid proteins
affect viral infectivity: lack of an RGD integrin-binding motif,"
J. Gen. Virol., 75:3385-92, 1994. [0983] Ruffing et al., "Assembly
of viruslike particles by recombinant structural proteins of
adeno-associated virus type 2 in insect cells," J. Virol.,
66:6922-30, 1992. [0984] Russell et al., "DNA synthesis and
topoisomerase inhibitors increase transduction by adeno-associated
virus vectors," Proc. Nat'l. Acad. Sci. USA, 92:5719-23, 1995.
[0985] Rutledge et al., "Infectious clones and vectors derived from
adeno-associated virus (AAV) serotypes other than AAV type 2," J.
Virol., 72:309-19, 1998. [0986] Saito et al., "Experimental
preretinal neovascularization by laser-induced venous thrombosis in
rats," Curr. Eye Res., 16:26-33, 1997. [0987] Sakimura et al.,
"Upstream and intron regulatory regions for expression of the rat
neuron-specific enolase gene," Brain Res. Mol. Brain. Res.,
1:19-28, 1993. [0988] Salceda and Caro, "Hypoxia-inducible factor
1alpha (HIF-1a) protein is rapidly degraded by the
ubiquitin-proteasome system under normoxic conditions. Its
stabilization by hypoxia depends on redox-induced changes," J.
Biol. Chem., 272:22642-47, 1997. [0989] Sallenave and Ryle,
"Purification and characterization of elastase-specific inhibitor.
Sequence homology with mucus proteinase inhibitor," Biol. Chem.
Hoppe-Seyler, 372:13-21, 1991. [0990] Salvetti, "Factors
influencing recombinant adeno-associated virus production," Hum.
Gene Ther., 9:695-706, 1998. [0991] Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989. [0992] Samulski and Shenk, "Adenovirus
E1B 55-M, polypeptide facilitates timely cytoplasmic accumulation
of adeno-associated virus mRNAs," J. Virol., 62:206-10, 1988.
[0993] Samulski et al., "A recombinant plasmid from which an
infectious adeno-associated virus genome can be excised in vitro
and its use to study viral replication," J. Virol., 61:3096-101,
1987. [0994] Samulski, "Adeno-associated virus: integration at a
specific chromosomal locus," Curr. Opin. Genet. Dev., 3:74-80,
1993. [0995] Samulski et al., "Cloning of adeno-associated virus
into pBR322: rescue of intact virus from the recombinant plasmid in
human cells," Proc. Nat'l. Acad. Sci. USA, 79(6):2077-2080, 1982.
[0996] Samulski et al., "Helper-free stocks of recombinant
adeno-associated viruses: normal integration does not require viral
gene expression," J. Virol., 63:3822-28, 1989. [0997] Samulski et
al., "Rescue of adeno-associated virus from recombinant plasmids:
gene correction within the terminal repeats of AAV," Cell,
33:135-43, 1983. [0998] Samulski et al., "Targeted integration of
adeno-associated virus (AAV) into human chromosome 19," Embo. J.,
10:3941-50, (published erratum appears in Embo. J., 11:1228, 1992)
1991. [0999] Sandelain et al., "Prevention of type 1 Type I
diabetes in NOD mice by adjuvant immunotherapy," Diabetes, 39:583,
1990. [1000] Sandri-Goldin and Mendoza,
"A herpesvirus regulatory protein appears to act
post-transcriptionally by affecting mRNA processing," Genes Dev.,
6:848-63, 1992. [1001] Sanes et al., "Use of a recombinant
retrovirus to study post-implantation cell lineage in mouse
embryos," EMBO J., 5:3133-42, 1986. [1002] Sarver et al.,
"Ribozymes as potential anti-HIV-1 therapeutic agents," Science,
247(4947):1222-1225, 1990. [1003] Saville and Collins, "A
site-specific self-cleavage reaction performed by a novel RNA in
Neurospora mitochondria," Cell, 61(4):685-696, 1990. [1004] Saville
and Collins, "RNA-mediated ligation of self-cleavage products of a
Neurospora mitochondrial plasmid transcript," Proc. Nat'l. Acad.
Sci. USA, 88(19):8826-8830, 1991. [1005] Sawicki et al., "A
composite CMV-IE enhancer/beta-actin promoter is ubiquitously
expressed in mouse cutaneous epithelium," Exp. Cell Res.,
10:367-369, 1998. [1006] Scanlon et al., "Ribozyme-mediated
cleavage of c-fos mRNA reduces gene expression of DNA synthesis
enzymes and metallothionein," Proc. Nat'l. Acad. Sci. USA, 88(23):
10591-10595, 1991. [1007] Scaringe et al., "Chemical synthesis of
biologically active oligoribonucleotides using beta-cyanoethyl
protected ribonucleoside phosphoramidites," Nucl. Acids Res.,
18(18): 5433-5441, 1990. [1008] Scharp et al., "Results of our
first nine intraportal islet allografts in Type 1 Type I,
insulin-dependent diabetic patients," Transplantation, 51:76-85,
1991. [1009] Schmidt-Wolf and Schmidt-Wolf, "Cytokines and gene
therapy," Immunol. Today, 16:173-75, 1995. [1010] Sculier et al.,
"Pilot study of amphotericin B entrapped in sonicated liposomes in
cancer patients with fungal infections," J. Cancer Clin. Oncol.,
24:527-38, 1988. [1011] Segal, BIOCHEMICAL CALCULATIONS, 2nd Ed.,
John Wiley & Sons, New York, 1976. [1012] Selden, "Transfection
using DEAE-Dextran," in Current Protocols in Molecular Biology,
Ausubel, et al. (Eds.), John Wiley & Sons: New York, pp.
9.2.1-9.2.6, 1993. [1013] Senaphthy et al., "Replication of
adeno-associated virus DNA. Complementation of naturally occurring
rep.sup.- mutants by a wild-type genome or an ori.sup.- mutant and
correction of terminal palindrome deletions," J. Mol. Biol.,
179:1-20, 1984. [1014] Serreze, "Autoimmune diabetes results from
genetic defects manifest by antigen presenting cells," FASEB J.,
7:1092-96, 1993. [1015] Sexl et al., "Stimulation of human
umbilical vein endothelial cell proliferation by A2-adenosine and
beta 2-adrenoceptors," Br. J. Pharmacol., 114:1577-86, 1995. [1016]
Shafron et al., "Reduced MK801 binding in neocortical neurons after
AAV-mediated transfections with NMDA-R1 antisense cDNA," Brain Res.
784:325-328, 1998. [1017] Shapiro et al., "Islet transplantation in
seven patients with type 1 diabetes mellitus using a
glucocorticoid-free immunosuppressive regimen," N. Engl. J. Med.,
343:230-38, 2000. [1018] Sharp, "The current status of
alpha-1-antityrpsin, a protease inhibitor, in gastrointestinal
disease," Gastroenterology, 70:611-21, 1976. [1019] Shaw and Lewin,
"Protein-induced folding of a group I intron in cytochrome b
pre-mRNA," J. Biol. Chem., 270(37):21552-62, 1995. [1020] Shaw et
al., "Ribozymes in the treatment of retinal disease," In:
Vertebrate Phototransduction and the Visual Cycle. Methods in
Enzymology 316 Palczewski, Ed., New York, Academic Press, in press,
2000. [1021] She et al., "Heterophile antibodies segregate in
families and are associated with protection from type 1 diabetes,"
Proc. Nat'l. Acad. Sci. USA, 96:8116-19, 1999. [1022] Shehadeh et
al., "Effect of adjuvant therapy on the development of diabetes in
mouse and man," Lancet, 343:706, 1994. [1023] Shelburne and Ryan,
"The role of Th2 cytokines in mast cell homeostasis," Immunol.
Rev., 179:82-93, 2001. [1024] Shepard et al., "A second-site
revertant of a defective herpes simplex virus ICP4 protein with
restored regulatory activities and impaired DNA-binding
properties," J. Virol., 65:787-95, 1991. [1025] Shepard et al.,
"Separation of primary structural components conferring
autoregulation, transactivation, and DNA-binding properties to the
herpes simplex virus transcriptional regulatory protein ICP4," J.
Virol., 63:3714-28, 1989. [1026] Shi et al., "Insertional
mutagenesis of the adeno-associated virus type 2 (AAV2) capsid
gene, and generation of AAV2 vectors targeted to alternative
cell-surface receptors," Hum. Gene Ther., 12:1697-1711, 2001.
[1027] Shima et al., "The mouse gene for vascular endothelial
growth factor. Genomic structure, definition of the transcriptional
unit, and characterization of transcriptional and
post-transcriptional regulatory sequences," J. Biol. Chem.,
271:3877-83, 1996. [1028] Shimayama et al., "Generality of the NUX
rule: kinetic analysis of the results of systematic mutations in
the trinucleotide at the cleavage site of hammerhead ribozymes,"
Biochem., 34:3649-3654, 1995. [1029] Shryock and Belardinelli,
"Adenosine and adenosine receptors in the cardiovascular system:
biochemistry, physiology, and pharmacology," Am. J. Cardiol.,
79:2-10, 1997. [1030] Sibley and Sutherland, "Pancreas
transplantation. An immunohistologic and histopathologic
examination of 100 grafts," Am. J. Pathol., 128:151-70, 1987.
[1031] Simmons et al., "A Complete Protocol for In Situ
Hybridization of Messenger RNAs in Brain and Other Tissues With
Radio-labeled Single-Stranded RNA Probes," J. Histotechnol.,
12:169-181, 1989. [1032] Sleigh and Lockett, "SV40 enhancer
activation during retinoic-acid-induced differentiation of F9
embryonal carcinoma cells," J. EMBO, 4:3831, 1985. [1033] Smith et
al., "Essential role of growth hormone in ischemia-induced retinal
neovascularization," Science, 276:1706-09, 1997. [1034] Smith et
al., "Interleukin-4 or interleukin-10 expressed from
adenovirus-transduced syngeneic islet grafts fails to prevent
.beta. cell destruction in diabetic NOD mice," Transplantation,
64:1040-49, 1997. [1035] Smith et al., "Regulation of vascular
endothelial growth factor-dependent retinal neovascularization by
insulin-like growth factor-1 receptor," Nat. Med., 5:1390-95, 1999.
[1036] Smith et al., "Oxygen-induced retinopathy in the mouse,"
Invest. Ophthalmol. Vis. Sci., 35:101-11, 1994. [1037] Snyder et
al., "Correction of hemophilia B in canine and murine models using
recombinant adeno-associated viral vectors," Nat. Med., 5:64-70,
1999. [1038] Snyder et al., "Persistent and therapeutic
concentrations of human factor IX in mice after hepatic gene
transfer of recombinant AAV vectors," Nat. Genet., 16:270-76,
1997b. [1039] Snyder et al., "Efficient and stable adeno-associated
virus-mediated transduction in the skeletal muscle of adult
immunocompetent mice," Hum. Gene Ther., 8:1891-900, 1997a. [1040]
Socci et al., "Fresh human islet transplantation to replace
pancreatic endocrine function in Type 1 Type I diabetic patients.
Report of six cases," Acta Diabetol., 28:151-57, 1991. [1041] Song
et al., "Stable therapeutic serum levels of human alpha-1
antitrypsin (AAT) after portal vein injection of recombinant
adeno-associated virus (rAAV) vectors," Gene Ther., 8:1299-306,
2001a. [1042] Song et al., "Effect of DNA-dependent protein kinase
on the molecular fate of the rAAV2 genome in skeletal muscle,"
Proc. Nat'l. Acad. Sci. USA, 98:4084-88, 2001b. [1043] Song et al.,
"Sustained secretion of human .alpha.1-antitrypsin from murine
muscle transduced with adeno-associated virus vectors," Proc.
Nat'l. Acad. Sci. USA, 95:14384-88, 1998. [1044] Sonksen et al.,
"Growth hormone and diabetes mellitus: a review of sixty-three
years of medical research and a glimpse into the future?" Horm.
Res., 40:68-79, 1993. [1045] Srivastava et al., "Nucleotide
sequence and organization of the adeno-associated virus 2 genome,"
J. Virol., 45:555-64, 1983. [1046] Stein and Carrell, "What do
dysfunctional serpins tell us about molecular mobility and
disease?," Nat. Struct. Biol., 2:96-113, 1995. [1047] Stein and
Stein, "Bovine aortic endothelial cells display macrophage-like
properties towards acetylated 125I-labelled low density
lipoprotein," Biochem. Biophys. Acta, 620:631-35, 1980. [1048]
Stelzner et al., "Role of cyclic adenosine monophosphate in the
induction of endothelial barrier properties," J. Cell Physiol.,
139:157-66, 1989. [1049] Stephenson and Gibson, "In vitro cleavage
of an N-ras messenger-like RNA by a ribozyme," Antisense Res. Dev.,
1:261-68, 1991. [1050] Stevens et al., "Role of nitric oxide in the
pathogenesis of early pancreatic islet dysfunction during rat and
human intraportal islet transplantation," Transplant Proc., 26:692,
1994. [1051] Stewart et al., "Cloning of human RTEF-1, a
transcriptional enhancer factor-1-related gene preferentially
expressed in skeletal muscle: evidence for an ancient multigene
family," Genomics, 37(1):68-76, 1996. [1052] Studier et al., "Use
of T7 RNA polymerase to direct expression of cloned genes," Methods
Enzymol., 185:60-89, 1990. [1053] Summerford and Samulski,
"Membrane-associated heparan sulfate proteoglycan is a receptor for
adeno-associated virus type 2 virions," J. Virol., 72:1438-45,
1998. [1054] Summerford et al., ".alpha..sub.v.beta..sub.5
integrin: a co-receptor for adeno-associated virus type 2
infection," Nat. Med., 5:78-82, 1999. [1055] Suzuki et al., "Direct
gene transfer into rat liver cells by in vivo electroporation,"
FEBS Lett., 425:436-40, 1998. [1056] Tahara et al., "Islet cell
transplantation facilitated by gene transfer," Transplant Proc.,
24:2975-76, 1992. [1057] Taira et al., "Construction of a novel
RNA-transcript-trimming plasmid which can be used both in vitro in
place of run-off and (G)-free transcriptions and in vivo as
multi-sequences transcription vectors," Nucl. Acids Res.,
19(19):5125-5130, 1991. [1058] Takagi et al., "Hypoxia regulates
vascular endothelial growth factor receptor KDR/Flk gene expression
through adenosine A2 receptors in retinal capillary endothelial
cells," Invest. Ophthalmol. Vis. Sci., 37:1311-21, 1996a. [1059]
Takagi et al., "Adenosine mediates hypoxic induction of vascular
endothelial growth factor in retinal pericytes and endothelial
cells," Invest. Ophthalmol. Vis. Sci., 37:2165-76, 1996b. [1060]
Takahashi et al., "Ischemia- and cytokine-induced mobilization of
bone marrow-derived endothelial progenitor cells for
neovascularization," Nat. Med., 5:434-38, 1999. [1061] Takahashi et
al., "Spontaneous transformation and immortalization of human
endothelial cells," In Vitro Cell Dev. Biol., 26:265-74, 1990.
[1062] Takakura, "Drug delivery systems in gene therapy," Nippon
Rinsho, 56:691-95, 1998. [1063] Tamayose et al., "A new strategy
for large-scale preparation of high-titer recombinant
adeno-associated virus vectors by using packaging cell lines and
sulfonated cellulose column chromatography," Hum. Gene Ther.,
7:507-13, 1997. [1064] Taomoto et al., "Localization of adenosine
A2a receptor in retinal development and oxygen-induced
retinopathy," Invest. Ophthalmol. Vis. Sci., 41:230-43, 2000.
[1065] Taylor and Rossi, "Ribozyme-mediated cleavage of an HIV-1
gag RNA: the effects of nontargeted sequences and secondary
structure on ribozyme cleavage activity," Antisense Res. Dev.,
1:173-86, 1991. [1066] Taylor-Robinson and Phillips, "Expression of
IL-1 receptor discriminates Th2 from Th1 cloned CD4+ T cells
specific for Plasmodium chabaudi," Immunology, 81:216, 1994. [1067]
Thomson and Efstathiou, "Acquisition of the human adeno-associated
virus type-2 rep gene by human herpesvirus type-6," Nature,
351:78-80, 1991. [1068] Thomson et al., "Human herpesvirus 6
(HHV-6) is a helper virus for adeno-associated virus type 2 (AAV-2)
and the AAV-2 rep gene homologue in HHV-6 can mediate AAV-2 DNA
replication and regulate gene expression," Virology, 204:304-11,
1994. [1069] Tian et al., "Infectious Th1 and Th2 autoimmunity in
diabetes-prone mice," Immunol. Rev., 164:119-27, 1998. [1070]
Timmers et al., "Synthesis and stability of retinal photoreceptor
mRNAs are coordinately regulated during bovine fetal development,"
Exp. Eye Res., 56:251-265, 1993. [1071] Tratschin et al., "Genetic
analysis of adeno-associated virus: properties of delection mutants
constructed in vitro and evidence for an adeno-associated virus
replication function," J. Virol., 51:611-19, 1984. [1072] Tratschin
et al., "A human parvovirus, adeno-associated virus, as a
eucaryotic vector: transient expression and encapsidation of the
procaryotic gene for chloramphenicol acetyltransferase," Mol. Cell
Biol., 4:2072-81, 1984. [1073] Tremblay et al.,
"Elafin/elastase-specific inhibitor in bronchoalveolar lavage of
normal subjects and farmer's lung," Am. J. Respir. Crit. Care Med.,
154:1092-98, 1996. [1074] Trempe and Carter, "Alternate mRNA
splicing is required for synthesis of adeno-associated virus VP1
capsid protein," J. Virol., 62:3356-63, 1988. [1075] Tresnan et
al., "Analysis of the cell and erythrocyte binding activities of
the dimple and canyon regions of the canine parvovirus capsid,"
Virol., 211:123-32, 1995. [1076] Trudeau et al., "Neonatal
.beta.-cell apoptosis: a trigger for autoimmune diabetes?"
Diabetes, 49:1-7, 2000. [1077] Tsao et al., "The three-dimensional
structure of canine parvovirus and its functional implications,"
Science, 251:1456-64, 1991. [1078] Tsao et al., "Structure
determination of monoclinic canine parvovirus," Acta. Crystallogr.
B, 48:75-88, 1992. [1079] Tuder et al., "Cyclic adenosine
monophosphate levels and the function of skin microvascular
endothelial cells," J. Cell Physiol., 142:272-83, 1990. [1080]
Tur-Kaspa et al., "Use of electroporation to introduce biologically
active foreign genes into primary rat hepatocytes,"Mol. Cell Biol.,
6:716-18, 1986. [1081] Usman and Cedergren, "Exploiting the
chemical synthesis of RNA," Trends Biochem. Sci., 17(9):334, 1992.
[1082] Usman et al., "The automated chemical synthesis of long
oligoribuncleotides using 2'-O-silylated ribonucleoside
3'-O-phosphoramidites on a controlled-pore glass support: synthesis
of a 43-nucleotide sequence similar to the 3'-half molecule of an
Escherichia coli formylmethionine tRNA," J. Am. Chem. Soc.,
109:7845-7854, 1987. [1083] Van Cott et al., "Phenotypic and
genotypic stability of multiple lines of transgenic pigs expressing
recombinant human protein C," Transgenic Res., 6(3):203-212, 1997.
[1084] van Ginkel and Hauswirth, "Parallel regulation of fetal gene
expression in different photoreceptor cell types," J. Biol. Chem.,
269:4986-92, 1994. [1085] Vanbever et al., "In vivo noninvasive
evaluation of hairless rat skin after high-voltage pulse exposure,"
Skin Parmacol. Appl. Skin Physiol., 11:23-34, 1998. [1086] Varban
et al., "Targeted mutation reveals a central role for SR-BI in
hepatic selective uptake of high density lipoprotein cholesterol,"
Proc. Nat'l. Acad. Sci. USA, 95:4619-24, 1998. [1087] Veldwijk et
al., "Development and optimization of a real-time quantitative
PCR-based method for the titration of AAV-2 vector stocks," Mol.
Ther., 6:272-78, 2002. [1088] Venkatesan et al., "Possible role for
the glucose-fatty acid cycle in dexamethasone-induced insulin
antagonism in rats,
" Metabolism, 36:883-91, 1987. [1089] Ventura et al., "Activation
of HIV-specific ribozyme activity by self-cleavage," Nucl. Acids
Res., 21:3249-3255, 1993. [1090] Vestweber and Blanks, "Mechanisms
that regulate the function of the selectins and their ligands,"
Physiol. Rev., 79:181-213, 1999. [1091] Vincent et al., "Analysis
of recombinant adeno-associated virus packaging and requirements
for rep and cap gene products," J. Virol. 71:1897-905, 1997a.
[1092] Vincent et al., "Preclinical testing of recombinant
adenoviral herpes simplex virus-thymidine kinase gene therapy for
central nervous system malignancies," Neurosurgery, 41:442-51,
1997b. [1093] Vincent et al., "Replication and packaging of HIV
envelope genes in a novel adeno-associated virus vector system,"
Vaccine, 90:353-59, 1990. [1094] Virella-Lowell et al., "A
CMV/.beta.-actin hybrid promoter greatly improves recombinant
adeno-associated virus (rAAV) vector expression in the murine
lung," Ped. Pulmonol., S19:231, 1999. [1095] von Weizsacker et al.,
"Cleavage of hepatitis B virus RNA by three ribozymes transcribed
from a single DNA template," Biochem. Biophys. Res. Commun.,
189:743-48, 1992. [1096] Voyta et al., "Identification and
isolation of endothelial cells based on their increased uptake of
acetylated-low density lipoprotein," J. Cell Biol., 99:2034-40,
1984. [1097] Wagner et al., "Efficient and persistent gene transfer
of AAV-CFTR in maxillary sinus," Lancet, 351:1702-03, 1998. [1098]
Wagner et al., "Coupling of adenovirus to
transferrin-polylysine/DNA complexes greatly enhances
receptor-mediated gene delivery and expression of transfected
genes," Proc. Natl. Acad. Sci. USA, 89:6099-103, 1992. [1099]
Walker et al., "Strand displacement amplification--an isothermal,
in vitro DNA amplification technique," Nucleic Acids Res.,
20(7):1691-6, 1992. [1100] Walters et al., "Binding of
adeno-associated virus type 5 to 2,3-linked sialic acid is required
for gene transfer," J. Biol. Chem., 276:20610-16, 2001. [1101] Wang
et al., "NGF gene expression in dividing and non-dividing cells
from AAV-derived constructs," Neurochem Res., 23(5):779-86, 1998.
[1102] Wang et al., "Autoimmune diabetes in NOD mouse is L3T4
T-lymphocyte dependent," Diabetes, 36:535-38, 1987. [1103] Wang et
al., "Efficient CFTR expression from AAV vectors packaged with
promoters--the second generation," Gene Ther., 6(4):667-675, 1999.
[1104] Warnock et al., "Normoglycaemia after transplantation of
freshly isolated and cryopreserved pancreatic islets in Type 1 Type
I (insulin-dependent) diabetes mellitus," Diabetologia, 34:55-58,
1991. [1105] Watson, "Fluid and electrolyte disorders in
cardiovascular patients," Nurs. Clin. North Am., 22:797-803, 1987.
[1106] Waugh et al., "Therapeutic elastase inhibition by
.alpha.-1-antitrypsin gene transfer limits neointima formation in
normal rabbits," J. Vasc. Interv. Radiol., 12:1203-09, 2001. [1107]
Weerasinghe et al., "Resistance to human immunodeficiency virus
type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell
lines conferred by using retroviral vectors expression an HIV-1
RNA-specific ribozyme," J. Virol., 65(10):5531-5534, 1991. [1108]
Weger et al., "The adeno-associated virus type 2 regulatory
proteins Rep78 and Rep68 interact with the transcriptional
coactivator PC4," J. Virol., 73:260-69, 1999. [1109] Wegmann and
Eisenbarth, "It's insulin," J. Autoimmun., 15:286-91, 2000. [1110]
Wei et al., "Isolation and comparison of two molecular species of
the BAL 31 nuclease from Alteromonas espejiana with distinct
kinetic properties," J. Biol. Chem., 258:13506-512, 1983. [1111]
Weindler and Heilbronn, "A subset of herpes simplex virus
replication genes provides helper functions for productive
adeno-associated virus replication," J. Virol., 65:2476-83, 1991.
[1112] Weir and Bonner-Weir, "Islet transplantation as a treatment
for diabetes," J. Am. Optom. Assoc., 69:727-32, 1998. [1113] Weir
et al., "Islet mass and function in diabetes and transplantation,"
Diabetes, 39:401-05, 1990. [1114] Weitzman et al., "Interaction of
wild-type and mutant adeno-associated virus (AAV) Rep proteins on
AAV hairpin DNA," J. Virol., 70:2440-48, 1996a. [1115] Weitzman et
al., "Recruitment of wild-type and recombinant adeno-associated
virus into adenovirus replication centers," J. Virol., 70:1845-54,
1996b. [1116] Weitzman et al., "Adeno-associated virus (AAV) Rep
proteins mediate complex formation between AAV DNA and its
integration site in human DNA," Proc. Nat'l. Acad. Sci. USA,
91:5808-12, 1994. [1117] Weller, "Genetic analysis of HSV-1 gene
required for genome replication," In: Herpes virus transcription
and its regulation, Wagner (ed.), Boca Raton, Fla.: CRC Press, pp.
105-136, 1991. [1118] Wiedow et al., "Elafin: an elastase-specific
inhibitor of human skin. Purification, characterization, and
complete amino acid sequence," J. Biol. Chem., 265:14791-95, 1990.
[1119] Wistuba et al., "Subcellular compartmentalization of
adeno-associated virus type 2 assembly," J. Virol., 71:1341-52,
1997. [1120] Wistuba et al., "Intermediates of adeno-associated
virus type 2 assembly: identification of soluble complexes
containing Rep and Cap proteins," J. Virol., 69:5311-19, 1995.
[1121] Wobus et al., "Monoclonal antibodies against the
adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and
identification of capsid domains involved in AAV-2-cell interaction
and neutralization of AAV-2 infection," J. Virol., 74:9281-93,
2000. [1122] Wogensen et al., "Leukocyte extravasation into the
pancreatic tissue in transgenic mice expressing interleukin 10 in
the islets of Langerhans," J. Exp. Med., 178:175-85, 1993. [1123]
Wogensen et al., "Production of interleukin 10 by islet cells
accelerates immune-mediated destruction of cells in nonobese
diabetic mice," J. Exp. Med., 179:1379-84, 1994. [1124] Wong and
Janeway, "The role of CD4 vs. CD8 T cells in IDDM," J. Autoimmun.,
13:290-95, 1999. [1125] Wong and Neumann, "Electric field mediated
gene transfer," Biochim. Biophys. Res. Commun., 107:584-87, 1982.
[1126] Wong et al., "Appearance of .beta.-lactamase activity in
animal cells upon liposome mediated gene transfer," Gene, 10:87-94,
1980. [1127] Woolf et al., "Specificity of antisense
oligonucleotides in vivo," Proc. Natl. Acad. Sci. USA,
89(16):7305-7309, 1992. [1128] Wu and Dean, "Functional
significance of loops in the receptor binding domain of Bacillus
thuringiensis CryIIIA delta-endotoxin," J. Mol. Biol.,
255(4):628-640, 1996. [1129] Wu and Wu, "Evidence for targeted gene
delivery to HepG2 hepatoma cells in vitro," Biochemistry,
27:887-92, 1988. [1130] Wu and Wu, "Receptor-mediated in vitro gene
transfections by a soluble DNA carrier system," J. Biol. Chem.,
262:4429-32, 1987. [1131] Wu et al., "Identification of herpes
simples virus type 1 genes required for origin-dependent DNA
synthesis," J. Virol., 62:435, 1988. [1132] Wu et al., "Inhibition
of the EGF-activated MAP kinase signaling pathway by adenosine
3',5'-monophosphate," Science, 262:1065-69, 1993. [1133] Wu et al.,
"Mutational analysis of the adeno-associated virus type 2 (AAV2)
capsid gene and construction of AAV2 vectors with altered tropism,"
J. Virol., 74:8635-47, 2000. [1134] Wyble et al., "TNF-.alpha. and
IL-1 upregulate membrane-bound and soluble E-selectin through a
common pathway," J. Surg. Res., 73:107-12, 1997. [1135] Xiao et
al., "Adeno-associated virus (AAV) vector antisense gene transfer
in vivo decreases GABA(A) alpha1 containing receptors and increases
inferior collicular seizure sensitivity," Brain Res., 756:76-83,
1997. [1136] Xiao et al., "Adeno-associated virus as a vector for
liver-directed gene therapy," J. Virol., 72:10222-26, 1998. [1137]
Xiao et al., "Efficient long-term gene transfer into muscle tissue
of immunocompetent mice by adeno-associated virus vector," J.
Virol., 70:8098-108, 1996. [1138] Xiao et al., "Production of
high-titer recombinant adeno-associated virus vectors in the
absence of helper adenovirus," J. Virol. 72:2224-32, 1998. [1139]
Xiao et al., "Gene transfer by adeno-associated virus vectors into
the central nervous system," Exp. Neurol., 144:113-124, 1997.
[1140] Xie et al., "The atomic structure of adeno-associated virus
(AAV-2), a vector for human gene therapy," Proc. Nat'l. Acad. Sci.
USA, 99:10405-10, 2002. [1141] Xing and Whitton, "An
anti-lymphocytic choriomeningitis virus ribozyme expressed in
tissue culture cells diminishes viral RNA levels and leads to a
reduction in infectious virus yield," J. Virol., 67:1840-47, 1993.
[1142] Xu and Gong, "Adaptation of inverse PCR to generate an
internal deletion," Biotechniques, 26:639-41, 1999. [1143] Xu et
al., "CMV-.beta.-actin promoter directs higher expression from an
adeno-associated viral vector in the liver than the cytomegalovirus
or elongation factor 1.alpha. promoter and results in therapeutic
levels of human factor X in mice," Hum. Gene Ther., 12:563-73,
2001. [1144] Yan et al., "Selective degradation of nonsense
beta-phosphodiesterase mRNA in the heterozygous rd mouse," Invest.
Ophthalmol. Vis. Sci., 39:2529-2536, 1998. [1145] Yan et al., "From
the cover: trans-splicing vectors expand the utility of
adeno-associated virus for gene therapy," Proc. Nat'l. Acad. Sci.
USA, 97:6716-21, 2000. [1146] Yang and Kotin, "Glucose-responsive
gene delivery in pancreatic Islet cells via recombinant
adeno-associated viral vectors," Pharm. Res., 17:1056-61, 2000.
[1147] Yang et al., "In vivo and in vitro gene transfer to
mammalian somatic cells by particle bombardment," Proc. Nat'l.
Acad. Sci. USA, 87:9568-72, 1990. [1148] Yang et al., "Development
of novel cell surface CD34-targeted recombinant adenoassociated
virus vectors for gene therapy," Hum. Gene Ther., 9:1929-37, 1998.
[1149] Yang et al., "Effects of streptozotocin-induced diabetes
mellitus on growth and hepatic insulin-like growth factor I gene
expression in the rat," Metabolism, 39:295-301, 1990. [1150]
Yarfitz and Hurley, "Transduction mechanisms of vertebrate and
invertebrate photoreceptors," J. Biol. Chem., 269:14329-14332,
1994. [1151] Yoon et al., "Cellular and molecular mechanisms for
the initiation and progression of cell destruction resulting from
the collaboration between macrophages and T cells," Autoimmunity,
27:109-22, 1998. [1152] Yu et al., "A hairpin ribozyme inhibits
expression of diverse strains of human immunodeficiency virus type
1," Proc. Nat'l. Acad. Sci. USA, 90:6340-6344, 1993. [1153] Yu et
al., "In vitro and in vivo characterization of a second functional
hairpin ribozyme against HIV-1," Virology, 206(1):381-86, 1995.
[1154] Yu et al., "Early expression of antiinsulin autoantibodies
of humans and the NOD mouse: evidence for early determination of
subsequent diabetes," Proc. Nat'l. Acad. Sci. USA, 97:1701-06,
2000. [1155] Zabner et al., "Adeno-associated virus type 5 (AAV5)
but not AAV2 binds to the apical surfaces of airway epithelia and
facilitates gene transfer," J. Virol., 74:3852-58, 2000. [1156]
Zaidi et al., "Targeted overexpression of claim protects mice
against cardiac dysfunction and mortality following viral
myocarditis," J. Clin. Invest., 103:1211-19, 1999. [1157] Zambaux
et al., "Influence of experimental paparmeters on the
characteristics of poly(lactic acid) nanoparticles prepared by a
double emulsion method," J. Control. Rel., 50(1-3):31-40, 1998.
[1158] Zhang et al., "Adeno-associated virus transduction of islets
with interleukin-4 results in impaired metabolic function in
syngeneic marginal islet mass transplantation," Transplantation,
74(8):1184-6, 2002b. [1159] Zhang et al., "Genetic predisposition
to autoimmunity specifically imparts responsiveness to transgenes
delivered by recombinant adeno-associated virus," Mol. Ther.,
5:S430 (Abstr. 1317), 2002a. [1160] Zhong and Hayward, "Assembly of
complete functionally active herpes simplex virus DNA replication
compartments and recruitment of associated viral and cellular
proteins in transient cotransfection assays," J. Virol.,
71:3146-60, 1997. [1161] Zhou and Muzyczka, "In vitro packaging of
adeno-associated virus DNA," J. Virol., 72:3241-47, 1998. [1162]
Zhou et al., "Adeno-associated virus 2-mediated high efficiency
gene transfer into immature and mature subsets of hematopoietic
progenitor cells in human umbilical cord blood," J. Exp. Med.,
179:1867-75, 1994. [1163] Zhou et al., "Synthesis of functional
mRNA in mammalian cells by bacteriophage T3 RNA polymerase," Mol.
Cell Biol., 10(9):4529-4537, 1990. [1164] Ziady et al., "Chain
length of the polylysine in receptor-targeted gene transfer
complexes affects duration of reporter gene expression both in
vitro and in vivo," J. Biol. Chem., 274:4908-16, 1999. [1165] Ziady
et al., "Gene transfer into hepatoma cell lines via the serpin
enzyme complex receptor," Am. J. Physiol., 273(2 Pt 1):G545-52,
1997. [1166] Zolotukhin et al., "Recombinant adeno-associated virus
purification using novel methods improves infectious titer and
yield," Gene Ther., 6:973-985, 1999. [1167] Zolotukhin et al., "A
`humanized` green fluorescent protein cDNA adapted for high-level
expression in mammalian cells," J. Virol., 70:4646-54, 1996. [1168]
Zolotukhin et al., "Production and purification of serotype 1, 2,
and 5 recombinant adeno-associated viral vectors," Methods,
28:158-67, 2002. [1169] zur Muhlen et al., "Solid lipid
nanoparticles (SLN) for controlled drug delivery--drug release and
release mechanism," Eur. J. Pharm. Biopharm., 45:149-155, 1998.
[1170] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
Sequence CWU 1
1
516PRTadeno-associated virus 2 1Arg Gly Asn Arg Gln Ala 1 5
26PRTadeno-associated virus 5 2Ser Asn Ser Asn Leu Pro 1 5
323DNAArtificial SequenceSynthetic oligonucleotide primer
3ttcaaagatg acgggaacta caa 23420DNAArtificial SequenceSynthetic
oligonucleotide primer 4tcaatgccct tcagctcgat 20524DNAArtificial
SequenceSynthetic oligonucleotide primer 5cccgcgctga agtcaagttc
gaag 24
* * * * *