U.S. patent application number 12/976334 was filed with the patent office on 2011-12-01 for viral vectors and methods for producing and using the same.
Invention is credited to Andrea Amalfitano, Dwight D. Koeberl, Baodong Sun.
Application Number | 20110294193 12/976334 |
Document ID | / |
Family ID | 29401341 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110294193 |
Kind Code |
A1 |
Amalfitano; Andrea ; et
al. |
December 1, 2011 |
VIRAL VECTORS AND METHODS FOR PRODUCING AND USING THE SAME
Abstract
A recombinant hybrid virus, including: (a) a deleted adenovirus
vector genome comprising the adenovirus 5' and 3' cis-elements for
viral replication and encapsidation, and further comprising a
deletion in an adenovirus genomic region selected from the group
consisting of: (i) the polymerase region, wherein said deletion
essentially prevents the expression of a functional polymerase
protein from said deleted region and said hybrid virus does not
otherwise express a functional polymerase protein, (ii) the
preterminal protein region, wherein said deletion essentially
prevents the expression of a functional preterminal protein from
said deleted region, and said hybrid virus does not otherwise
express a functional preterminal protein, and (iii) both the
regions of (i) and (ii); and (b) a recombinant adeno-associated
virus (AAV) vector genome flanked by the adenovirus vector genome
sequences of (a), said recombinant AAV vector genome comprising (i)
AAV 5' and 3' inverted terminal repeats, (ii) an AAV packaging
sequence, and (iii) a heterologous nucleic acid sequence, wherein
said heterologous nucleic acid sequence is flanked by the 5' and 3'
AAV inverted terminal repeats of (i). Methods of making and using
the recombinant hybrid virus are also disclosed.
Inventors: |
Amalfitano; Andrea; (Durham,
NC) ; Koeberl; Dwight D.; (Durham, NC) ; Sun;
Baodong; (Morrisville, NC) |
Family ID: |
29401341 |
Appl. No.: |
12/976334 |
Filed: |
December 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10511980 |
Apr 7, 2005 |
7858367 |
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PCT/US03/13323 |
Apr 30, 2003 |
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12976334 |
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60376397 |
Apr 30, 2002 |
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Current U.S.
Class: |
435/235.1 |
Current CPC
Class: |
A61K 48/0083 20130101;
A61K 48/0066 20130101; A61K 2039/5256 20130101; A61K 48/0075
20130101; C12N 2710/10344 20130101; C12Y 302/0102 20130101; C12N
9/2411 20130101; C12N 9/2408 20130101; A61K 38/47 20130101; C12N
15/86 20130101; A61K 48/00 20130101; C12N 2750/14143 20130101 |
Class at
Publication: |
435/235.1 |
International
Class: |
C12N 7/01 20060101
C12N007/01 |
Goverment Interests
GRANT STATEMENT
[0002] This work was supported by grant R01-DK 52925 from the U.S.
National Institute of Health. Thus, the U.S. government has certain
rights in the invention.
Claims
1-36. (canceled)
37. A method of producing a recombinant hybrid adeno-associated
virus (AAV) particle, comprising providing to a cell: (a) a
recombinant hybrid virus; comprising: (i) a deleted adenovirus
vector genome comprising the adenovirus 5' and 3' cis-elements for
viral replication and encapsidation; a functional adenovirus
genomic region selected from the group consisting of an adenovirus
E1a region, E2a region, E4orf6 region, VA RNA region, and any
combination of the foregoing; and further comprising a deletion in
an adenovirus genomic region selected from the group consisting of:
(1) the polymerase region, wherein said deletion essentially
prevents the expression of a functional polymerase protein from
said deleted region and said hybrid virus does not otherwise
express a functional polymerase protein, (2) the preterminal
protein region, wherein said deletion essentially prevents the
expression of a functional preterminal protein from said deleted
region, and said hybrid virus does not otherwise express a
functional preterminal protein, and (3) both the regions of (1) and
(2); and (ii) a recombinant adeno-associated virus (AAV) vector
genome flanked by the adenovirus vector genome sequences of (i),
said recombinant AAV vector genome comprising: (4) AAV 5' and 3'
inverted terminal repeats; (5) an AAV packaging sequence; and (6) a
heterologous nucleic acid sequence, wherein said heterologous
nucleic acid sequence is flanked by the 5' and 3' AAV inverted
terminal repeats of (4), and further wherein the AAV vector genome
does not encode the AAV Rep or AAV capsid proteins; (b) AAV
sequences sufficient for replication and packaging of the AAV
vector genome; and (c) AAV sequences sufficient to produce a
functional AAV capsid, wherein (a), (b), and (c) are provided to
the cell under conditions sufficient for replication of the AAV
vector genome and packaging thereof in the AAV capsid such that AAV
particles comprising the AAV vector genome encapsidated within the
AAV capsid are produced in the cell essentially without producing
contaminating adenovirus.
38. The method of claim 37, further comprising collecting the
recombinant AAV particle.
39. (canceled)
40. The method of claim 37, wherein each of adenovirus E1a, E2a,
E4orf6, and VA RNA helper sequences are provided.
41. The method of claim 37, wherein the cell is selected from the
group consisting of a HeLa cell, a 293 cell, a muscle cell, and a
liver cell.
42. (canceled)
43. The method of claim 37, wherein the yield of recombinant AAV
particles is at least 5-fold greater than in the presence of the
adenovirus polymerase and/or preterminal proteins.
44. The method of claim 37, wherein sequences encoding an AAV Rep
protein and/or sequences encoding the AAV capsid protein are stably
expressed by the cell.
45. The method of claim 37, wherein sequences encoding an AAV Rep
protein and/or sequences encoding the AAV capsid protein are
provided by a vector other than the recombinant hybrid virus.
46. The method of claim 45, wherein the vector is selected from the
group consisting of a plasmid, an adenovirus, an Epstein Barr
virus, and a herpesvirus vector.
47. The method of claim 37, wherein the AAV inverted terminal
repeats and the AAV capsid are derived from different AAV
serotypes.
48. The method of claim 37, wherein the AAV capsid is an AAV-6
capsid.
49. The method of claim 37, wherein the AAV inverted terminal
repeats are AAV-2 inverted terminal repeats.
50. A method of producing a recombinant hybrid adeno-associated
virus (AAV) particle, comprising providing to a cell a hybrid virus
particle produced by the method of claim 37, said recombinant
hybrid virus particle expressing the adenovirus helper functions
for AAV replication and packaging; wherein the cell: (i) expresses
AAV rep sequences sufficient for replication and packaging of the
AAV vector genome; (ii) expresses AAV cap sequences sufficient to
produce a functional AAV capsid; and (iii) does not express
sequences sufficient to produce a functional adenovirus E1a
protein; and further wherein the hybrid virus particle is provided
under conditions sufficient for replication of the AAV vector
genome and packaging thereof in the AAV capsid such that AAV
particles comprising the AAV vector genome encapsidated within the
AAV capsid are produced in the cell.
51. A method of producing a recombinant hybrid adeno-associated
virus (AAV) particle, comprising providing to a cell a recombinant
hybrid AAV particle produced by the method of claim 37, the
recombinant hybrid AAV particle expressing: (i) adenovirus helper
functions for AAV replication and packaging except the hybrid virus
particle does not express a functional adenovirus E1a gene product,
(ii) AAV rep sequences sufficient for replication and packaging of
the AAV vector genome, and (iii) AAV cap sequences sufficient to
produce a functional AAV capsid, wherein the cell expresses
functional adenovirus E1a gene products; and further wherein the
hybrid virus particle is provided to the cell under conditions
sufficient for replication of the AAV vector genome and packaging
thereof in the AAV capsid such that AAV particles comprising the
AAV vector genome encapsidated within the AAV capsid are produced
in the cell.
52. A method of producing a recombinant hybrid adeno-associated
virus (AAV) particle, comprising providing to a cell: (a) a
recombinant hybrid AAV particle produced by the method of claim 37,
the recombinant hybrid virus AAV particle expressing adenovirus
helper functions for AAV replication and packaging except the
hybrid virus particle does not express a functional adenovirus E1a
gene product, (b) a separate vector comprising inducible AAV rep
sequences sufficient for replication and packaging of the AAV
vector genome, and AAV cap sequences sufficient to produce a
functional AAV capsid, wherein: (i) the cell expresses a functional
adenovirus E1a gene product; and (ii) (a) and (b) are provided to
the cell under conditions sufficient for replication of the AAV
vector genome and packaging thereof in the AAV capsid such that AAV
particles comprising the AAV vector genome encapsidated within the
AAV capsid are produced in the cell.
53. A method of producing a recombinant hybrid adeno-associated
virus (AAV) particle, comprising providing to a cell: (a) a
recombinant hybrid AAV particle produced by the method of claim 37,
the recombinant hybrid virus AAV particle expressing adenovirus
helper functions for AAV replication and packaging, (b) a separate
vector comprising AAV rep sequences sufficient for replication and
packaging of the AAV vector genome, and AAV cap sequences
sufficient to produce a functional AAV capsid, wherein: (i) the
cell does not express a functional adenovirus E1a gene product; and
(ii) (a) and (b) are provided to the cell under conditions
sufficient for replication of the AAV vector genome and packaging
thereof in the AAV capsid such that AAV particles comprising the
AAV vector genome encapsidated within the AAV capsid are produced
in the cell.
54. The method of claim 52, wherein the separate vector is a
plasmid vector.
55. The method of claim 52, wherein the separate vector is an
adenovirus vector.
56-142. (canceled)
143. The method of claim 53, wherein the separate vector is a
plasmid vector.
144. The method of claim 53, wherein the separate vector is an
adenovirus vector.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/511,980, filed Oct. 20, 2004, which is a national phase
filing under 35 U.S.C Section 371 based on PCT International
Application No. PCT/US03/13323, filed Apr. 30, 2003, which is based
on and claims priority to U.S. Provisional Application Ser. No.
60/376,397, filed Apr. 30, 2002; the disclosures of each of which
are herein incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to reagents and methods for
producing viral vectors, in particular stocks of adeno-associated
virus (AAV). The invention further relates to novel viral vectors
and methods of administering the same in vitro and in vivo.
BACKGROUND OF THE INVENTION
[0004] Limitations of AAV vectors include inefficient production
methods, packaging size constraints (introduced gene no larger than
4.5 kb), and a high level of immunity to AAV among adults (although
AAV infection is not associated with any disease). The first AAV
vectors were produced by transfection of 293 cells with two
plasmids (an AAV vector plasmid and an AAV helper plasmid), and
infection with adenovirus (reviewed in Muzyczka, (1992) Curr.
Topics Microbiol. Immunol. 158:97-129). This method provided the
essential elements needed for AAV vector production, including AAV
terminal repeat (TR) sequences flanking a gene of interest, AAV
helper functions consisting of the rep and cap genes, and
adenovirus genes.
[0005] Improvements to the basic method have included: delivery of
adenovirus genes by transfection to eliminate contaminating
adenovirus (Grimm et al. (1998) Hum. Gene Ther. 9:2745-2760,
Matsushita et al. (1998) Gene Ther. 5:938-945, Xiao et al. (1998)
J. Virol. 72:10222-10226); delivery of AAV vector sequences within
an Ad/AAV hybrid vector to increase vector production (Gao et al.
(1998) Gene Ther. 9:2353-2362, Liu et al. (1999) Gene Ther.
6:293-299); and construction of first generation packaging cell
lines containing the AAV rep and cap genes (Clark et al. (1995)
Hum. Gene Ther. 6:1329-1341, Gao et al. (1998) Gene Ther.
9:2353-2362, Inoue & Russell (1998) J. Virol. 72:7024-7031, Liu
et al. (1999) Gene Ther. 6:293-299, Tamayose et al. (1996) Hum.
Gene Ther. 7:507-513, Yang et al. (1994) J. Virol.
68:4847-4856).
[0006] Glycogen storage disease type II (GSD II) presents as a
classical lysosomal storage disorder, characterized by lysosomal
accumulation of glycogen and tissue damage, primarily in muscle and
heart (Hirschhorn et al. (2001) Glycogen Storage Disease Type II:
Acid .alpha.-Glucosidase (Acid Maltase) Deficiency, p. 3389-3419.
In C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle (eds.),
The Metabolic and Molecular Basis for Inherited Disease.
McGraw-Hill, New York). Administration of a modified adenovirus
vector encoding murine GAA or hGAA that was targeted to mouse liver
reversed the glycogen accumulation in a GAA-knockout (GAA-KO) mouse
model for Pompe disease within days, although the effect diminished
with time (Amalfitano et al. (1999) Proc. Natl. Acad. Sci. U.S.A.
96:8861-8866, Ding et al. (2001) Hum. Gene Ther. 12:955-965, Pauly
et al. (2001) Human Gene Ther. 12:527-538). AAV vectors have
reversed the abnormalities in mouse models for hemophilia B (Snyder
et al. (1999) Nat. Med. 5:64-70 [see comments]), Sly disease (Daly
et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2296-2300), and for
Fabry disease (Jung et al. (2001) Proc. Natl. Acad. Sci. U.S.A.
98:2676-2681) with long-term benefits, but not yet for GSD II.
[0007] In accordance with the present invention, highly increased
AAV vector packaging with a hybrid Ad-AAV vector has been observed,
and a modified adenovirus has been utilized, such that no
contaminating Ad particles are produced during AAV packaging. Both
the Ad and AAV versions of the vector encoding hGAA have been
administered to the GAA-KO mouse model for GSD II. The hybrid
Ad-AAV vector provides advantages for the development of gene
therapy for GSD II, including but not limited to: (a) transgene
delivery in vivo; (2) improved packaging of an AAV vector that
delivered human GAA in the GAA-KO mouse; and (3) a combination
thereof.
SUMMARY OF INVENTION
[0008] One aspect of the present invention is based on an improved
method for producing stocks of adeno-associated virus (AAV) using a
novel hybrid adenovirus comprising a recombinant AAV vector genome
embedded within the adenovirus backbone. Traditional methods of AAV
production are known to have a number of drawbacks, including, low
yield, contamination with adenovirus particles, and reliance on
E1a+ packaging cell lines (e.g., 293 cells). The novel hybrid
adenovirus comprises an adenovirus backbone that does not express a
functional polymerase and/or preterminal protein (pTP), for
example, by deletion within these regions. The resulting virus is
replication-incompetent and cannot complete viral replication or
produce new virus particles. The hybrid adenovirus may contain
deletions in regions other than the polymerase or pTP regions.
Moreover, the hybrid adenovirus may be E1a+ or E1a-.
[0009] Use of the inventive reagents and methods may improve AAV
production titers, reduce or even essentially eliminate
contamination with adenovirus, and allow for the use of an E1a+
hybrid adenovirus (or an additional E1a+ adenovirus), thereby
decreasing reliance on an E1a+ packaging cell. Moreover, improved
packaging cell lines expressing the AAV Rep proteins (and,
optionally, capsid protein) may be possible if the E1a genes are
provided by a vector rather than the cell.
[0010] Thus, the present invention provides a recombinant hybrid
virus that can be used to prepare an AAV gene therapy vector,
including AAV pseudotype vectors. In a representative embodiment of
the invention, a recombinant hybrid virus comprises: (a) a deleted
adenovirus vector genome comprising the adenovirus 5' and 3'
cis-elements for viral replication and encapsidation, and further
comprising a deletion in an adenovirus genomic region selected from
the group consisting of: (i) the polymerase region, wherein said
deletion essentially prevents the expression of a functional
polymerase protein from said deleted region and said hybrid virus
does not otherwise express a functional polymerase protein, (ii)
the preterminal protein region, wherein said deletion essentially
prevents the expression of a functional preterminal protein from
said deleted region, and said hybrid virus does not otherwise
express a functional preterminal protein, and (iii) both the
regions of (i) and (ii); and (b) a recombinant adeno-associated
virus (AAV) vector genome flanked by the adenovirus vector genome
sequences of (a), said recombinant AAV vector genome comprising (i)
AAV 5' and 3' inverted terminal repeats, (ii) and AAV packaging
sequence, and (iii) a heterologous nucleic acid sequence, wherein
said heterologous nucleic acid sequence is flanked by the 5' and 3'
AAV inverted terminal repeats of (i).
[0011] Thus, the invention provides novel AAV vectors for delivery
of a heterologous nucleic acid sequence of interest (e.g., GAA) to
a target cell (e.g., a skeletal muscle, smooth muscle, cardiac
muscle or diaphragm cell) in vitro or in vivo. In particular
embodiments, the novel rAAV vector is a rAAV1, rAAV5, rAAV6 vector
comprising a heterologous nucleic acid sequence encoding GAA. In
other embodiments, the novel rAAV vector is a pseudotyped vector
comprising a capsid from one AAV serotype (e.g., AAV1, AAV5, or
AAV6); ITRs from another serotype (e.g., AAV2 ITRs); and/or a
heterologous nucleic acid sequence encoding GAA. The vector may be
delivered to a desired target cell in vitro or in vivo (e.g., a
skeletal muscle, cardiac muscle, smooth muscle or diaphragm cell).
In preferred embodiments, the invention provides a rAAV6 or
pseudotyped rAAV6 (e.g., with AAV2 terminal repeat sequences)
vector comprising a heterologous nucleic acid sequence encoding a
polypeptide or antisense sequence of interest for delivery to
skeletal, cardiac or diaphragm muscle (e.g., a nucleic acid
sequence encoding GAA, dystrophin, or a sarcoglycan).
[0012] A recombinant hybrid virus of the invention can also be used
directly for gene therapy applications. Thus, the present invention
provides a method for delivering a nucleic acid sequence to a cell
(in vitro or in vivo) using the inventive hybrid adenovirus
particles comprising a recombinant AAV vector genome. Typically,
the recombinant AAV (rAAV) vector genome will comprise the 5' and
3' AAV inverted terminal repeat (ITR) sequences flanking the
transgene of interested. In turn, the rAAV genome is inserted into
the inventive adenovirus backbone (e.g., in the E1a region) to
produce the hybrid virus. Gene delivery using the hybrid adenovirus
may advantageously combine benefits associated with both AAV (e.g.,
possibility for site-specific integration, persistent expression)
and adenovirus (e.g., large carrying capacity for transgenes, high
level infection of target cells). The method may be carried out to
deliver any transgene of interest. Methods of treating subjects
with lysosomal acid .alpha.-glycosidase (GAA) deficiency are of
particular interest.
[0013] Accordingly, it is an object of the present invention to
provide a recombinant hybrid virus, and methods for preparing the
same. The recombinant hybrid virus is useful for therapeutic
applications and for large scale AAV production, among other
applications. This and other objects are achieved in whole or in
part by the present invention.
[0014] An object of the invention having been stated above, other
objects and advantages of the present invention will become
apparent to those skilled in the art after a study of the following
description of the invention and non-limiting Examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1A is a schematic of a hybrid Ad-AAV vector containing
the chicken beta-actin (CB) promoter driving the hGAA cDNA. The
hybrid vector, AdAAVCBGAApA, was constructed by bacterial
recombination. The packaging size for the AAV vector sequence is
4.4 kb.
[0016] FIG. 1B is an autoradiograph depicting Southern blot
analysis of DNase I-resistant hybrid Ad-AAV vector particles
(Ad-AAV hybrid), the plasmid containing the AAV vector sequences
prior to bacterial recombination to produce Ad particles
(pShuttle-AAV), and the AAV vector plasmid (pAAVCBGAApA). DNA was
analyzed with AhdI and BssHII to determine that the AAV TR
sequences were present (not deleted during recombination). AhdI
cuts once in each terminal repeat and BssHII cuts twice in each
terminal repeat, and each restriction digest gives unique fragments
that were present in the recombinant Ad-AAV DNA.
[0017] FIG. 1C is a photograph of a cesium chloride gradient of
hybrid Ad-AAV vector particles. Two viral bands were present, which
equilibrated at positions below a layer of protein at the top of
the gradient.
[0018] FIG. 1D is an autoradiograph depicting Southern blot
analysis of the two viral bands in FIG. 1C. Vector DNA was treated
with DNase I and extracted prior to restriction enzyme analysis and
Southern blotting. Each sample was 10 .mu.l. Lanes (11)-(16)
contain linearized Ads-containing plasmid representing the
indicated number of double-stranded (ds) Ad particles. Therefore,
the vector stock purified from the lower band contained
3.1.times.10.sup.11 DNase I-resistant Ad-AAV vector particles per
ml (lanes 6-9).
[0019] FIG. 1E is a schematic of a hybrid Ad-AAV vector packaging
method for AAV vector purification. 293 cells were transfected with
split AAV helper plasmids and transduced with a hybrid Ad-AAV
vector containing the AAV vector sequences. No contaminating
modified Ad vector is replicated by 293 cells (Amalfitano et al.
(1998) J. Virol. 72:926-933). The AAV vector was purified by
heparin-agarose column method (Zolotukhin et al. (1999) Gene Ther.
6:973-985).
[0020] FIG. 2A is an autoradiograph of a Southern blot depicting
AAV vector packaging with an Ad-AAV hybrid vector. For the
transfection-only method, 293 cells were transfected with plasmids
containing the AAV rep and cap genes driven by heterologous
promoters (Allen et al. (2000) Mol. Ther. 1:88-95) and with the AAV
vector plasmid and pLNCorf6 (Scaria et al. (1995) Gene Ther.
2:295-298) (lane 1 only). For the hybrid Ad-AAV method of AAV
vector packaging, the cells were transduced with the indicated
number of hybrid Ad-AAV vector DNase-I resistant particles, and
transfected with plasmids containing the AAV rep and cap genes (as
shown in FIG. 1D). The Southern blot shows the yield of DNase
I-resistant single-stranded AAV vector genomes per cell for each
condition. Each sample represented 6.times.10.sup.5 293 cells.
Lanes (7) to (11) contained vector plasmid, digested with BglII to
release the double-stranded AAV vector sequences, representing the
indicated number of single-stranded (ss) AAV vector particles.
[0021] FIG. 2B is a bar graph showing that AAV-CBGAApA was packaged
with different Ad and AAV helpers. Five conditions for packaging of
AAV-CBGAApA were evaluated, including transfection of pAAV-CBGAApA
plus split AAV helper plasmids and pLNCorf6 (adapted from Allen et
al., 2000), hybrid Ad-AAV transduction plus transfection of split
AAV helper plasmids, modified Ad ([E1-, polymerase-]AdCMVLacZ)
transduction plus split transfection of split AAV helper plasmids,
wild-type Ad5 infection plus transfection of pACG2 (Xiao et al.
(1998) J. Virol. 72:10222-10226) and pAAV-CBGAApA, and hybrid
Ad-AAV transduction plus transfection of pACG2.
[0022] FIG. 2C is an autoradiograph of a Southern blot that was
performed to quantify the contaminating Ad-AAV genomes. Lanes as
follows: (1) untreated 293 cells, (2) transfection of pAAV-CBGAApA
plus split AAV helper plasmids and pLNCorf6 (adapted from Allen et
al. 2000), (3) hybrid Ad-AAV transduction plus transfection of
split AAV helper plasmids, (4) modified Ad ([E1-,
polymerase-]AdCMVLacZ) transduction plus split transfection of
split AAV helper plasmids, (5) wild-type Ad5 infection plus
transfection of pACG2 (Xiao et al. (1998) J. Virol. 72:10222-10226)
and pAAV-CBGAApA, and (6) hybrid Ad-AAV transduction plus
transfection of pACG2, (7) no sample, and (8)-(12) linearized
Ad5-containing plasmid representing the indicated number of
double-stranded (ds) Ad particles. Each sample represented
6.times.10.sup.5 293 cells.
[0023] FIG. 3A is a bar graph depicting analysis of large-scale AAV
vector packaging with an Ad-AAV hybrid vector. The yield of DNase
I-resistant AAV vector particles for AAV-CBGAApA packaged by
transfection of pLNCorf6, or transduction with the Ad-AAV hybrid to
provide Ad helper functions, compared to a vector encoding
glucose-6-phosphatase, AAV-CBcG6PpA, packaged with pLNCorf6. Twenty
to 40 plate vector preparations were purified (3 vector
preparations per condition), and the yield was calculated per cell
plated. The mean number of AAV vector particles per cell is shown
with the standard deviation indicated.
[0024] FIG. 3B is an autoradiograph of a Southern blot analysis of
AAV-CBGAApA purification, quantified versus titrated vector plasmid
DNA. The samples represent vector DNA extracted from 25 microliters
of sample. Standard amounts of vector plasmids were loaded for
quantitation of vector particles. Lanes represent the following
samples: (1) Crude cell lysate, (2) 40% iodoxinal fraction (3)
Heparin-agarose (HA) column flow-through, (4) HA column wash, (5)
HA column eluate fraction (ef) 1, (6) HA column ef 2, (7) HA column
ef 3, (8) HA column ef 4, (9) HA column ef 2 after dialysis (10) HA
column ef 2 plus 2.5.times.10.sup.10 particles AAV vector plasmid,
(11) HA column ef 2 plus 2.5.times.10.sup.10 particles AAV vector
plasmid, no DNase I added, (12)-(18) vector plasmid representing
the indicated number of single-stranded (ss) AAV vector particles.
Therefore, the purified AAV vector stock contained
4.8.times.10.sup.11 DNase I-resistant vector particles per ml.
[0025] FIG. 3C is an autoradiograph of a Southern blot analysis
that quantitated the contaminating Ad-AAV genomes in the samples
described in FIG. 3B above. Lanes 10-18 differed as follows: (10)
HA column ef 2 plus 2.5.times.10.sup.9 particles Ad-containing
plasmid, (11) HA column ef 2 plus 2.5.times.10.sup.9 particles
Ad-containing plasmid, no DNase I added, (12) No sample, (13)-(17)
linearized Ad5-containing plasmid representing the indicated number
of double-stranded (ds) Ad particles. The residual Ad-AAV in the
AAV vector stock was reduced to less than 1 infectious particle per
10.sup.10 AAV vector particles.
[0026] FIG. 4A depicts Western blot analysis of plasma that was
performed at 3 days following intravenous administration of the
hybrid Ad-AAV (essentially Ad) vector encoding hGAA
(2.times.10.sup.10 vector particles/mouse). Recombinant hGAA
(rhGAA) is shown for reference (2 ng total), and the .about.110 kD
hGAA precursor was detected as expected (Amalfitano et al. (1999)
Proc. Natl. Acad. Sci. U.S.A. 96:8861-8866, Ding et al. (2001) Hum.
Gene Ther. 12:955-965).
[0027] FIG. 4B depicts Western blot analysis of GAA-KO mice that
received a hybrid Ad-AAV vector (2.times.10.sup.10 DNase
I-resistant vector particles) or an AAV vector (4.times.10.sup.10
DNase I-resistant vector particles or 1.times.10.sup.12 DNase
I-resistant vector particles) encoding hGAA by intravenous
administration. Western blot analysis of liver is shown at 2 and 6
weeks after vector administration for each group (n=3 for each
group). (Note: hGAA in mouse liver migrates slightly faster than
rhGAA.) GAA-KO mice that received an AAV vector (1.times.10.sup.12
vector particles) encoding hGAA by intravenous administration (n=2)
shown 6 weeks after vector administration. Untreated, affected
GAA-KO mouse liver is shown for comparison (No vector, n=2). For
the higher number of AAV vector particles, the .about.67 kD,
.about.76 kD, and .about.110 kD hGAA species were detected as
expected (Amalfitano et al. (1999) Proc. Natl. Acad. Sci. U.S.A.
96:8861-8866, Ding et al. (2001) Hum. Gene Ther. 12:955-965).
[0028] FIGS. 5A-5C depict human GAA secretion and uptake following
portal vein injection of an AAV vector in GAA-KO mice.
[0029] FIG. 5A depicts Western blot analysis of plasma from
GAAKO\SCID mice at the indicated times following portal vein
injection of the indicated AAV vector encoding hGAA, and from
untreated, GAA-KO\SCID mice (Controls). Each lane represents an
individual mouse.
[0030] FIG. 5B is a bar graph that summarizes GAA analysis for
tissues following portal vein injection of an AAV vector.
GAA-KO/SCID mice received the vector packaged as AAV2 (n=1) or AAV6
(n=1). Controls were age-matched, untreated GAA-KO/SCID mice (n=2).
The GAA level was analyzed twice, independently, and the average
and range are shown.
[0031] FIG. 5C is a photomicrograph depicting periodic acid Schiff
(PAS) staining of the heart for a GAA-KO/SCID mouse that received
an AAV vector (left panel--AAV-CBGAApA) and for an untreated
GAA-KO/SCID mouse (right panel) and HE staining (lower panels).
Magnification 100.times..
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention is explained in greater detail below.
This description is not intended to be a detailed catalog of all
the different ways in which the invention may be implemented, or
all the features that may be added, to the instant invention. For
example, features illustrated with respect to one embodiment may be
incorporated into other embodiments, and features illustrated with
respect to a particular embodiment may be deleted from that
embodiment. In addition, numerous variations and additions to the
various embodiments suggested herein will be apparent to those
skilled in the art in light of the instant disclosure that do not
depart from the instant invention. Hence, the following
specification is intended to illustrate some particular embodiments
of the invention, and not to exhaustively specify all permutations,
combinations and variations thereof.
[0033] The present invention will now be described with reference
to the accompanying drawings, in which preferred embodiments of the
invention are shown. This invention may, however, be embodied in
different forms and should not be construed as limited to the
embodiments set forth herein. Rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the invention to those skilled in
the art.
[0034] The references cited in the specification are incorporated
herein by reference to the extent that they supplement, explain,
provide a background for or teach methodology, techniques and/or
compositions employed herein.
[0035] The following U.S. patents are herein incorporated by
reference in their entirety: U.S. Pat. Nos. 6,328,958; 6,258,595;
6,294,370; 5,872,005; 6,270,996; 6,329,181; 6,251,677; 5,871,982;
and 6,156,303.
I. DEFINITIONS
[0036] While the following terms are believed to be well understood
by one of ordinary skill in the art, the following definitions are
set forth to facilitate explanation of the invention. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention.
[0037] The term "adenovirus" as used herein is intended to
encompass all adenoviruses, including the Mastadenovirus and
Aviadenovirus genera. To date, at least forty-seven human serotypes
of adenoviruses have been identified (see, e.g., FIELDS et al.,
VIROLOGY, volume 2, chapter 67 (3d ed., Lippincott-Raven
Publishers). Preferably, the adenovirus is a serogroup C
adenovirus, still more preferably the adenovirus is serotype 2
(Ad2) or serotype 5 (Ad5).
[0038] Except as otherwise indicated, standard methods may be used
for the construction of the recombinant adenovirus genomes, helper
adenoviruses, and packaging cells according to the present
invention. Such techniques are known to those skilled in the art.
See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL
2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al.
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing
Associates, Inc. and John Wiley & Sons, Inc., New York).
[0039] Those skilled in the art will appreciate that the inventive
adenovirus vectors may be modified or "targeted" as described in
Douglas et al., (1996) Nature Biotechnology 14:1574; U.S. Pat. No.
5,922,315 to Roy et al.; U.S. Pat. No. 5,770,442 to Wickham et al.;
and/or U.S. Pat. No. 5,712,136 to Wickham et al. (the disclosures
of which are all incorporated herein in their entirety).
[0040] As used herein, the term "vector" or "gene delivery vector"
may refer to an Ad particle that functions as a gene delivery
vehicle, and which comprises vDNA (i.e., the vector genome)
packaged within an Ad capsid. Alternatively, in some contexts, the
term "vector" may be used to refer to the vector genome/vDNA.
[0041] An "Ad vector genome" refers to the viral genomic DNA, in
either its naturally occurring or modified form. Thus, the term "Ad
vector genome" also refers to nucleic acids derived from an Ad
genome. A "rAd vector genome" is a recombinant Ad genome (i.e.;
vDNA) that comprises one or more heterologous nucleotide
sequence(s). The Ad vector genome or rAd vector genome will
typically comprise the Ad terminal repeat sequences and packaging
signal. An "Ad particle" or "rAd particle" comprises an Ad vector
genome or rAd vector genome, respectively, packaged within an Ad
capsid. Generally, the Ad vector genome is most stable at sizes of
about 28 kb to 38 kb. (approximately 75% to 105% of the native
genome size). In the case of an adenovirus vector containing large
deletions and a relatively small transgene, "stuffer DNA" can be
used to maintain the total size of the vector within the desired
range by methods known in the art.
[0042] By "infectious", as used herein, it is meant that the
adenovirus can enter the cell by natural transduction mechanisms
and express the transgene therein. Alternatively, an "infectious"
adenovirus is one that can enter the cell by other mechanisms and
express the transgene therein. As one illustrative example, the
vector can enter a target cell by expressing a ligand or binding
protein for a cell-surface receptor in the adenovirus capsid or by
using an antibody(ies) directed against molecules on the
cell-surface followed by internalization of the complex, as is
described hereinbelow.
[0043] The term "replication" or "Ad replication" as used herein,
refers specifically to replication (i.e., making new copies of) of
the Ad vector genome (i.e., virion DNA).
[0044] The term "propagation" as used herein refers to a productive
viral infection wherein the viral genome is replicated and packaged
to produce new virions, which typically can "spread" by infection
of cells beyond the initially infected cell. A
"propagation-defective" virus is impaired in its ability to produce
a productive viral infection and spread beyond the initially
infected cell.
[0045] The terms "nucleic acid molecule" or "nucleic acid" each
refer to deoxyribonucleotides or ribonucleotides and polymers
thereof in single-stranded, double-stranded, or triplexed form.
Unless specifically limited, the term encompasses nucleic acids
containing known analogues of natural nucleotides that have similar
properties as the reference natural nucleic acid. The terms
"nucleic acid molecule" or "nucleic acid" can also be used in place
of "gene", "cDNA", or "mRNA". Nucleic acids can be synthesized, or
can be derived from any biological source, including any
organism.
[0046] The term "heterologous nucleic acids" refers to a sequence
that originates from a source foreign to an intended host cell or,
if from the same source, is modified from its original form. Thus,
a heterologous nucleic acid in a host cell includes a gene that is
endogenous to the particular host cell, but which has been
modified, for example by mutagenesis or by isolation from native
cis-regulatory sequences. The term "heterologous nucleic acid" also
includes non-naturally occurring multiple copies of a native
nucleotide sequence. The term "heterologous nucleic acid" also
encompasses a nucleic acid that is incorporated into a host cell's
nucleic acids, however at a position wherein such nucleic acids are
not ordinarily found. The term "transgene" is also used herein
interchangeably with the term "heterologous nucleic acid."
[0047] The term "recombinant" generally refers to an isolated
nucleic acid that is replicable in a non-native environment. Thus,
a recombinant nucleic acid can comprise a non-replicable nucleic
acid in combination with additional nucleic acids, for example
vector nucleic acids, which enable its replication in a host cell.
The term "recombinant" is also used to describe a vector (e.g., an
adenovirus or an adeno-associated virus) comprising recombinant
nucleic acids.
[0048] The term "gene" refers broadly to any segment of DNA
associated with a biological function. A gene can comprise
sequences including but not limited to a coding sequence, a
promoter region, a cis-regulatory sequence, a non-expressed DNA
segment that is a specific recognition sequence for regulatory
proteins, a non-expressed DNA segment that contributes to gene
expression, a DNA segment designed to have desired parameters, or
combinations thereof. A gene can be obtained by a variety of
methods, including cloning from a biological sample, synthesis
based on known or predicted sequence information, and recombinant
derivation of an existing sequence.
[0049] The term "operatively linked", as used herein, refers to a
functional combination between a promoter region and a nucleic acid
molecule such that the transcription of the nucleic acid molecule
is controlled and regulated by the promoter region. Techniques for
operatively linking a promoter region to a nucleic acid molecule
are known in the art.
[0050] The term "vector" is used herein to refer to a nucleic acid
molecule having nucleotide sequences that enable its replication in
a host cell. A vector can also include nucleic acids to permit
ligation of nucleotide sequences within the vector, wherein such
nucleic acids are also replicated in a host cell. Representative
vectors include plasmids, cosmids, and viral vectors. The term
"vector" is also used to describe an expression construct, wherein
the expression construct comprises a vector and a nucleic acid
operatively inserted with the vector, such that the nucleic acid is
expressed.
[0051] Vectors can also comprise nucleic acids including expression
control elements, such as transcription/translation control
signals, origins of replication, polyadenylation signals, internal
ribosome entry sites, promoters, enhancers, etc., wherein the
control elements are operatively associated with a nucleic acid
encoding a gene product. Selection of these and other common vector
elements are conventional and many such sequences can be derived
from commercially available vectors. See e.g., Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, and references cited
therein.
[0052] The terms "cis-acting regulatory sequence" or
"cis-regulatory motif" or "response element", as used herein, each
refer to a nucleotide sequence within a promoter region that
enables responsiveness to a regulatory transcription factor.
Responsiveness can encompass a decrease or an increase in
transcriptional output and is mediated by binding of the
transcription factor to the DNA molecule comprising the response
element.
[0053] The term "transcription factor" generally refers to a
protein that modulates gene expression by interaction with the
cis-regulatory element and cellular components for transcription,
including RNA Polymerase, Transcription Associated Factors (TAFs),
chromatin-remodeling proteins, reverse tet-responsive
transcriptional activator, and any other relevant protein that
impacts gene transcription.
[0054] The term "promoter" defines a region within a gene that is
positioned 5' to a coding region of a same gene and functions to
direct transcription of the coding region. The promoter region
includes a transcriptional start site and at least one
cis-regulatory element. The term "promoter" also includes
functional portions of a promoter region, wherein the functional
portion is sufficient for gene transcription. To determine
nucleotide sequences that are functional, the expression of a
reporter gene is assayed when variably placed under the direction
of a promoter region fragment.
[0055] Promoter region fragments can be conveniently made by
enzymatic digestion of a larger fragment using restriction
endonucleases or DNAse I. Preferably, a functional promoter region
fragment comprises about 5000 nucleotides, more preferably 2000
nucleotides, more preferably about 1000 nucleotides. Even more
preferably a functional promoter region fragment comprises about
500 nucleotides, even more preferably a functional promoter region
fragment comprises about 100 nucleotides, and even more preferably
a functional promoter region fragment comprises about 20
nucleotides.
[0056] The term "about", as used herein when referring to a
measurable value such as an amount of virus (e.g., titer), dose
(e.g. an amount of a chemical inducer), time, temperature (e.g., a
temperature for induction of a heat-inducible promoter), etc., is
meant to encompass variations of .+-.20% or .+-.10%, more
preferably .+-.5%, even more preferably .+-.1%, and still more
preferably .+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed method.
[0057] As used in the description of the invention and the appended
claims, the singular forms "a", "an" and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise.
II. HYBRID VECTORS
[0058] In a preferred embodiment, a hybrid vector in accordance
with the present invention comprises a deleted adenovirus vector
genome comprising the adenovirus 5' and 3' cis-elements for viral
replication and encapsidation, and further comprising a deletion in
an adenovirus genomic region selected from the group consisting of:
(i) the polymerase region, wherein said deletion essentially
prevents the expression of a functional polymerase protein from
said deleted region and said hybrid virus does not otherwise
express a functional polymerase protein, (ii) the preterminal
protein region, wherein said deletion essentially prevents the
expression of a functional preterminal protein from said deleted
region, and said hybrid virus does not otherwise express a
functional preterminal protein, and (iii) both the regions of (i)
and (ii).
[0059] Continuing with a preferred embodiment, a hybrid vector of
the present invention further comprises a recombinant
adeno-associated virus (AAV) vector genome flanked by the
adenovirus vector genome sequences disclosed above. The recombinant
AAV vector genome comprise (i) AAV 5' and 3' inverted terminal
repeats, (ii) an AAV packaging sequence, and (iii) a heterologous
nucleic acid sequence, wherein the heterologous nucleic acid
sequence is flanked by the 5' and 3' AAV inverted terminal repeats
of (i).
[0060] A hybrid virus particle comprising a recombinant hybrid
virus of the present invention is also provided. The hybrid virus
particle is encapsidated within an adenovirus capsid. A cell
comprising the recombinant hybrid virus or the hybrid virus
particle of the present invention is also provided.
[0061] II.A. Deleted Adenovirus Vectors
[0062] As noted above, a deleted adenovirus vector genome of the
present invention preferably comprises the adenovirus 5' and 3'
cis-elements for viral replication and encapsidation, and further
comprises a deletion in an adenovirus genomic region selected from
the group consisting of: (i) the polymerase region, wherein the
deletion essentially prevents the expression of a functional
polymerase protein from the deleted region and the hybrid virus
does not otherwise express a functional polymerase protein, (ii)
the preterminal protein region, wherein the deletion essentially
prevents the expression of a functional preterminal protein from
the deleted region, and the hybrid virus does not otherwise express
a functional preterminal protein, and (iii) both the regions of (i)
and (ii).
[0063] Optionally, the adenovirus 5' and 3' cis-elements comprise
5' and 3' adenovirus inverted terminal repeats and an adenovirus
packaging sequence. In this case, the adenovirus packaging sequence
can further comprise the E1A enhancer, which is embedded in the
packaging signal.
[0064] The adenovirus vectors of the invention have an impairment
in polymerase (pol) activity, preterminal protein region (pTP)
activity, or a combination thereof (e.g., produced reduced levels
of functional pol protein and/or functional pTP protein).
Preferably, the Ad vector produces essentially no detectable pol or
pTP activity. The [pol-], [rTP-], and [pol-, pTP-] adenoviral
vectors of the invention are replication competent, but impaired in
their ability to propagate (as defined above). Stated another way,
a deleted Ad vector of the present invention is impaired in its
ability to package new virions in the absence of
transcomplementation, which is provided, for example, by a
packaging cell that expresses the Ad pol protein, by a packaging
cell that expresses the Ad pTP protein, or by a packaging cell that
expresses both the Ad pol protein and the Ad pTP protein.
[0065] A [pol-] Ad of the invention preferably has essentially no
detectable pol activity (alternatively, essentially no detectable
pol transcript or protein). Alternatively, in other embodiments,
pol activity (alternatively, pol transcripts or protein) can be
reduced by about 70%, 80%, 90%, 95%, 98%, 99% or more as compared
with wild-type Ad or [pol+] Ad.
[0066] A [pTP-] Ad of the invention preferably has essentially no
detectable pTP activity (alternatively, essentially no detectable
pTP transcript or protein). Alternatively, in other embodiments,
pTP activity (alternatively, pTP transcripts or protein) can be
reduced by about 70%, 80%, 90%, 95%, 98%, 99% or more as compared
with wild-type Ad or [pTP+] Ad.
[0067] A [pol-, pTP-] Ad of the invention preferably has
essentially no detectable pol activity and have essentially no
detectable pTP activity (alternatively, essentially no detectable
pol transcript or protein and essentially no detectable pTP
transcript or protein). Alternatively, in other embodiments, pol
and pTP activity (alternatively, pol transcripts or protein and pTP
transcripts or protein) can be reduced by about 70%, 80%, 90%, 95%,
98%, 99% or more as compared with wild-type Ad or [pol+, pTP+]
Ad.
[0068] Alternatively stated, a propagation-defective Ad of the
invention is impaired in their ability to produce a productive
infection in the absence of transcomplementation. Preferably,
essentially no new virions are detected following infection with
the inventive [pol-], [rTP-], and [pol-, pTP-] adenoviral vectors.
Alternatively, production of new virions in infected cells may be
reduced by at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or
more as compared with a wild-type Ad infection or, alternatively,
as compared with new virion production in a cell line that can
transcomplement the loss of pol function and/or the loss of pTP
function.
[0069] The Ad genome can be modified by any mutation known in the
art (e.g., an insertion, missense, nonsense and/or deletion
mutation), so as to result in an impairment in pol activity
expressed by the Ad genome and/or so as to produce an impairment in
pTP activity expressed in the Ad genome. Preferably, the mutation
or alteration to the pol coding region is a deletion mutation, more
preferably a deletion mutation that essentially ablates (e.g.,
essentially eliminates) pol activity. Preferably, the mutation in
the pol and/or pTP coding regions are not temperature-sensitive
mutations.
[0070] The term "deleted vector," as used herein to describe a type
of Ad, refers to an Ad wherein one or more, but not all Ad genes
have been deleted. Thus, the [pol-], [rTP-], and [pot-, pTP-]
adenoviral vectors of the invention specifically exclude "gutted"
adenovirus vectors (as that term is understood in the art, see
e.g., Lieber, et al., (1996) J. Virol. 70:8944-60) in which
essentially all of the adenovirus genomic sequences are
deleted.
[0071] Thus, in preferred embodiments, the vector genome packaged
within [pol-] particle has one or more deletions in the pol coding
region. Similarly, a vector genome packaged within a [pTP-]
particle has one or more deletions in the pTP coding region. The
deletion(s) preferably prevents, or essentially prevents, the
expression of a functional form of a pol protein and/or a pTP
protein from the deleted region.
[0072] The term "produces essentially no functional protein," as
used herein, means that essentially no pol and/or pTP protein or
activity is detectable (e.g., at most, only an insignificant amount
is detectable) following infection of non-complementing cells with
the inventive [pol-], [pTP-], and [pol-, pTP-] adenoviral vectors.
The defect resulting in essentially no functional protein can be at
the level of transcription, translation and/or post-translational
processes. Thus, even if there is transcription and translation of
the pol and/or pTP gene, the resulting protein has essentially no
detectable biological activity. Pol and pTP activities can be
evaluated by any method known in the art.
[0073] As used herein, a "functional" protein is one that retains
at least one biological activity normally associated with that
protein. Preferably, a "functional" protein retains all of the
activities possessed by the unmodified protein. A "non-functional"
protein is one that exhibits essentially no detectable biological
activity normally associated with the protein (e.g., at most, only
an insignificant amount).
[0074] The term "deleted" as used herein refers to the omission of
at least one nucleotide from the relevant coding region of the
adenovirus genome. Deletions can be greater than about 1, 2, 3, 5,
7, 10, 15, 20, 50, 75, 100, 150, 200, or even 500 nucleotides, or
more. Deletions in the relevant coding region of the adenovirus
genome can be at least about 1%, 5%, 10%, 25%, 50%, 75%, 85%, 90%,
95%, 99%, or more of the coding region. Alternately, the entire
coding region of interest (e.g., the entire pol coding region
and/or the entire pTP coding region) of the adenovirus genome is
deleted. Preferably, the deletion prevents or essentially prevents
the expression of a functional protein from the coding region.
[0075] In a preferred embodiment of the invention, a deletion
comprises a pol region including about nucleotide 7274 to about
nucleotide 7881 of an adenovirus serotype 5 genome. Those skilled
in the art will appreciate that similar deletions can be made in
the homologous regions of the adenovirus genomes from other
serotypes.
[0076] In another preferred embodiment of the invention, a deletion
comprises a pTP region including about nucleotide 9198 to about
nucleotide 9630 of an adenovirus serotype 5 genome. Those skilled
in the art will appreciate that similar deletions can be made in
the homologous regions of the adenovirus genomes from other
serotypes.
[0077] The various regions of the adenovirus genome have been
mapped and are understood by those skilled in the art (see, e.g.,
FIELDS et al., VIROLOGY, volume 2, chapters 67 and 68 (3d ed.,
Lippincott-Raven Publishers). The genomic sequences of the various
Ad serotypes, as well as the nucleotide sequence of the particular
coding regions of the Ad genome, are known in the art and may be
accessed, e.g., from GenBank.
[0078] In general, larger deletions are preferred as these have the
additional advantage that they will increase the capacity of the
deleted adenovirus to carry a heterologous nucleotide sequence of
interest.
[0079] In particular embodiments, [pol-], [ptP-], and [pol-, ptP-]
adenoviral vectors of the invention contain mutations or deletions
in other regions of the Ad genome. Additional deletions will
advantageously increase the carrying capacity of the vector and
reduce the likelihood of recombination to generate replication
competent virus. Preferably, the additional deletions do not unduly
impair the ability of the resulting virus to replicate in desired
target cells (e.g., does not reduce replication by more than about
40%, 50%, 60%, 70% or more). For example, the E3 coding region can
be deleted, without the provision of E3 by
transcomplementation.
[0080] In one embodiment, the adenovirus vector genome further
comprises a deletion in an adenovirus E1 region. In this case, the
deletion preferably essentially prevents the expression of one or
more functional E1 proteins from the deleted region. More
preferably, the adenovirus vector genome does not otherwise express
a functional E1 gene product. Optionally, a recombinant AAV genome
as disclosed herein is inserted into the deleted adenovirus E1
region of the adenovirus vector genome.
[0081] Additionally, the adenovirus vector genome can further
comprise a deletion in an adenovirus region selected from the group
consisting of the IVa2 region, the 100K region, the E2a region, the
E4 region, the L1 region, the L2 region, the L3 region, the L4
region, the L5 region, the intermediate gene IX region, and any
combination of the foregoing. In this case it is preferred that the
adenovirus vector genome does not otherwise express a gene product
associated with the deleted region. By way of particular example,
open reading frames (orf's) 1-5 of the E4 region can be
deleted.
[0082] It should be noted that the E2a region and the E4orf6 region
facilitate high level AAV production. Thus, if these regions are
deleted, it is preferred that these regions be provided elsewhere,
e.g., by a second Ad, by a packaging cell, or by a plasmid. Also,
preferably, the deletion(s) in the Ad genome are selected so as not
to interfere with other Ad functions essential for viral
replication in target cells of interest.
[0083] The inventive deleted adenoviruses are impaired in their
ability to propagate (i.e., produce new virions) without
complementation to compensate for the loss of pol and/or pTP
function, e.g., by a packaging cell. As described in more detail
hereinbelow, the packaging cell will typically be stably modified
to express a functional pol and/or pTP protein. In the presence of
transcomplementing functions, the [pol-], [pTP-], and [pol-, pTP-]
adenoviral vectors of the invention can replicate and package new
virions.
[0084] The adenovirus vector genome can further comprise nucleic
acids encoding an AAV capsid protein. The adenovirus vector genome
can also comprise nucleic acids encoding an AAV Rep protein. In
this case, the sequences encoding the AAV Rep protein can be
operably associated with an inducible promoter. The inducible
promoter can be selected from a group including but not limited to
a tetracycline response element, an ecdysone response element, a
heat shock promoter, an MMLV long terminal repeat sequence, a
bacteria phage T7 promoter, a metalothionein response element, and
an AAV p5 promoter. Alternatively, the sequences encoding said AAV
Rep protein are operably associated with a tissue-specific promoter
selected from a group including but not limited to a
liver-specific, muscle-specific, and brain-specific promoter.
[0085] The adenovirus vector genome can optionally comprise nucleic
acids encoding the adenovirus helper functions for AAV replication
and packaging. In this case, the adenovirus vector genome
preferably comprises a functional adenovirus genomic region
selected from the group consisting of an adenovirus E1a region, E2a
region, E4orf6 region, VA RNA region, and any combination of the
foregoing.
[0086] II.B. Recombinant Adeno-Associated Virus Vector Genome
[0087] In a preferred embodiment a hybrid vector in accordance with
the present invention further comprises a recombinant
adeno-associated virus (AAV) vector genome flanked by the
adenovirus vector genome sequences disclosed herein above. The
recombinant AAV vector genome preferably comprises: (i) AAV 5' and
3' inverted terminal repeats, (ii) an AAV packaging sequence, and
(iii) a heterologous nucleic acid sequence, wherein the
heterologous nucleic acid sequence is flanked by the 5' and 3' AAV
inverted terminal repeats of (i).
[0088] More preferably, the AAV inverted terminal repeats are
selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4,
AAV-5 and AAV-6 inverted terminal repeats. Optionally, the AAV
vector genome does not encode the AAV Rep or AAV capsid
proteins.
[0089] II.C. Heterologous Nucleotide Sequences
[0090] As described in more detail hereinbelow, any of the
inventive hybrid viruses described above can further comprise one
or more heterologous nucleotide sequences (e.g., two, three, four,
five, six or more sequences) of interest. For example, in one
embodiment, the heterologous nucleic acid sequence encodes an
antisense nucleic acid sequence.
[0091] In another embodiment the heterologous nucleic acid sequence
is operatively associated with an expression control sequence. A
representative expression control sequence comprises a promoter.
Representative promoters include but are not limited to a
liver-specific, muscle-specific, brain-specific promoter and a
glucose-responsive promoter. A preferred liver-specific promoter is
the alpha1 anti-trypsin promoter. A preferred glucose-responsive
promoter is the canine glucose-6-phosphatase promoter. (Kishnani et
al. (2001) Vet. Path. 38:83-91). Optionally, the promoter is an
inducible promoter. Other representative promoters include but are
not limited to the CMV promoter, albumin promoter, EF1-.alpha.
promoter, P.gamma.K promoter, MFG promoter, and Rous sarcoma virus
promoter.
[0092] In another embodiment the heterologous nucleic acid sequence
encodes a polypeptide. Representative polypeptides include but are
not limited to a therapeutic polypeptide, an immunogenic
polypeptide, and a reporter polypeptide. Particular examples of
such polypeptides are set forth herein below.
III. METHODS FOR MAKING VIRAL VECTORS
[0093] Methods for making viral vectors are also provided in
accordance with the present invention. In a preferred embodiment, a
method of producing a recombinant adeno-associated virus (AAV)
particle is provided. The method comprises providing to a cell: (a)
a recombinant hybrid virus according to o the present invention or
a hybrid virus particle comprising a recombinant hybrid virus
encapsidated with an adenovirus capsid; (b) AAV sequences
sufficient for replication and packaging of the AAV vector genome;
(c) AAV sequences sufficient to produce a functional AAV capsid,
wherein (a), (b) and (c) are provided to the cell under conditions
sufficient for replication of the AAV vector genome and packaging
thereof in the AAV capsid such that AAV particles comprising the
AAV vector genome encapsidated within the AAV capsid are produced
in the cell. Representative cells include but are not limited to a
HeLa cell, a 293 cell, a muscle cell, and a liver cell. The
recombinant AAV particle can be collected in any desired manner. In
a preferred embodiment recombinant AAV particles are collected by
column purification.
[0094] Optionally, the method further comprises providing to the
cell the adenovirus helper functions for AAV replication and
packaging. Representative sequences that provide adenovirus helper
functions include but are not limited to adenovirus E1a, E2a, E4 or
16, and VA RNA helper sequences.
[0095] In one embodiment, the cell stably expresses sequences
encoding an AAV Rep protein and/or sequences encoding the AAV
capsid protein. The term "stable", as used herein with respect to
gene expression; is meant to refer to and encompass inducible
and/or constitutive expression. In another embodiment, a vector
other than the recombinant hybrid virus provides sequences encoding
an AAV Rep protein and/or sequences encoding the AAV capsid
protein. Representative such vectors include but are not limited to
a plasmid, an adenovirus, an Epstein Barr virus, and a herpesvirus
vector.
[0096] Optionally, the AAV inverted terminal repeats and the AAV
capsid are derived from different AAV serotypes. In one embodiment,
the AAV inverted terminal repeats are AAV-2 inverted terminal
repeats. In another embodiment, the AAV capsid is an AAV-6 capsid.
In yet another embodiment, the AAV inverted terminal repeats are
AAV-2 inverted terminal repeats and the AAV capsid is an AAV-6
capsid. The term AAV-6 capsid refers to an AAV6 capsid comprising
one or more AAV6 capsid proteins.
[0097] Preferably, essentially no adenovirus particles are produced
when AAV are prepared using a deleted Ad or the present invention.
Also preferably, the yield of recombinant AAV particles is at least
5-fold greater than AAV prepared using a pol+ and/or a pTP+ Ad.
[0098] A method of producing a recombinant adeno-associated virus
(AAV) particle is also provided. The method comprises providing to
a cell a hybrid virus particle according to the present invention,
the recombinant hybrid virus particle expressing the adenovirus
helper functions for AAV replication and packaging; wherein the
cell (i) expresses AAV rep sequences sufficient for replication and
packaging of the AAV vector genome, (ii) expresses AAV cap
sequences sufficient to produce a functional AAV capsid, and (iii)
does not express sequences sufficient to produce a functional
adenovirus E1a protein; and further wherein the hybrid virus
particle is provided under conditions sufficient for replication of
the AAV vector genome and packaging thereof in the AAV capsid such
that AAV particles comprising the AAV vector genome encapsidated
within the AAV capsid are produced in the cell.
[0099] In another embodiment, the method of producing a recombinant
adeno-associated virus (AAV) particle comprises providing to a cell
a hybrid virus particle according to the present invention. In this
embodiment, the hybrid virus particle expresses: (i) adenovirus
helper functions for AAV replication and packaging except the
hybrid virus particle does not express a functional adenovirus E1a
gene product, (ii) AAV rep sequences sufficient for replication and
packaging of the AAV vector genome, and (iii) AAV cap sequences
sufficient to produce a functional AAV capsid. The cell expresses
functional adenovirus E1a gene products; and the hybrid virus
particle is provided to the cell under conditions sufficient for
replication of the AAV vector genome and packaging thereof in the
AAV capsid such that AAV particles comprising the AAV vector genome
encapsidated within the AAV capsid are produced in the cell.
[0100] In yet another embodiment, the method of producing a
recombinant adeno-associated virus (AAV) particle comprises
providing to a cell: (a) a hybrid virus particle according to the
present invention; and (b) a separate vector comprising inducible
AAV rep sequences sufficient for replication and packaging of the
AAV vector genome, and AAV cap sequences sufficient to produce a
functional AAV capsid. In this embodiment, the hybrid virus
particle expresses adenovirus helper functions for AAV replication
and packaging except the hybrid virus particle does not express a
functional adenovirus E1a gene product. The cell expresses a
functional adenovirus E1a gene product; and the hybrid virus
particle and separate vector are provided to the cell under
conditions sufficient for replication of the AAV vector genome and
packaging thereof in the AAV capsid such that AAV particles
comprising the AAV vector genome encapsidated within the AAV capsid
are produced in the cell.
[0101] In yet a further embodiment, a method of producing a
recombinant adeno-associated virus (AAV) particle, comprising
providing to a cell: (a) a hybrid virus particle according to the
present invention; and (b) a separate vector comprising AAV rep
sequences sufficient for replication and packaging of the AAV
vector genome, and AAV cap sequences sufficient to produce a
functional AAV capsid. The hybrid virus particle expresses
adenovirus helper functions for AAV replication and packaging. The
cell does not express a functional adenovirus E1a gene product; and
the hybric virus particle and the separate vector are provided to
the cell under conditions sufficient for replication of the AAV
vector genome and packaging thereof in the AAV capsid such that AAV
particles comprising the AAV vector genome encapsidated within the
AAV capsid are produced in the cell.
[0102] In each of the two immediately preceeding embodiments the
separate vector is preferably one of a plasmid vector and an
adenovirus vector.
IV. METHODS FOR USING VIRAL VECTORS
[0103] The present invention provides methods that employ the viral
vectors disclosed herein, including hybrid Ad vectors and AAV
vectors, in particular AAV6 vectors.
[0104] As used herein, a "viral vector" is a virus that carries one
or more heterologous nucleotide sequences (i.e., transgenes), e.g.,
two, three, four, five or more heterologous nucleotide sequences.
The viral vectors of the present invention are useful for the
delivery of nucleic acids to cells in vitro, ex vivo, and in vivo.
In particular, the inventive vectors can be advantageously employed
to deliver or transfer nucleic acids to animal cells, more
preferably to mammalian cells. Preferably, the sequence is
expressed in the cell. Nucleic acids of interest include nucleic
acids encoding polypeptides, preferably therapeutic (e.g., for
medical or veterinary uses) or immunogenic (e.g., for vaccines)
polypeptides. In one embodiment, site-specific integration into
human chromosome 19 in cells or in vivo can be accomplished by
providing AAV Rep 68/78 protein (or sequences encoding the same)
along with a hybrid vector of the present invention.
[0105] Alternatively, in particular embodiments of the invention,
the nucleic acid of interest may encode therapeutic nucleic acids
such as an antisense nucleic acid, a ribozyme, (e.g., as described
in U.S. Pat. No. 5,877,022), RNAs that effect spliceosome-mediated
trans-splicing (see, Puttaraju et al., (1999) Nature Biotech.
17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702),
interfering RNAs (RNAi) that mediate gene silencing (see, Sharp et
al., (2000) Science 287:2431) or other non-translated RNAs, such as
"guide" RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA
95:4929; U.S. Pat. No. 5,869,248), and the like.
[0106] As a further alternative, the viral vectors of the present
invention can be used to infect a cell in culture to express a
desired gene product, e.g., to produce a polypeptide of interest
(for example, lysosomal acid .alpha.-glucosidase). Preferably, the
polypeptide is secreted into the medium and can be purified
therefrom using routine techniques known in the art. Signal peptide
sequences that direct extracellular secretion of proteins are known
in the art and nucleotide sequences encoding the same can be
operably linked to the nucleotide sequence encoding the polypeptide
of interest by routine techniques known in the art. Alternatively,
the cells can be lysed and the expressed recombinant protein can be
purified from the cell lysate. The cell can be a bacterial,
protozoan, plant, yeast, fungus, or animal cell. Preferably, the
cell is an animal cell (e.g., insect, avian or mammalian), more
preferably a mammalian cell. Also preferred are cells that are
permissive for transduction by viral vectors.
[0107] The inventive methods may be used to express any polypeptide
of interest, e.g., a therapeutic polypeptide, as described below.
Alternatively, the polypeptide can be for use in an industrial
process, in particular, an industrial enzyme. Industrial enzymes
are known in the art and include, but are not limited to,
cellulases, lipases, .beta.-glucanases, hemicellulases, alkaline
proteases, .alpha.-amylases, xylanases, catalases, lactases,
pectinases, isoamylases, amyloglucosidases, invertases, phytases,
rennet, and tannases.
[0108] Heterologous nucleotide sequences encoding polypeptides also
include those encoding reporter polypeptides (e.g., an enzyme).
Reporter polypeptides are known in the art and include, but are not
limited to, Green Fluorescent Protein, .beta.-galactosidase,
alkaline phosphatase, and chloramphenicol acetyltransferase
gene.
[0109] The present invention also provides vectors useful as
vaccines. The antigen can be presented in the adenovirus capsid,
alternatively, the antigen can be expressed from a heterologous
nucleic acid introduced into a viral vector. Any immunogen of
interest can be provided by the viral vector. Immunogens of
interest are well-known in the art and include, but are not limited
to, immunogens from human immunodeficiency virus (e.g., envelope
proteins), influenza virus, gag proteins, cancer antigens, HBV
surface antigen (to immunize against hepatitis), rabies
glycoproteins, and the like.
[0110] An immunogenic polypeptide, or immunogen, can be any
polypeptide suitable for protecting the subject against a
pathogenic disease, including but not limited to bacterial,
protozoal, fungal, and viral diseases. For example, the immunogen
can be an orthomyxovirus immunogen (e.g., an influenza virus
immunogen, such as the influenza virus hemagglutinin (HA) surface
protein or the influenza virus nucleoprotein gene, or an equine
influenza virus immunogen), or a lentivirus immunogen (e.g., an
equine infectious anemia virus immunogen, a Simian Immunodeficiency
Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV)
immunogen, such as the HIV or SIV envelope GP160 protein, the HIV
or SIV matrix/capsid proteins, and the HIV or SIV gag, pol and env
genes products).
[0111] The immunogen may also be an arenavirus immunogen (e.g.,
Lassa fever virus immunogen, such as the Lassa fever virus
nucleocapsid protein gene and the Lassa fever envelope glycoprotein
gene), a poxvirus immunogen (e.g., vaccinia, such as the vaccinia
L1 or L8 genes), a flavivirus immunogen (e.g., a yellow fever virus
immunogen or a Japanese encephalitis virus immunogen), a filovirus
immunogen (e.g., an Ebola virus immunogen, or a Marburg virus
immunogen, such as NP and GP genes), a bunyavirus immunogen (e.g.,
RVFV, CCHF, and SFS viruses), or a coronavirus immunogen (e.g., an
infectious human coronavirus immunogen, such as the human
coronavirus envelope glycoprotein gene, or a porcine transmissible
gastroenteritis virus immunogen, or an avian infectious bronchitis
virus immunogen). The immunogen can further be a polio immunogen,
herpes immunogen (e.g., CMV, EBV, HSV immunogens) mumps immunogen,
measles immunogen, rubella immunogen, diptheria toxin or other
diptheria immunogen, pertussis immunogen, hepatitis (e.g.,
hepatitis A or hepatitis B) immunogen, or any other vaccine
immunogen known in the art.
[0112] Alternatively, the immunogen can be any cancer cell antigen
(including tumor cell antigens), or any other antigen that induces
an immune response against cancer cells. A "cancer cell antigen,"
as used herein, is an antigen that is associated cancer in general
or with a particular cancer. Preferably, the cancer cell antigen is
expressed on the surface of the cancer cell. Exemplary cancer cell
antigens are described in S. A. Rosenberg, (1999) Immunity 10:281).
Other illustrative cancer cell antigens include, but are not
limited to: the BRCA1 gene product, BRCA2 gene product, gp100,
tyrosinase, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, .beta.-catenin,
MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma
tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA
91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et
al., (1994) Cancer Res. 54:3124), MART-1 (Coulie et al., (1991) J.
Exp. Med. 180:35), gp100 (Wick et al., (1988) J. Cutan. Pathol.
4:201), MAGE antigen, MAGE-1, MAGE-2 and MAGE-3 (Van der Bruggen et
al., (1991) Science, 254:1643), CEA, TRP-1, TRP-2, P-15, HER-2/neu
gene product (U.S. Pat. No. 4,968,603), CA 125, LK26, FB5
(endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1,
CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 proteins, PSA,
L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor
protein (Levine, (1993) Ann. Rev. Biochem. 62:623), mucin antigens
(international patent publication WO 90/05142), telomerases;
nuclear matrix proteins, prostatic acid phosphatase, papilloma
virus antigens, and antigens associated with the following cancers:
melanomas, metastases, adenocarcinoma, thymoma, lymphoma, sarcoma,
lung cancer, liver cancer, colon cancer, non-Hodgkins lymphoma,
Hodgkins lymphoma, leukemias; uterine cancer, breast cancer,
prostate cancer, ovarian cancer, cervical cancer, bladder cancer,
kidney cancer, pancreatic cancer and others (see, e.g., Rosenberg,
(1996) Ann. Rev. Med. 47:481-91).
[0113] The present invention can be further used to deliver a
therapeutic polypeptide. Representative polypeptides include but
are not limited to insulin, myophosphorylase (associated with
glycogen storage disease V), VEGF, interleukins, p53, Rb and other
anti-cancer proteins.
[0114] Other therapeutic polypeptides include, but are not limited
to, cystic fibrosis transmembrane regulator protein (CFTR),
dystrophin (including the protein product of dystrophin mini-genes,
see, e.g, Vincent et al., (1993) Nature Genetics 5:130),
dystrophin-associated polypeptides, sarcoglycans, glycogen
phosphorylase, utrophin (Tinsley et al., (1996) Nature 384:349),
clotting factors (e.g., Factor XIII, Factor IX, Factor X, etc.),
erythropoietin, angiostatin, endostatin, catalase, tyrosine
hydroxylase, superoxide dismutase, leptin, the LDL receptor,
lipoprotein lipase, ornithine transcarbamylase, .beta.-globin,
.alpha.-globin, spectrin, .alpha.-antitrypsin, adenosine deaminase,
hypoxanthine guanine phosphoribosyl transferase,
.beta.-glucocerebrosidase, sphingomyelinase, lysosomal
hexosaminidase, branched-chain keto acid dehydrogenase, cytokines
(e.g., .alpha.-interferon, .beta.-interferon, interferon-.gamma.,
interleukin-2, interleukin-4, granulocyte-macrophage colony
stimulating factor, lymphotoxin, and the like), peptide growth
factors and hormones (e.g., somatotropin, insulin, insulin-like
growth factors 1 and 2, platelet derived growth factor, epidermal
growth factor, fibroblast growth factor, nerve growth factor,
neurotrophic factor -3 and -4, brain-derived neurotrophic factor,
glial derived growth factor, transforming growth factor -.alpha.
and -.beta., and the like), receptors (e.g., the tumor necrosis
growth factor receptor), monoclonal antibodies (including single
chain monoclonal antibodies). Other illustrative heterologous
nucleotide sequences encode suicide gene products (e.g., thymidine
kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis
factor), proteins conferring resistance to a drug used in cancer
therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), and
any other polypeptide that has a therapeutic effect in a subject in
need thereof.
[0115] In a preferred embodiment of the present invention, the
heterologous nucleic acid encodes a polypeptide associated with
muscular dystrophy, for example a dystrophan polypeptide, a
dystrophin-associated polypeptide, a sarcoglycan polypeptide, and a
glycogen phosphorylase polypeptide.
[0116] In another preferred embodiment of the invention, the
heterologous nucleotide sequence encodes a polypeptide that is
associated with a metabolic disorder. By "associated with a
metabolic disorder", it is intended that the expressed polypeptide
is one that is deficient or defective in a metabolic disorder, or
is otherwise a causative agent in a metabolic disorder.
[0117] In another preferred embodiment, the polypeptide is a
lysosomal polypeptide, more preferably a precursor polypeptide that
retains the mannose-6-phosphate residues that are characteristic of
proteins targeted to the lysosomal compartment.
[0118] In still another preferred embodiment, the heterologous
nucleotide sequence encodes a polypeptide that is associated with a
lysosomal storage disease. By "associated with a lysosomal storage
disease", it is intended that the expressed polypeptide is one that
is deficient or defective in a lysosomal storage disorder, or is
otherwise a causative agent in a lysosomal storage disorder.
[0119] There are a multitude of lysosomal storage diseases, as is
recognized in the art. Exemplary lysosomal storage disease include,
but are not limited to, GM1 gangliosidosis, Tay-Sachs disease, GM2
gangliosidosis (AB variant), Sandhoff disease, Fabry disease,
Gaucher disease, metachromatic leukodystrophy, Krabbe disease,
Niemann-Pick disease (Types A-D), Farber disease, Wolman disease,
Hurler Syndrome (MPS III), Scheie Syndrome (MPS IS), Hurler-Scheie
Syndrome (MPS IH/S), Hunter Syndrome (MPS II), Sanfilippo A
Syndrome (MPS IIIA), Sanfilippo B Syndrome (MPS IIIB), Sanfilippo C
Syndrome (MPS IIIC), Sanfilippo D Syndrome (MPS IIID), Morquio A
disease (MPS IVA), Morquio B disease (MPS IV B), Maroteaux-Lamy
disease (MPS VI), Sly Syndrome (MPS VII), .alpha.-mannosidosis,
.beta.-mannosidosis, fucosidosis, aspartylglucosaminuria,
sialidosis (mucolipidosis I), galactosialidosis (Goldberg
Syndrome), Schindler disease, mucolipidosis II (1-Cell disease),
mucolipidosis III (pseudo-Hurler polydystrophy), cystinosis, Salla
disease, infantile sialic acid storage disease, Batten disease
(juvenile neuronal ceroid lipofuscinosis), infantile neuronal
ceroid lipofuscinosis, mucolipidosis IV, and prosaposin.
[0120] Polypeptides that are associated with lysosomal storage
diseases according to the present invention include, but are not
limited to, .beta.-galactosidase, .beta.-hexosaminidase A,
.beta.-hexosaminidase B, GM.sub.2 activator protein,
glucocerebrosidase, arylsulfatase A, galactosylceramidase, acid
sphingomyelinase, acid ceramidase, acid lipase,
.alpha.-L-iduronidase, iduronate sulfatase, heparan N-sulfatase,
.alpha.-N-acetylglucosaminidase acetyl-CoA, glucosaminide
acetyltransferase, N-acetylglucosamine-6-sulfatase, arylsulfatase
B, .beta.-glucuronidase, .alpha.-mannosidase, .beta.-mannosidase,
.alpha.-L-fucosidase, N-aspartyl-.beta.-glucosaminidase,
.alpha.-neuraminidase, lysosomal protective protein,
.alpha.-N-acetyl-galactosaminidase,
N-acetylglucosamine-1-phosphotransferase, cystine transport
protein, sialic acid transport protein, the CLN3 gene product,
palmitoyl-protein thioesterase, saposin A, saposin B, saposin C,
and saposin D.
[0121] The present invention further provides viral vectors
carrying a transgene encoding a polypeptide associated with a
glycogen storage disease. By "associated with a glycogen storage
disease", it is intended that the expressed polypeptide is one that
is deficient or defective in a glycogen storage disease, or is
otherwise a causative agent in a glycogen storage disease.
[0122] There are a multitude of glycogen storage diseases (GSD), as
is recognized in the art. Exemplary glycogen storage diseases
include, but are not limited to, Type Ia GSD (von Gierke disease),
Type Ib GSD, Type Ic GSD, Type Id GSD, Type II GSD (including Pompe
disease or infantile Type II GSD), Type IIIa GSD, Type IIIb GSD,
Type IV GSD, Type V GSD (McArdle disease), Type VI GSD, Type VII
GSD, glycogen synthase deficiency, hepatic glycogenosis with renal
Fanconi syndrome, phosphoglucoisomerase deficiency, muscle
phosphoglycerate kinase deficiency, phosphoglycerate mutase
deficiency, and lactate dehydrogenase deficiency.
[0123] Polypeptides that are associated with glycogen storage
diseases according to the present invention include, but are not
limited to, glucose 6-phosphatase, lysosomal acid a glucosidase,
glycogen debranching enzyme, branching enzyme, muscle
phosphorylase, liver phosphorylase, phosphorylase kinase, muscle
phosphofructokinase, glycogen synthase, phosphoglucoisomerase,
muscle phosphoglycerate kinase, phosphoglycerate mutase, and
lactate dehydrogenase.
[0124] In more preferred embodiments, a viral vector of the present
invention carries a transgene encoding a lysosomal acid
.alpha.-glucosidase (GAA), e.g., to treat Type II GSD including
infantile (Pompe disease), juvenile and adult onset forms of the
disease. More preferably, the lysosomal acid .alpha.-glucosidase is
a human lysosomal acid .alpha.-glucosidase (hGAA). The transgene
can encode either the mature GAA protein (e.g., the 76 kD form) or
a GAA precursor (e.g., the 110 kD form). Preferably, the transgene
encodes a GAA precursor. The term "GAA" as used herein encompasses
mature and precursor GAA proteins as well as modified (e.g.,
mutated) GAA proteins that retain biological function (i.e., have
at least one biological activity of the native GAA protein, e.g.,
can hydrolyze glycogen).
[0125] Lysosomal acid .alpha.-glucosidase (E.C. 3.2.1.20)
(1,4-.alpha.-D-glucan glucohydrolase), is an
exo-1,4-.alpha.-D-glucosidase that hydrolyses both .alpha.-1,4 and
.alpha.-1,6 linkages of oligosaccharides liberating glucose. It
catalyzes the complete degradation of glycogen with slowing at
branching points. The 28 kb acid .alpha.-glucosidase gene on human
chromosome 17 encodes a 3.6 kb mRNA which produces a 951 amino acid
polypeptide (Hoefsloot et al., (1988) EMBO J. 7:1697; Martiniuk et
al., (1990) DNA and Cell Biology 9:85). The nucleotide sequence of
a cDNA coding for a GAA polypeptide, as well as the deduced amino
acid sequence is provided in Hoefsloot et al. (Id.). The first 27
amino acids of the polypeptide are typical of a leader sequence of
a signal peptide of lysosomal and secretory proteins. The enzyme
receives co-translational N-linked glycosylation on the endoplasmic
reticulum. It is synthesized as a 110-kDa precursor form, which
matures by extensive modification of its glycosylation, by
phosphorylation and by proteolytic processing through an
approximately 90-kDa endosomal intermediate into the final
lysosomal 76 and 67 kDa forms (Hoefsloot, (1988) EMBO J. 7:1697;
Hoefsloot et al., (1990) Biochem. J. 272:485; Wisselaar et al.,
(1993) J. Biol. Chem. 268:2223; Hermans et al., (1993) Biochem. J.
289:681). The human GAA gene as described by Hoefsloot et al.,
(1988) EMBO J. 7:1697 and Van Hove et al., (1996) Proc. Natl. Acad.
Sci. USA 93:65, includes 5' untranslated sequences. In particular
preferred embodiments, the hGAA transgene includes the entire
approximately 3.8 kb sequence described by Van Hove et al.
Alternatively, a viral vector of the present invention can encode
more or less of the 5' and 3' untranslated regions of the GAA
gene.
[0126] Those skilled in the art will appreciate that the
heterologous nucleotide sequence(s) are preferably operably
associated with the appropriate expression control sequences. For
example, the viral vectors of the invention preferably contain
appropriate transcription/translation control signals and
polyadenylation signals operably associated with the heterologous
nucleic acid sequence(s) to be delivered to the target cell. Those
skilled in the art will appreciate that a variety of
promoter/enhancer elements can be used depending on the level and
tissue-specific expression desired. The promoter can be
constitutive or inducible (e.g., the metallothionine promoter or a
hormone inducible promoter), depending on the pattern of expression
desired. The promoter may be native or foreign and can be a natural
or a synthetic sequence. By foreign, it is intended that the
transcriptional initiation region is not found in the wild-type
host into which the transcriptional initiation region is
introduced. The promoter is chosen so that it will function in the
target cell(s) of interest. Brain-specific, hepatic-specific, and
muscle-specific (including skeletal, cardiac, smooth, and/or
diaphragm-specific) promoters are more preferred. Also preferred
are cancer cell specific promoters. Mammalian promoters are also
preferred.
[0127] More preferably, the heterologous nucleotide sequence(s) are
operatively associated with a cytomegalovirus (CMV) major
immediate-early promoter, an albumin promoter, an Elongation Factor
1-.alpha. (EF1-.alpha.) promoter, a P.gamma.K promoter, a MFG
promoter, or a Rous sarcoma virus promoter. It has been speculated
that driving heterologous nucleotide transcription with the CMV
promoter results in down-regulation of expression in
immunocompetent animals (see, e.g., Guo et al., (1996) Gene Therapy
3:802). Accordingly, it is also preferred to operably associate the
heterologous nucleotide sequence(s) with a modified CMV promoter
that does not result in this down-regulation of transgene
expression.
[0128] In embodiments wherein there is more than one heterologous
nucleotide sequence, those skilled in the art will appreciate that
the heterologous nucleotide sequences may be operatively associated
with a single upstream promoter and one or more downstream internal
ribosome entry site (IRES) sequences (e.g., the picornavirus EMC
IRES sequence).
[0129] In embodiments of the invention in which the heterologous
nucleotide sequence(s) will be transcribed and then translated in
the target cells, specific initiation signals are generally
required for efficient translation of inserted protein coding
sequences. These exogenous translational control sequences, which
may include the ATG initiation codon and adjacent sequences, can be
of a variety of origins, both natural and synthetic.
[0130] IV.A. Gene Transfer Technology
[0131] The methods of the present invention provide an approach for
delivering heterologous nucleotide sequences into a broad range of
host cells, including both dividing and non-dividing cells in vitro
or in vivo. The vectors, methods and pharmaceutical formulations of
the present invention are additionally useful in a method of
administering a therapeutic nucleic acid or therapeutic polypeptide
to a subject in need thereof, as a method of treatment or
otherwise. In this manner, the encoded nucleic acid or polypeptide
is produced in vivo in the subject. For example, the subject might
be in need of the polypeptide because the subject has a deficiency
of the polypeptide, or because the production of the polypeptide in
the subject imparts some therapeutic effect, as a method of
treatment or otherwise, and as explained further below.
[0132] In general, the present invention can be employed to deliver
any foreign nucleotide sequence to treat or ameliorate the symptoms
associated with any disorder related to gene expression.
Illustrative disease states include: lysosomal storage diseases,
glycogen storage diseases, hemophilias (e.g., hemophilia A and
hemophilia B) and other clotting disorders, Gaucher's Disease,
diabetes mellitus, cystic fibrosis (and other diseases of the
lung), muscular dystrophies (e.g., Duchenne, Becker), diseases of
the nervous system (e.g., Alzheimer's Disease, Parkinson's Disease,
amyotrophic lateral sclerosis, epilepsy), retinal degenerative
diseases (and other diseases of the eye), diseases of solid organs
(e.g., brain, liver, kidney, heart), and any other diseases having
an infectious or genetic basis.
[0133] Alternatively, a viral vector may be administered that
encodes any therapeutic polypeptide.
[0134] Gene transfer has substantial potential use in understanding
and providing therapy for disease states. There are a number of
inherited diseases in which defective genes are known and have been
cloned. In general, the above disease states fall into two classes:
deficiency states, usually of enzymes, which are generally
inherited in a recessive manner, and unbalanced states, which may
involve regulatory or structural proteins, and which are typically
inherited in a dominant manner. For deficiency state diseases, gene
transfer can be used to bring a normal gene into affected tissues
to thereby replace lost function. Gene transfer can be used as well
to create animal models for the disease using antisense mutations.
Conversely, a therapeutic polypeptide comprising a dominant
negative function can be used to treat disorders characterized by
excessive gene function. For unbalanced disease states, gene
transfer could be used to create a disease state in a model system,
which could then be used in efforts to counteract the disease
state. Thus the methods of the present invention permit the
treatment of genetic diseases. As used herein, a disease state is
treated by partially or wholly remedying the deficiency or
imbalance that causes the disease or makes it more severe. The use
of site-specific recombination of nucleic sequences to cause
mutations or to correct defects is also possible.
[0135] The instant invention can also be employed to provide an
antisense nucleic acid to a cell in vitro or in vivo. Expression of
the antisense nucleic acid in the target cell diminishes expression
of a particular protein by the cell. Accordingly, antisense nucleic
acids can be administered to decrease expression of a particular
protein in a subject in need thereof. Antisense nucleic acids can
also be administered to cells in vitro to regulate cell physiology,
e.g., to optimize cell or tissue culture systems. The present
invention is also useful to deliver other therapeutic nucleic
acids, including but not limited to non-translated RNAs, e.g.,
ribozymes or "guide" RNAs (see, e.g., Gorman et al., (1998) Proc.
Nat. Acad. Sci. USA 95:4929) to a target cell.
[0136] Finally, the instant invention finds further use in
diagnostic and screening methods, whereby a gene of interest is
transiently or stably expressed in a cell culture system.
[0137] The present invention is further useful for imaging methods.
Viral vectors of the invention can further comprise a detectable
label, preferably a label that is detectable in vivo. Thus, the
methods of the present invention can further comprise detecting the
detectable label, to thereby detect viral vectors following
administration to a subject.
[0138] IV.B. Immunization Methods
[0139] As a further aspect, the present invention provides a method
of producing an immune response in a subject, comprising
administering a viral vector carrying a nucleotide sequence
encoding an immunogen to a subject, and an active immune response
is mounted by the subject against the immunogen. Immunogens are as
described hereinabove. Preferably, a protective immune response is
elicited.
[0140] An "active immune response" or "active immunity" is
characterized by "participation of host tissues and cells after an
encounter with the immunogen. It involves differentiation and
proliferation of immunocompetent cells in lymphoreticular tissues,
which lead to synthesis of antibody or the development of
cell-mediated reactivity, or both." Herbert B. Herscowitz,
Immunophysiology: Cell Function and Cellular Interactions in
Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A.
Bellanti ed., 1985). Alternatively stated, an active immune
response is mounted by the host after exposure to immunogens by
infection or by vaccination. Active immunity can be contrasted with
passive immunity, which is acquired through the "transfer of
preformed substances (antibody, transfer factor, thymic graft,
interleukin-2) from an actively immunized host to a non-immune
host." Id.
[0141] A "protective" immune response or "protective" immunity as
used herein indicates that the immune response confers some benefit
to the subject in that it prevents or reduces the incidence of
disease. Alternatively, a protective immune response or protective
immunity may be useful in the treatment of disease, in particular
cancer or tumors (e.g., by causing regression of a cancer or tumor
and/or by preventing metastasis and/or by preventing growth of
metastatic nodules). The protective effects may be complete or
partial, as long as the benefits of the treatment outweigh any
disadvantages thereof.
[0142] A viral vector expressing an immunogen may be administered
directly to the subject, as described below.
[0143] Alternatively, a viral vector may be administered to a cell
ex vivo and the altered cell is administered to the subject. The
heterologous nucleotide sequence is permitted to be introduced into
the cell, and the cell is administered to the subject, where the
heterologous nucleotide sequence encoding the immunogen is
preferably expressed and induces an immune response in the subject
against the immunogen. Preferably, the cell is an antigen
presenting cell (e.g., a dendritic cell) or a cancer.
[0144] According to the foregoing methods of inducing an immune
response in a subject, it is preferred that a viral vector carrying
the heterologous nucleotide sequence is administered in an
immunogenically effective amount, as described below.
[0145] As described in more detail below, the present invention
also encompasses methods of treating cancer using immunotherapy by
administration of Ad vectors expressing cancer cell antigens or any
other immunogen that produces an immune response against a cancer
cell. In one particular embodiment, an immune response may be
produced against a cancer cell antigen in a subject by
administering a viral vector comprising a heterologous nucleotide
sequence encoding the cancer cell antigen, for example to treat a
patient with cancer. The viral vector may be administered to a
subject in vivo or by using ex vivo methods, as described
herein.
[0146] IV.C. Methods of Treating Cancer
[0147] As used herein, the term "cancer" encompasses tumor-forming
cancers. Likewise, the term "cancerous tissue" encompasses tumors.
A "cancer cell antigen" encompasses tumor antigens.
[0148] In particular embodiments, the inventive viral vectors are
administered as part of a method of treating cancer by
administering anti-cancer agents (e.g., cytokines) or a cancer cell
antigen or other immunogen that produces an immune response against
a cancer cell. A viral vector may be administered to a cell in
vitro or to a subject in vivo or by using ex vivo methods, as
described herein and known in the art.
[0149] The term "cancer" has its understood meaning in the art, for
example, an uncontrolled growth of tissue that has the potential to
spread to distant sites of the body (i.e., metastasize). Exemplary
cancers include, but are not limited to, leukemias, lymphomas,
colon cancer, renal cancer, liver cancer, breast cancer, lung
cancer, prostate cancer, ovarian cancer, melanoma, and the like.
Preferred are methods of treating and preventing tumor-forming
cancers.
[0150] The term "tumor" is also understood in the art, for example,
as an abnormal mass of undifferentiated cells within a
multicellular organism. Tumors can be malignant or benign.
Preferably, the inventive methods disclosed herein are used to
prevent and treat malignant tumors.
[0151] Cancer cell antigens according to the present invention have
been described hereinabove. By the terms "treating cancer" or
"treatment of cancer", it is intended that the severity of the
cancer is reduced or the cancer is at least partially eliminated.
Preferably, these terms indicate that metastasis of the cancer is
reduced or at least partially eliminated. It is further preferred
that these terms indicate that growth of metastatic nodules (e.g.,
after surgical removal of a primary tumor) is reduced or at least
partially eliminated. By the terms "prevention of cancer" or
"preventing cancer" it is intended that the inventive methods at
least partially eliminate or reduce the incidence or onset of
cancer. Alternatively stated, the present methods slow, control,
decrease the likelihood or probability, or delay the onset of
cancer in the subject.
[0152] In particular embodiments, cells may be removed from a
subject with cancer and contacted with the viral vectors of the
invention. The modified cell is then administered to the subject,
whereby an immune response against the cancer cell antigen is
elicited. This method is particularly advantageously employed with
immunocompromised subjects that cannot mount a sufficient immune
response in vivo (i.e., cannot produce enhancing antibodies in
sufficient quantities).
[0153] It is known in the art that immune responses may be enhanced
by immunomodulatory cytokines (a g., .alpha.-interferon,
.beta.-interferon, .gamma.-interferon, .omega.-interferon,
.tau.-interferon, interleukin-1.alpha., interleukin-1.beta.,
interleukin-2, interleukin-3, interleukin-4, interleukin 5,
interleukin-6, interleukin-7, interleukin-8, interleukin-9,
interleukin-10, interleukin-11, interleukin 12, interleukin-13,
interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand,
tumor necrosis factor-.alpha., tumor necrosis factor-.beta.,
monocyte chemoattractant protein-1, granulocyte-macrophage colony
stimulating factor, and lymphotoxin). Accordingly, in particular
embodiments of the invention, immunomodulatory cytokines
(preferably, CTL inductive cytokines) are administered to a subject
in conjunction with the methods described herein for producing an
immune response or providing immunotherapy.
[0154] Cytokines may be administered by any method known in the
art. Exogenous cytokines may be administered to the subject, or
alternatively, a nucleotide sequence encoding a cytokine may be
delivered to the subject using a suitable vector, and the cytokine
produced in vivo.
[0155] IV. D. Subjects, Pharmaceutical Formulations, Vaccine and
Modes of Administration
[0156] The present invention finds use in veterinary and medical
applications. Suitable subjects include both avians and mammals,
with mammals being preferred. The term "avian" as used herein
includes, but is not limited to, chickens, ducks, geese, quail,
turkeys and pheasants. The term "mammal" as used herein includes,
but is not limited to, humans, bovines, ovines, caprines, equines,
felines, canines, lagomorphs, etc. Human subjects are the most
preferred. Human subjects include neonates, infants, juveniles, and
adults.
[0157] In particular embodiments, the present invention provides a
pharmaceutical composition comprising a virus particle of the
invention in a pharmaceutically-acceptable carrier and, optionally,
other medicinal agents, pharmaceutical agents, carriers, adjuvants,
diluents, and the like. For injection, the carrier will typically
be a liquid. For other methods of administration, the carrier may
be either solid or liquid, such as sterile, pyrogen-free water or
sterile pyrogen-free phosphate-buffered saline solution. For
inhalation administration, the carrier will be respirable, and will
preferably be in solid or liquid particulate form. As an injection
medium, it is preferred to use water that contains the additives
usual for injection solutions, such as stabilizing agents, salts or
saline, and/or buffers.
[0158] In general, a "physiologically acceptable carrier" is one
that is not toxic or unduly detrimental to cells. Exemplary
physiologically acceptable carriers include sterile, pyrogen-free
water and sterile, pyrogen-free, phosphate buffered saline.
Physiologically-acceptable carriers include
pharmaceutically-acceptable carriers.
[0159] By "pharmaceutically acceptable" it is meant a material that
is not biologically or otherwise undesirable, i.e., the material
may be administered to a subject along with the viral vector
without causing any undesirable biological effects. Thus, such a
pharmaceutical composition can be used, for example, in
transfection of a cell ex vivo or in administering a viral particle
directly to a subject.
[0160] One aspect of the present invention is a method of
transferring a nucleotide sequence to a cell. The virus particles
may be added to the cells at the appropriate multiplicity of
infection according to standard transduction methods appropriate
for the particular target cells. Titers of virus to administer can
vary, depending upon the target cell type and the particular virus
vector, and can be determined by those of skill in the art without
undue experimentation. Preferably, at least about 10.sup.3
infectious units, more preferably 10.sup.4 infectious units, even
more preferably at least about 10.sup.5 infectious units, are
administered to the cell. In another embodiment quantities for the
number of AAV vector particles (e.g. AAV6 particles) administered
per cell can range from about 100 to about 10,000 particles per
cell, including about 250, about 500, about 1,000, and about
5,000.
[0161] Alternatively, administration of a viral vector of the
present invention can be accomplished by any other means known in
the art. For example, viral vectors can be targeted to cells,
including cells that are not normally competent for transduction by
adenoviruses using antibodies, e.g., as described in U.S. Pat. No.
5,861,156 to George et al.; U.S. Pat. No. 5,521,291 to Curiel et
al., the disclosures of which are incorporated herein in their
entirety by reference. Alternatively, adenoviruses can be targeted
to cell-surface proteins (e.g., receptors) by expressing a binding
protein or ligand on the surface of the adenovirus, e.g., as
described by Douglas et al., (1996) Nature Biotechnology 14:1574;
U.S. Pat. No. 5,770,442 to Wickham et al.; and U.S. Pat. No.
5,712,136 to Wickham et al. (the disclosures of which are all
incorporated herein in their entirety).
[0162] The cell to be administered the inventive virus vectors can
be of any type, including but not limited to neuronal cells
(including cells of the peripheral and central nervous systems),
retinal cells, epithelial cells (including dermal, gut,
respiratory, bladder and breast tissue epithelium), muscle cells
(including cardiac, smooth muscle, skeletal muscle, and diaphragm
muscle), pancreatic cells (including islet cells), hepatic cells
(e.g., parenchyma), fibroblasts, endothelial cells, germ cells,
lung cells (including bronchial cells and alveolar cells), prostate
cells, stem cells, progenitor cells, dendritic cells, and the like.
Alternatively, the cell is a cancer cell (including tumor cells).
Moreover, the cells can be from any species of origin, as indicated
above. Preferred are cells that are naturally transduced by
adenoviruses.
[0163] The viral vectors of the invention may be employed to
produce polypeptides of interest by cells in vitro. The adenovirus
comprises a heterologous nucleotide sequence(s) that may encode any
polypeptide of interest, as described hereinabove. The nucleotide
sequence preferably encodes a therapeutic polypeptide or an
industrial protein (i.e., for use in an industrial process). In
more preferred embodiments, the heterologous nucleotide sequence
encodes a GAA, more preferably human GAA, which may be isolated
from the cells using standard techniques and administered to
subjects with GAA deficiency using enzyme replacement protocols
(see, e.g., Van der Ploeg et al., (1991) J. Clin. Invest.
87:513).
[0164] In particular embodiments of the invention, the cell has
been removed from a subject, the viral vector is introduced
therein, and the cells are then replaced back into the subject.
Methods of removing cells from subjects for treatment ex vivo,
followed by introduction back into the subject are known in the art
(see, e.g., U.S. Pat. No. 5,399,346; the disclosure of which is
incorporated herein in its entirety). As a further alternative, the
cells that are manipulated and then introduced into the subject are
provided from another subject or cell line.
[0165] A further aspect of the invention is a method of treating
subjects in vivo with the inventive virus particles. Administration
of the viral particles of the present invention to a human subject
or an animal in need thereof can be by any approach known in the
art for administering virus vectors. Preferably, at least about
1000, more preferably, at least about 10,000 infectious units are
administered to the subject per treatment. Preferably, the subject
is a mammalian subject, more preferably a human subject. Also
preferred are subjects that have been diagnosed with a lysosomal
storage disease or a glycogen storage disease. More preferred are
subjects who have been diagnosed with GAA deficiency. Also
preferred are subjects with cancer.
[0166] In one embodiment, a method of introducing a nucleic acid in
accordance with the present invention comprises contacting a cell
with a recombinant hybrid virus of the present invention under
conditions sufficient for entry of the recombinant virus particle
into the cell, and further comprises introducing an AAV Rep 68/78
protein or sequences encoding an AAV Rep 68/78 protein into the
cell. Preferably, the cell comprises a human cell present in vitro,
ex vivo, or in vivo. Thus, in this embodiment, site-specific
integration into human chromosome 19 in cells or in vivo can be
accomplished by providing AAV Rep 68/78 protein (or sequences
encoding the same) as part of or along with a hybrid vector of the
present invention. General guidance as to the practice of this
embodiment can be found in Recchia, A., et al., Proc Nat Acad Sci
USA 96:2615-2620.
[0167] Continuing with this embodiment, a hybrid vector is
optionally c-administered with a vector comprising inducible AAV
rep68/78 sequences. Preferably, the second vector does not include
AAV rep48/52 sequences. Thus, the sequences encoding the AAV Rep
68/78 protein are operably associated with an inducible promoter.
The inducible promoter can comprise any of the inducible promoters
disclosed herein, but preferably comprises a tetracycline response
element. A vector encoding an inducible AAV Rep 68/78 can comprise
any of the vectors disclosed herein, but is preferably one of a
plasmid vector and an adenovirus vector. In the case of the use of
an adenovirus vector, it is preferred that the hybrid vector and
the vector encoding Rep 68/78 be delivered to a liver cell in
accordance with techniques disclosed herein. Indeed, through the
use of an adenovirus vector as the second vector, long-term
delivery of a gene to about 100% of cells in the liver is
provided.
[0168] Dosages will depend upon the mode of administration, the
disease or condition to be treated, the individual subject's
condition, the particular virus vector, and the gene to be
delivered, and can be determined in a routine manner. Preferably,
at least about 10.sup.5 infectious units of the inventive viral
vectors are administered to the subject. Exemplary doses for
achieving therapeutic effects are virus titers of
10.sup.8-10.sup.14 particles, preferably 10.sup.10-10.sup.13
particles, yet more preferably about 10.sup.12 particles.
[0169] In another embodiment quantities for the number of AAV
vector particles (e.g. AAV6 particles) administered per cell can
range from about 100 to about 10,000 particles per cell, including
about 250, about 500, about 1,000, and about 5,000.
[0170] A "therapeutically-effective" amount as used herein is an
amount that provides sufficient expression of the heterologous
nucleotide sequence delivered by the vector to provide some
improvement or benefit to the subject. Alternatively stated, a
"therapeutically-effective" amount is an amount that will provide
some alleviation, mitigation, or decrease in at least one clinical
symptom in the subject. Those skilled in the art will appreciate
that the therapeutic effects need not be complete or curative, as
long as some benefit is provided to the subject.
[0171] In particular embodiments of the invention, more than one
administration (e.g., two, three, four, or more administrations)
may be employed to achieve therapeutic levels of gene
expression.
[0172] Vaccines of the present invention comprise an immunogenic
amount of infectious virus particles as disclosed herein in
combination with a pharmaceutically-acceptable carrier. An
"immunogenic amount" is an amount of the infectious virus particles
that is sufficient to evoke an immune response in the subject to
which the pharmaceutical formulation is administered. An amount of
from about 10.sup.2 to about 10.sup.9 virus particles, preferably
from about 10.sup.3 to about 10.sup.7 virus particles, and more
preferably about 10.sup.4 to 10.sup.6 virus particles per dose may
be suitable, depending upon the age and species of the subject
being treated, and the immunogen against which the immune response
is desired. Other appropriate doses of the inventive virus
particles for producing a desired immune response may be routinely
determined by those skilled in the art.
[0173] Exemplary modes of administration include oral, rectal,
transmucosal, topical, transdermal, inhalation, parenteral (e.g.,
intravenous, subcutaneous, intradermal, intramuscular, and
intraarticular) administration, and the like, as well as direct
tissue (e.g., muscle) or organ injection (e.g., into the liver,
into the brain for delivery to the central nervous system),
alternatively, intrathecal, direct intramuscular, intraventricular,
intravenous, intraperitoneal, intranasal, or intraocular
injections. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions. Alternatively, one may administer the virus in a local
rather than systemic manner, for example, in a depot or
sustained-release formulation.
[0174] In particularly preferred embodiments of the invention, the
viral vector comprising a heterologous nucleic acid sequence of
interest is delivered to the liver of the subject. Administration
to the liver can be achieved by any method known in the art,
including, but not limited to intravenous administration,
intraportal administration, intrabiliary administration,
intra-arterial administration, and direct injection into the liver
parenchyma. Intramuscular delivery to skeletal muscle is also
preferred.
[0175] The viral vectors disclosed herein may alternatively be
administered to the lungs of a subject by any suitable means, but
are preferably administered by administering an aerosol suspension
of respirable particles comprised of the inventive viral vectors,
which the subject inhales. The respirable particles may be liquid
or solid. Aerosols of liquid particles comprising the inventive
viral vectors may be produced by any suitable means, such as with a
pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is
known to those of skill in the art. See, e.g., U.S. Pat. No.
4,501,729. Aerosols of solid particles comprising the inventive
virus vectors may likewise be produced with any solid particulate
medicament aerosol generator, by techniques known in the
pharmaceutical art.
[0176] In particular embodiments, a viral vector encoding a
polypeptide is introduced into target cells (e.g., liver cells or
skeletal muscle cells) and the polypeptide is expressed therein,
and optionally secreted into the circulatory system, where it is
delivered to target tissues, preferably, in a therapeutic amount.
Intramuscular delivery to skeletal muscle or delivery to the liver
are preferred in the practice of this embodiment of the
invention.
EXAMPLES
[0177] The following Examples have been included to illustrate
modes of the invention. Certain aspects of the following Examples
are described in terms of techniques and procedures found or
contemplated by the present co-inventors to work well in the
practice of the invention. These Examples illustrate standard
laboratory practices of the co-inventors. In light of the present
disclosure and the general level of skill in the art, those of
skill will appreciate that the following Examples are intended to
be exemplary only and that numerous changes, modifications, and
alterations can be employed without departing from the scope of the
invention.
Example 1
Cell Culture
[0178] 293 cells, C-7 cells (Amalfitano et al. (1999) Proc. Natl.
Acad. Sci. U.S.A.
[0179] 96:8861-8866), and GSD II patient fibroblasts were
maintained in Dulbecco's modified Eagle medium supplemented with
10% fetal bovine serum, 100 U penicillin per milliliter, and 100
.mu.g streptomycin per milliliter at 37.degree. C. in a 5%
CO.sub.2-air atmosphere. C-7 cells were grown in the presence of
hygromycin, 50 .mu.g/ml. HeLa cells were maintained in minimum
essential medium Eagle supplemented with 10% fetal bovine serum, 1
mm minimum essential medium sodium pyruvate, 0.1 mm minimum
essential medium non-essential amino acids, 100 U penicillin per
milliliter, and 100 .mu.g streptomycin per milliliter at 37.degree.
C. in a 5% CO.sub.2-air atmosphere.
Example 2
Transduction of Cultured Cells
[0180] HeLa cells and GSD II fibroblasts were plated at
1.times.10.sup.6 cells per 150 mm tissue culture dish. Cells were
transduced the next day by adding a volume of the respective vector
stock containing 1,000, 10,000, or 50,000 DNase I-resistant vector
particles (as defined by Southern blot analysis) per cell. Cells
were harvested five days later for hGAA measurement and Western
blotting analysis.
Example 3
Construction of an AAV Vector Plasmid Encoding hGAA
[0181] The hGAA cDNA was subcloned with the CMV promoter from
pcDNA3-hGAA (Van Hove et al. (1996) Proc. Natl. Acad. Sci. U.S.A.
93:65-70) into an AAV vector plasmid to generate pAAV-CBGAApA. The
vector sequences from pAAV-CBGAApA were isolated as a 4.4 kbp
fragment from a partial BglII digest, and ligated with the calf
intestinal alkaline phosphatase-dephosphorylated BglII site of
pShuttle (He et al. (1998) Proc. Nat. Acad. Sci. U.S.A.
95:2509-2514).
Example 4
Construction of a Hybrid [E1-, Polymerase-, Preterminal Protein-]
Ad-AAV Vector Encoding hGAA
[0182] Kanamycin-resistant shuttle plasmids were constructed to
contain within the Ad E1 region the CB promoter+hGAA cDNA+polyA
transgene cassette flanked by the AAV2 TR sequences. The shuttle
plasmid was digested with Pmel, and electroporated into the BJ5183
recombinogenic strain of E. coli with the pAd[E1-, polymerase-,
preterminal protein-] plasmid (Amalfitano et al. (1998) J. Virol.
72:926-933). Recombinant kanamycin-resistant clones were screened
by restriction enzyme digestion (BstXI) to confirm successful
generation of the full length recombinant Ad vector genomes. These
clones were digested with PacI and transfected as previously
described into the E1, and E2b expressing cell line, C-7
(Amalfitano et al. (1998) J. Virol. 72:926-933). The vectors was
amplified and confirmed to have the correct construction by
restriction enzyme mapping of vector genomes, and subsequent
functional assays in vitro and in vivo.
[0183] Once isolated, the respective Ad vectors are serially
propagated in increasing numbers of C-7 cells (Hodges et al. (2000)
J. Gene Med. 2:250-259). Forty-eight hours after infection,
infected cell pellets were harvested by low speed centrifugation,
resuspended in 10 mM Tris-HCl pH=8.0, vector released from the
cells by repeated freeze-thawing (3 times) of the lysate and by
ultrasonification, and the vector containing supernatant subjected
to two rounds of equilibrium density CsCl centrifugation
(Amalfitano et al. (1998) J. Virol. 72:926-933). Two virus bands
were visible. The virus bands were then removed, dialyzed
extensively against 10 mmTris-HCl pH=8.0 (or PBS), sucrose added to
1%, and aliquots stored at -80.degree. C. The number of vector
particles was quantified based on the OD.sub.260 of vector
contained in dialysis buffer with sodium dodecyl sulfate [SDS]
disruption (Ding et al. (2001) Hum. Gene Ther. 12:955-965), and by
DNase I digestion, DNA extraction, and Southern blot analysis.
[0184] Hybrid Ad-AAV vector DNA analysis included vector DNA
isolation and restriction enzyme digestion followed by Southern
blotting to verify the presence of intact AAV vector sequences,
including restriction enzymes that demonstrate the presence of AAV
terminal repeat sequences flanking the transgene (AhdI and
BssHII).
Example 5
Preparation of AAV Vectors
[0185] AAV vector stocks were prepared as described with
modifications (Allen et al. (2000) Mol. Ther. 1:88-95, Conway et
al. (1999) Gene Ther. 6:986-993). 293 cells were transduced with
the hybrid Ad-AAV vector (2000 DNase I-resistant vector
particles/cell as quantitated by Southern blot analysis) containing
the AAV vector sequences 15-30 minutes before transfection with a
AAV packaging plasmids containing the AAV2 Rep and Cap genes driven
by heterologous promoters, which typically generate no detectable
replication-competent AAV (rcAAV) (Allen et al. (2000) Mol. Ther.
1:88-95). For the transfection-only method (modified from Allen et
al. (2000) Mol. Ther. 1:88-95), pLNCorf6 (Hadjigeorgiou et al.
(1999) J. Inherit. Metab. Dis. 22:762-763) provided E4orf6 gene
which is an essential Ad helper function for AAV packaging, and no
Ad or Ad-AAV vector was required. Cell lysate was harvested 48
hours following infection and freeze-thawed 3 times, isolated by
iodoxinal step gradient centrifugation before heparin affinity
column purification (Zolotukhin et al. (1999) Gene Ther.
6:973-985), and aliquots stored were at -80.degree. C.
[0186] The number of vector DNA containing-particles was determined
by DNase I digestion, DNA extraction, and Southern blot analysis.
Contaminating wt AAV particles were detected in recombinant AAV
vector preparations by Southern blot analysis of extracted vector
DNA, and by a sensitive PCR assay utilizing primers spanning the
junction between the rep and cap genes. The level of rcAAV was less
than 1 particle in 10.sup.5 AAV vector particles. All viral vector
stocks were handled according to Biohazard Safety Level 2
guidelines published by the NIH.
Example 6
In Vivo Administration of Hybrid Ad-AAV and AAV Vector Stocks
[0187] The vector was administered intravenously (via the
retroorbital sinus) into 6-week-old GAA-KO mice (Raben et al.
(1998) J. Biol. Chem. 273:19086-19092). Either 2.times.10.sup.10
DNase I-resistant Ad-AAV, 4.times.10.sup.10 AAV, or
1.times.10.sup.12 AAV vector particles were injected per animal. At
the respective time points post-injection, plasma or tissue samples
were obtained and processed as described below. All animal
procedures were done in accordance with Duke University
Institutional Animal Care and Use Committee-approved guidelines
(Duke University, Durham, N.C., United States of America).
Example 7
Determination of hGAA Activity
[0188] Liver hGAA activity was measured following removal of the
liver from control or treated mice, flash-freezing on dry ice,
homogenization and sonication in distilled water, and pelleting of
insoluble membranes/proteins by centrifugation. The protein
concentrations of the clarified suspensions were quantified via the
Bradford assay. hGAA activity in the liver was determined as
described (Amalfitano et al. (1999) Proc. Natl. Acad. Sci. U.S.A.
96:8861-8866). Cellular hGAA was measured in transduced and control
HeLa cells following scraping, washing with PBS, suspension in and
sonic disruption in distilled water, and pelleting of insoluble
membranes/proteins by centrifugation. The protein concentrations of
the clarified suspensions were quantified via the Bradford assay,
and hGAA activity was determined as described (Amalfitano et al.
(1999) Proc. Natl. Acad. Sci. U.S.A. 96:8861-8866).
Example 8
Western Blotting Analysis of hGAA
[0189] For direct detection of hGAA in liver, the liver was
flash-frozen on dry ice, homogenized and sonically disrupted in
distilled water, and insoluble membranes/proteins were pelleted by
centrifugation. The protein content of the supernatants were
measured by the Bradford assay. Samples (100 .mu.g of protein) were
electrophoresed overnight in a 6% polyacrylamide gel to separate
proteins, and transferred to a nylon membrane. The blots were
blocked with 5% nonfat milk solution, incubated with primary and
secondary antibodies and visualized via the enhanced
chemiluminescence (ECL) detection system (Amersham Pharmacia,
Piscataway, N.J., United States of America) (Ding et al. (2001)
Hum. Gene Ther. 12:955-965).
Example 9
Markedly Enhanced AAV Vector Packaging with a Hybrid Ad-AAV
Vector
[0190] An AAV vector sequence was cloned into a multiply-deleted,
replication-deficient adenovirus, such that it is packaged as an
adenovirus vector (FIG. 1A). The hybrid vector, Ad-AAV-CBGAApA, was
constructed with the bacterial recombination system (He et al.
(1998) Proc. Nat. Acad. Sci. U.S.A. 95:2509-2514). The plasmid
containing the long arm of adenovirus was modified by deletion of
the E1, polymerase (pol), and preterminal protein (pTP) genes, such
that the Ad-AAV vector carried the deletion and was
replication-deficient in 293 cells (Amalfitano et al. (1998) J.
Virol. 72:926-933). Following two rounds of equilibrium density
cesium chloride centrifugation, 2 Ad-AAV bands were visible;
however, no vector DNA could be isolated from the upper,
lower-density Ad-AAV band in the cesium gradient (FIG. 1C), as
detected by Southern blot analysis with either Ad5 or hGAA probes
(FIG. 1D). The restriction sites within the TR sequences were
intact as determined by restriction enzyme digestion with AhdI and
BssHII (FIG. 1B and FIG. 1D), and DNase-I resistant hybrid Ad-AAV
vector particles were quantitated versus a standard curve of
plasmid DNA (FIG. 1D). The Ad-AAV vector generated high levels of
hGAA in transduced HeLa cells (Table 1). Thus, it was confirmed
that the hybrid Ad-AAV vector contained both the AAV vector
sequences and the adenovirus helper functions required to package
an AAV vector (FIG. 1E).
Example 10
TABLE-US-00001 [0191] TABLE 1 Enzyme analysis of transduced HeLa
cells. 10.sup.2 vector 10.sup.3 vector 10.sup.4 vector 5 .times.
10.sup.4 vector Vector No vector particles/cell particles/cell
particles/cell particles/cell No vector 34.5 +/- 17.7 N.A..sup.1
N.A. N.A. N.A. Ad-AAV-CBGAApA N.A. 43.4 +/- 2.5 189.3 +/- 27.1 N.D.
N.D. AAV-CBGAApA N.A. N.D. 74.4 +/- 10.2 106.3 +/- 25.5 394.2 +/-
79.4 .sup.1Not applicable. .sup.2Not done.
Analysis of Different Packaging Conditions for an AAV Vector in 293
Cells
[0192] The AAV vector packaging efficiency increased approximately
30-fold for the hybrid Ad-AAV method (as shown in FIG. 1D), because
the yield for this particular vector increased from 1,300 particles
per cell with a transfection-only method (modified from Allen et
al. (1997) J. Virol. 71:6816-6822) to 38,000 DNase I-resistant
particles per cell (FIG. 2A). Southern blot analysis of AAV vector
DNA demonstrated an identical signal for the vector DNA fragment
packaged with either the hybrid Ad-AAV or transfection-only method
(FIG. 2A).
[0193] The relative efficiency of AAV vector packaging with 5
different helper-gene combinations in 293 cells was evaluated in
order to elucidate factors underlying the hybrid Ad-AAV vector
method (FIG. 2B). High levels of AAV vector packaging was observed
for transduction with the hybrid Ad-AAV vector combined with
transfection of split AAV helper plasmids (Allen et al. (1997) J.
Virol. 71:6816-6822), which again showed a 33-fold increase in
vector particles/cell compared to the transfection-only method.
Transduction with an [E1-, polymerase-]Ad vector encoding
.beta.-galactosidase (Amalfitano, A., et al. (1998) J. Virol. 72:
926-933) also generated high numbers of AAV vector particles, when
combined with transfection with split AAV helper plasmids encoding
Rep and Cap (Allen et al. (2000) Mol. Ther. 1:88-95) and the AAV
vector plasmid (pAAV-CBGAApA). Either infection with a wild-type
Ad5 or transduction with the hybrid Ad-AAV vector, combined with
transfection with pACG2 (Xiao et al. (1998) J. Virol.
72:10222-10226) (an AAV packaging plasmid) and the AAV vector
plasmid (with wild-type Ad 5 only), demonstrated an intermediate
efficiency of AAV vector packaging (FIG. 2B). Northern blot
analysis of E4orf6 and E1A transcripts showed equivalent levels of
E1a and E4orf6RNAs under the 5 conditions analyzed, suggesting a
critical role for at least one additional Ad gene in the packaging
of AAV-CBGAApA. The Ad gene(s) in question were provided by the
second-generation Ad-AAV, [E1a-, polymerase-] Ad vector or
wild-type Ad5, but not by pLNCorf6 alone (Scaria et al. (1995) Gene
Ther. 2:295-298) during the transfection-only method.
[0194] High molecular weight Ad-AAV DNA was present at low levels
in the AAV vector stock at less than 1 Ad-AAV particle in 9,800
DNase I-resistant AAV particles, which hybridized with a probe
containing sequence from the right arm of Ad5 (FIG. 2C, lanes 3, 4
and 6). By contrast, Ad5 produced >8,000 contaminating Ad
DNase-resistant particles/cell during AAV vector packaging (FIG.
2C, lane 5). Thus, the level of contaminating Ad was reduced
markedly by the use of a second-generation hybrid Ad-AAV (or Ad)
vector that did not replicate in 293 cells to provide Ad helper
functions.
[0195] The yields for large-scale preparations of AAV-CBGAApA with
the hybrid Ad-AAV packaging method were compared to previous
results for the transfection-only method (FIG. 3A). The number of
particles/cell for AAV-CBGAApA was elevated approximately 14-fold
for the hybrid Ad-AAV packaging method (AAV-CBGAApA/Ad-AAV)
compared to the transfection-only method (AAV-CBGAApA/pLNCorf6).
The yield of AAV-CBcG6PpA for the transfection-only method was
higher than expected based on the small-scale vector preparations
described above. These variations could reflect higher-efficiency
transfections with the transfection-only method during large-scale
vector preparations. The packaging of another vector encoding
canine G6 Pase, AAV-CBcG6PpA, was increased 5.8-fold compared to
packaging AAV-CBGAApA with the transfection-only method. The
relatively low yield of AAV-CBGAApA with the transfection-only
method compared to AAV-CBcG6PpA suggested an effect related to
packaging size constraints, since the packaging size for
AAV-CBGAApA is approximately 1 kbp larger than for AAV-CBcG6PpA.
However, the packaging size for AAV-CBGAApA was 4.4 kbp, within the
optimal size for packaging in an AAV vector (Dong et al. (1996)
Gene Ther. 7:2101-2112).
[0196] AAV-CBGAApA was purified by the heparin-agarose column
method (FIG. 3B) as described by Zolotukhin et al. (1999) Gene
Ther. 6:973-985. AAV-CBGAApA DNase-I resistant particles were
recovered efficiently from the 40% iodoxinal fraction (FIG. 3B,
lane 2) by heparin-agarose column purification (FIG. 3B, lane 6).
The signal for Ad-AAV in the vector preparation was reduced
markedly by column purification of the AAV vector to less than the
limit of detection (<0.5 Ad-AAV particle/cell), and even gross
overexposure of the autoradiograph hybridized with an Ad5 probe did
not reveal a signal for Ad-AAV genomes in the purified AAV vector
stock (FIG. 3C, lane 1 versus lane 6). The DNase I-resistance of
AAV vector particles was confirmed by the elimination of the signal
for 2.5.times.10.sup.10 added vector plasmid particles when DNase I
was present (FIG. 2B, lane 10 versus lane 11).
Example 11
hGAA Expression with Ad-AAV and AAV Vectors in GAA-KO Mice
[0197] The hybrid Ad-AAV vector encoding hGAA was administered in
vivo to demonstrate hGAA secretion as shown previously for a
similar, modified Ad vector in GAA-KO mice (Amalfitano, A., et al.
(1999) Proc. Natl. Acad. Sci. USA 96:8861-8866). The hybrid Ad-AAV
vector encoding hGAA (6.times.10.sup.10 vector particles, according
to quantitation by spectrophotometry (Ding et al. (2001) Hum. Gene
Ther. 12:955-965)) was administered intravenously to GAA-KO mice.
To allow comparison to experiments with AAV vectors, quantitation
by Southern blot analysis revealed 3-fold fewer vector particles
(2.times.10.sup.10 DNase 1-resistant vector particles/GAA-KO mouse)
than when the vector stock was quantified by spectroscopy.
Secretion of hGAA was demonstrated in plasma by Western blot
analysis on day 3 following vector administration (FIG. 4A);
however, no hGAA was detected in plasma by Western blotting on day
7 following vector administration. The hGAA levels were
sufficiently elevated in liver to generate detectable plasma levels
of the .about.110 kD precursor hGAA as have been reported for an Ad
vector encoding hGAA (Amalfitano, A., et al. (1999) Proc. Natl.
Acad. Sci. USA 96:8861-8866). The number of hybrid Ad-AAV vector
particles administered was relatively low, indicating that higher
systemic hGAA levels could be achieved by administration of higher
numbers of vector particles (Amalfitano, A., et al. (1999) Proc.
Natl. Acad. Sci. USA 96:8861-8866).
[0198] AAV vector stocks were administered intravenously to GAA-KO
mice for in vivo analysis of the AAV vector encoding hGAA. In order
to deliver the same number of introduced genes encoding hGAA as
mice that received the double-strand Ad-AAV vector, GAA-KO mice
received twice as many (+ and - strand) single-stranded AAV vector
genomes (4.times.10.sup.10 DNase I-resistant vector particles). No
hGAA was detectable in plasma samples by Western blotting at 1, 2,
or 6 weeks following administration of the AAV vector. The
expression of hGAA in liver with Ad-AAV or the AAV vector was
compared at 2 and 6 weeks following vector administration (FIG. 4B
and Table 1). At 6 weeks the signal for hGAA with the lower dose of
the AAV vector (4.times.10.sup.10 DNase I-resistant vector
particle) was higher than for the hybrid Ad-AAV vector at the same
time point (FIG. 4B; Table 2). Liver-targeted administration of a
higher number of AAV vector particles (1.times.10.sup.12 DNase
I-resistant vector particle/GAA-KO mouse) produced markedly higher
levels of human GAA in liver at 6 weeks following vector
administration than for a lower number of Ad-AAV or AAV vector
particles (FIG. 4B; Table 2), approximately equivalent to the GAA
level in wild-type mice (Ding et al. (2001) Hum. Gene Ther.
12:955-965).
TABLE-US-00002 TABLE 2 Enzyme analysis of GAA-KO mouse liver
(nmol/hr/mg protein). Time following intravenous vector
Ad-AAV.sup.2, AAV.sup.2, AAV.sup.2, administration.sup.1 2 .times.
10.sup.10 (n = 3) 4 .times. 10.sup.10 (n = 3) 1 .times. 10.sup.12
(n = 2) 2 weeks 5.02 +/- 2.30 2.09 +/- 0.35 Not done 6 weeks 1.55
+/- 0.48 3.07 +/- 0.77 71.28 +/- 2.60 .sup.1hGAA in age-matched, 3
month-old GAA-KO mouse liver = 1.35 +/- 0.15 (n = 2)
.sup.2Ad-AAV-CBGAApA, hybrid Ad-AAV vector (DNase I-resistant
particles) .sup.3AAV-CBGAApA, AAV vector (DNase I-resistant
particles)
Example 12
Long-Term Correction of Glycogen Storage Disease, Type II, with a
Hybrid Adeno-Associated Virus-Adenovirus (Ad-AAV) Hybrid Vector
[0199] A replication-defective Ad-AAV hybrid virus was developed,
and was evaluated in muscle-targeted gene therapy in glycogen
storage disease type II (GSD II). Patients with GSD II exhibit a
progressive myopathy related to glycogen storage, that ultimately
leads to death from cardiac or respiratory failure. In the Ad-AAV
hybrid vector the AAV vector sequence has been cloned into an E1,
polymerase/preterminal protein-deleted adenovirus, such that it is
packaged as an adenovirus vector. The AAV vector sequence contains
a human acid glucosidase (hGAA) cDNA driven by a hybrid CMV-chicken
.beta.-actin promoter. The hybrid was administered to acid
glucosidase-knockout (GAA-KO) mice on day 3 of life by
gastrocnemius injection. Subsequently the glycogen content and hGAA
levels were analyzed in skeletal muscles, heart, diaphragm and
liver at various time points. The muscles of the hindlimb showed
reduced glycogen content and persistent hGAA for up to 6 months
following vector administration. Vector RNA was detected by
Northern blot analysis of the hindlimb muscles for up to 6 months.
Vector RNA and hGAA were present in the heart for 3 of 13 GAA-KO
mice for up to 6 months. Surprisingly, an antibody response to hGAA
was found in 10 of 13 mice; moreover, vector DNA and RNA and hGAA
persisted at low levels in the hearts of 3 GAA-KO mice that did not
exhibit an antibody response to hGAA. The presence of antibodies
against hGAA did not reverse the correction of glycogen storage in
the skeletal muscle of GAA-KO mice, implying that gene therapy has
a potential beneficial effect in patients with inhibitor antibodies
that would preclude successful enzyme therapy.
Example 13
hGAA Expression with AAV6 Vectors in GAA-KO Mice
[0200] The AAV2 pseudotyped vector detailed above demonstrated
wild-type levels of hGAA expression after muscle-targeted delivery
in GAA-knockout mice, and glycogen content was reduced in
transduced muscle signifying a partial correction of GSD II: To
increase hGAA expression of the introduced gene in muscle, the AAV
vector was subsequently packaged with AAV6 capsids utilizing the
hybrid Ad-AAV method. The hybrid Ad-AAV method achieved
approximately 100-fold higher packaging efficiency of the AAV6
pseudotyped vector as compared to the transfection-only method,
approximately 7000 DNase-I resistant particles/cell. The AAV6
pseudotyped vector is targeted to muscle in the GAA-KO mouse, and
the levels of hGAA expression for that vector are compared to the
AAV2 pseudotyped vector. AAV1 pseudotyped vectors produced much
higher levels of introduced proteins than AAV2 pseudotyped vectors
in muscle, and AAV6 capsids are essentially identical to AAV1
capsids. Therefore, much improved hGAA expression for the AAV6
pseudotyped vector in muscle is likely, and further development of
Ad-AAV and AAV vectors encoding hGAA offers distinct advantages for
gene therapy in GSD II.
Example 14
hGAA Secretion Following Portal Vein Injection with AAV Vectors in
GAA-KO/SCID Mice
[0201] In order to maximize the transduction of liver with the AAV
vector and increase the likelihood of secretion of hGAA, the AAV
vector was delivered by portal vein injection in GAA-KO/SCID mice.
Anti-hGAA antibodies previously abbreviated the secretion of hGAA
with Ad vectors in the liver of GAA-KO mice, and anti-hGAA
antibodies were not expected after vector administration in
GAA-KO/SCID mice (Ding, E. Y., et al. (2001), Hum. Gene Ther.
12:955-965). Western blot analysis of plasma demonstrated hGAA at 2
and 4 weeks following portal vein administration of AAV-CBGAApA
packaged either as AAV2 or as AAV6 (FIG. 5A), and was still
detected 3 months following injection of the AAV vector. Liver
production of hGAA was elevated approximately 10-fold compared to
wild-type levels (1110+/-120 mm/mg/hr) for a GAA-KO/SCID mouse at 3
months following AAV2 vector administration. For the vector
pseudotyped as AAV6, hGAA was elevated approximately twice the
level seen in normal mice. Untreated GAA-KO/SCID control mice had
low GAA activity in liver (1.4+/-0.3 mm/mg/hr).
[0202] Distal uptake of hGAA at the 3-month time point was
demonstrated in mice by GAA analysis of spleen, heart, diaphragm,
and the gastrocnemius, where GAA was clearly above the background
activity for untreated, GAA-KO/SCID mice (FIG. 5B). An advantage
from hGAA delivery to the heart, a primary site of pathology in
infantile GSD II (also known as Pompe disease) was shown by reduced
glycogen staining for the mouse that received the AAV2 vector, as
compared to an untreated control (FIG. 5C). Furthermore, glycogen
content in heart was reduced to 0.21 mmol glucose/gram protein
(range 0.14 to 0.28), compared to 1.2+/-0.25 mmol glucose/gm
protein for untreated controls.
[0203] It will be understood that various details of the invention
can be changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation.
* * * * *