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Journal of Virology, May 1999, p. 3994-4003, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Gene Therapy Vectors Based on Adeno-Associated Virus Type
1
Weidong
Xiao,
Narendra
Chirmule,
Scott C.
Berta,
Beth
McCullough,
Guangping
Gao, and
James M.
Wilson*
Institute for Human Gene Therapy and
Departments of Molecular and Cellular Engineering and of Medicine,
University of Pennsylvania, and The Wistar Institute, Philadelphia,
Pennsylvania 19104
Received 15 December 1998/Accepted 29 January 1999
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ABSTRACT |
The complete sequence of adeno-associated virus type 1 (AAV-1) was
defined. Its genome of 4,718 nucleotides demonstrates high homology
with those of other AAV serotypes, including AAV-6, which appears to
have arisen from homologous recombination between AAV-1 and AAV-2.
Analysis of sera from nonhuman and human primates for neutralizing
antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i)
NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates,
while the reverse is true in humans; and (ii) sera from 36% of
nonhuman primates neutralized AAV-1 but not AAV-2, while sera from 8%
of humans neutralized AAV-2 but not AAV-1. An infectious clone of AAV-1
was isolated from a replicated monomer form, and vectors were created
with AAV-2 inverted terminal repeats and AAV-1 Rep and Cap functions.
Both AAV-1- and AAV-2-based vectors transduced murine liver and
muscle in vivo; AAV-1 was more efficient for muscle, while AAV-2
transduced liver more efficiently. Strong NAB responses were detected
for each vector administered to murine skeletal muscle; these responses
prevented readministration of the same serotype but did not
substantially cross-neutralize the other serotype. Similar results were
observed in the context of liver-directed gene transfer, except for a
significant, but incomplete, neutralization of AAV-1 from a
previous treatment with AAV-2. Vectors based on AAV-1 may be
preferred in some applications of human gene therapy.
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INTRODUCTION |
Adeno-associated viruses (AAV) are
small, nonenveloped, single-stranded DNA viruses which require helper
virus to facilitate efficient replication (3). The 4.7-kb
genome of AAV is characterized by two inverted terminal repeats (ITRs)
and two sets of open reading frames, which encode the Rep and Cap
proteins. The Rep open reading frames encode four proteins with
molecular masses of 78, 68, 52, and 40 kDa. These proteins function
mainly in regulating AAV replication and integration. The Cap open
reading frames encode three structural proteins with molecular masses
of 85 kDa (VP1), 72 kDa (VP2), and 61 kDa (VP3) (3). The two
ITRs are the only cis elements essential for all steps in
the AAV life cycle.
AAV have been found in many animal species, including nonhuman
primates, canines, fowl, and humans (18). A total of six serotypes of AAV, including AAV type 1 (AAV-1), have been isolated from primates, and two have been isolated from nonhuman primates; AAV-2, AAV-3, and AAV-5 are from humans, and AAV-6 is
from a human adenovirus preparation. AAV-2 is the most
characterized primate serotype, since its infectious clone was the
first one made (24). The full sequences for AAV-3A,
AAV-3B, AAV-4, and AAV-6 recently were determined (4,
17, 22). Generally, all primate AAV show more than 80% homology
in nucleotide sequence.
A number of unique properties make AAV a very promising vector for
human gene therapy (19). AAV are not associated with any
known human diseases and are generally not considered pathogenic. Wild-type AAV are capable of integrating into the host chromosome in a
site-specific manner (14, 26). Recombinant AAV (rAAV) vectors can integrate into tissue culture cells at chromosome 19 if the
Rep proteins are supplied in trans (1, 29). The transduced genomes of AAV have been shown to confer long-term gene
expression in a number of tissues, including muscle, liver, brain, and
retina (8, 13, 27, 28, 30, 31). The development of new
methods for producing high-titer rAAV has largely removed the hurdles
which prevented AAV vectors from being tested in large-animal models of
human diseases and in human clinical trials (5, 6, 11, 32).
Among AAV-1 to AAV-6, only AAV-1 and AAV-4 are
considered to be simian viruses, since they were isolated from nonhuman
primates and monospecific antibodies to the viruses have not been
detected in human serum (20). They may have advantages for
use in human gene therapy to replace or augment the use of AAV-2
vectors. For example, AAV-1 vectors could be used in patients who
develop anti-AAV-2 neutralizing antibodies (NAB) due to a
naturally acquired infection or previous treatment with AAV-2
vectors. To study the possibility of using AAV-1 as a gene therapy
vector, we constructed an AAV-1 infectious clone and determined its
full sequence. Vectors derived from this infected clone were evaluated
in murine models of liver and muscle gene transfer.
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MATERIALS AND METHODS |
Murine studies.
C57BL/6 mice (6- to 8-week-old males)
were obtained from Jackson Laboratory. AAV vectors were administered by
either intramuscular or intrasplenic injection as described before
(8, 30).
Nonhuman primates.
Wild-caught juvenile rhesus monkeys were
purchased from Covance (Alice, Tex.) and LABS of Virginia (Yemassee,
S.C.) and kept in full quarantine. The monkeys weighed approximately 3 to 4 kg. The nonhuman primates used in the Institute for Human Gene
Therapy research program are purposefully bred in the United States
from specific-pathogen-free closed colonies. All vendors are U.S.
Department of Agriculture class A dealers. The rhesus macaques are
therefore not infected with important simian pathogens, including the
tuberculosis agent, major simian lentiviruses (simian immunodeficiency
virus and simian retroviruses), and cercopithecine herpesvirus. The animals are also free of internal and external parasites. The excellent
health status of these premium animals minimizes the potential for
extraneous variables. The use of these monkeys in protocols was
approved by the Infection Control Committee of the Hospital of the
University of Pennsylvania and the Environmental Health and Safety
Office, the Institutional Biosafety Committee, and the Institutional
Animal Care and Use Committee of the University of Pennsylvania. For
this study, serum was obtained from monkeys prior to initiation of any protocol.
Human subjects.
Normal volunteers (n, 77) were
analyzed for immune reactivity to AAV. Individuals were members of the
University of Pennsylvania community. The study was approved by the
Institutional Review Board of the University of Pennsylvania. The
median age was 27 years (range, 18 to 54 years), and the age
distributions in the two groups were similar.
Cell culture and virus.
AAV-free 293 cells and 84-31 cells were obtained from the Human Applications Laboratory of the
University of Pennsylvania. These cells were cultured in Dulbecco's
modified Eagle medium with 10% fetal bovine serum (Hyclone),
penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a
moisturized environment supplied with 5% CO2. The 84-31 cell line constitutively expresses adenovirus E4 proteins and has been
described previously (7). AAV-1 (ATCC VR-645) seed stock
was purchased from the American Type Culture Collection (Rockville,
Md.). AAV were propagated in 293 cells with wild-type adenovirus type 5 as a helper virus.
rAAV generation.
rAAV were generated by transfection with an
adenovirus-free method described elsewhere (30a). Briefly,
the cis plasmid (with AAV ITRs), the trans
plasmid (with the AAV Rep gene and Cap gene), and
a helper plasmid (pF
13, with an essential region from the adenovirus
genome) were cotransfected into 293 cells at a ratio of 1:1:2 by
calcium phosphate precipitation. The AAV vectors used in this study
express murine erythropoietin or human 
1-antitrypsin from a chicken
-actin promoter enhanced by sequences from cytomegalovirus. pF13
has an 8-kb deletion in the adenovirus E2B region and most of the late
genes. The cells were harvested 96 h posttransfection and
subjected to two rounds of CsCl gradient purification. For the
generation of rAAV based on AAV-2, p5E18 was used as the
trans plasmid, since it greatly improved the rAAV yield. To
make rAAV with AAV-1 virions, pAV1H or p5E18(2/1) was used as the
trans plasmid to provide Rep and Cap functions.
DNA techniques.
Hirt DNA extraction was performed as
described before with minor modifications (25).
To construct AAV-1 infectious clones, the Hirt DNA from
AAV-1-infected 293 cells was repaired with the Klenow enzyme (New England BioLabs) to make sure that the ends were blunt. The treated AAV-1 Hirt DNA was then digested with BamHI and cloned
into three vectors. The internal BamHI fragment was cloned
into pBluescript II-SK(+) cut with BamHI to produce pAV1-BM.
The left and right fragments were cloned into pBluescript II-SK(+) cut
with BamHI and EcoRV to obtain pAV1-BL and
pAV1-BR, respectively. The AAV sequences in these three plasmids were
subsequently assembled into the same vector to produce AAV-1
infectious clone pAAV-1.
For sequencing, an ABI 373 automatic sequencer was used to determine
the sequences for all plasmids and PCR fragments used
in this study by
fluorescence sequencing (FS) dye chemistry. They
were confirmed by
sequencing both plus and minus strands. For
sequencing the AAV terminal
repeats, the fragment containing AAV
ITRs was amplified from
pAAV-1, pAV1-BR, or pAV1-BL with M13F
(GTAAAACGACGGCCAGT) and CAP1a
(GAA CCG TGC TGG TAA GGT TAT T)
or M13F and V1Rep1s (CCA TGC CGG GCT
TCT ACG AGA TCG TTA TCA GGG
TG). The PCR was performed with a
GC-advantage kit (Clontech Laboratories,
Inc.). The 50-µl reaction
mixture contained 1× reaction buffer
(40 mM Tricine-KOH [pH 9.02] at
25°C, 15 mM KOAc, 3.5 mM Mg(OAc)
2,
5% dimethyl
sulfoxide, 3.75 µg of bovine serum albumin per ml);
1× GC melt; 10 mM dATP, dCTP, 7-deazaGTP-dGTP (3:1), and dTTP;
10 ng of template; 500 ng of each primer; and 2.5 U of polymerase.
The purified PCR product
was sequenced as described
above.
A plasmid expressing AAV-2 Rep and Cap, p5E18(2/2), was constructed
to maximize vector production. This was accomplished by
relocating the
p5 promoter to a position 3' to the Cap gene, thereby
minimizing
expression of Rep78 and Rep68. This strategy was initially
described by
Li et al. (
15). p5E18(2/2) was constructed in the
following
way. The previously described pMMTV-trans vector (
30a)
(i.e., the mouse mammary tumor virus promoter substituted for
the p5
promoter in an AAV-2-based vector) was digested with
SmaI
and
ClaI, filled in with the Klenow enzyme,
and then recircularized
with DNA ligase. The resulting construct was
digested with
XbaI,
filled in, and ligated to the
blunt-ended
BamHI-
XhaI fragment
from pCR-p5,
constructed in the following way. The p5 promoter
of AAV was amplified
by PCR with the following oligonucleotides:
TGT AGT TAA TTA ACC CGC CAT
GCT ACT TAT C and GGC GGC TGC GCG
TTC AAA CCT CCC GCT TCA AAA TG. The
amplified fragment was subsequently
cloned into pCR2.1 (Invitrogen) to
yield pCR-p5. The helper plasmid
pAV1H was constructed by cloning the
BfaI fragment of pAAV-1 into
pBluescript II-SK(+) at the
BcorV and
SmaI sites. The 3.0-kb
XbaI-
KpnI
fragment from p5E18(2/2), the 2.3-kb
XbaI-
KpnI fragment from pAV1H,
and the 1.7-kb
KpnI fragment from p5E18(2/2) were incorporated
into a
separate plasmid, p5E18(2/1), which contains AAV-2 Rep,
AAV-1
Cap, and the AAV-2 p5 promoter located 3' to the Cap
gene.
For Southern blot analysis, Hirt DNA was digested with
DpnI
to remove bacterium-borne plasmid and probed with the internal
BamHI fragment of AAV-1. The membrane was then washed
under high-stringency
conditions: twice for 30 min each time with 2×
SSC (1× SSC is
0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium
dodecyl sulfate
(SDS) at 65°C and twice for 30 min each time with
0.1× SSC-0.1%
SDS at 65°C. The membrane was then analyzed by both
phosphorimager
analysis and X-ray
autoradiography.
Western blot analyses.
Serum samples were analyzed for
reactivity to various AAV-2 capsid proteins by Western blotting as
described previously (8). Purified AAV-2 vectors were
used as antigens for the capsid proteins, and a monoclonal antibody
that recognizes shared epitopes on VP1, VP2, and VP3 (clone B1;
American Research Products) was used as a positive control. AAV-2
antigens were electrophoresed by SDS-10% polyacrylamide gel
electrophoresis and transferred to a nitrocellulose membrane
(Hybond-ECL; Amersham). The reactivity of the serum was measured by
incubating membrane containing AAV antigen with test serum samples,
followed by peroxidase anti-human immunoglobulin G antibody. The
reaction was detected with an ECL kit (Amersham).
Anti-AAV NAB.
NAB titers were analyzed by assessing the
ability of serum antibody to inhibit the transduction of reporter virus
(AAV1-GFP or AAV2-GFP) into 84-31 cells, which are subclones of 293 cells that stably express adenovirus E4 proteins, which render them permissive for AAV transduction. Various dilutions of antibodies preincubated with reporter virus for 1 h at 37°C were added to 90% confluent cell cultures. Cells were incubated for 48 h, and the expression of green fluorescent protein (GFP) was measured by
FluoroImaging (Molecular Dynamics). NAB titers were calculated as the
highest dilution at which 50% of the cells stained green.
Human 1-antitrypsin assay.
The concentration of
human 1-antitrypsin in mouse serum was measured by an enzyme-linked
immunosorbent assay (ELISA). The coating antibody was rabbit
anti-human 1-antitrypsin (Sigma). We used goat anti-human
1-antitrypsin (Sigma) as the primary detection antibody
(30). The sensitivity of the assay is 0.3 to 30 ng/ml.
Nucleotide sequence accession number.
The AAV-1 sequence
is available through GenBank under accession no. AF063497.
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RESULTS |
Generation of an infectious clone of AAV-1.
The replicated
form of AAV-1 was extracted from 293 cells infected with AAV-1
and wild-type adenovirus type 5. Analysis of Hirt DNA revealed three
bands with apparent molecular sizes equivalent to a double-stranded
dimer (9.4 kb), a double-stranded monomer (4.7 kb), and single-stranded
DNA (1.7 kb). The monomer band was excised from the gel and digested
with BamHI, revealing a pattern of three fragments (1.1, 0.8, and 2.8 kb), in accordance with the original description of
AAV-1 by Bantel-Schaal and zur Hausen (2). The
three fragments were subcloned into one plasmid-based construct to
obtain pAAV-1.
This clone was tested for its abilities to rescue AAV-1 genomes
from the plasmid backbone and to replicate and package infectious
virus. Plasmid pAAV-1 was transfected into 293 cells infected
with
adenovirus type 5 at a multiplicity of infection of 10, and
the virus
supernatant was used to reinfect 293 cells. DNA hybridization
analysis
of
DpnI-digested Hirt DNA from infected 293 cells
demonstrated
the rescue and replication of AAV-1 genomes (Fig.
1).
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FIG. 1.
DNA hybridization analysis of the rescue and replication
of the AAV-1 infectious clone. Two independent clones of pAAV-1
(Clone-1 and Clone-2) were transfected into 293 cells, which were
then superinfected with adenovirus type 5. Hirt DNA was extracted
48 h postinfection and digested with DpnI before
electrophoresis in an 0.8% agarose gel. An internal BamHI
fragment of pAAV-1 was used as a probe. The autoradiograph shows
the locations of replicated dimers and monomers and their molecular
sizes. The analysis included cells not infected (negative control) or
infected with clone-1 or clone-2.
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Genomic structure of AAV-1.
The entire AAV-1 genome
was sequenced from pAAV-1 (Fig.
2). PCR
products derived from the original AAV-1 seed stock were also sequenced to confirm the structure of the replicated form. No major
differences were noted. The length of AAV-1 (4,718 nucleotides) is
very close to that of AAV-3 (4,726 nucleotides), which is shorter than AAV-4 (4,774 nucleotides) but longer than AAV-2 (4,681 nucleotides) and AAV-6 (4,683 nucleotides).
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FIG. 2.
Alignment of nucleotides for AAV-1, AAV-2, and
AAV-6. The full sequence of AAV-1 is indicated in the top line;
the AAV-2 and AAV-6 sequences are shown below with nucleotide
differences. Homology is indicated by dots. Critical landmarks in the
structures of the AAV genomes are shown. Gaps are indicated by dashes.
The 3' ITR of AAV-1 is shown in the same configuration as in the
published AAV-2 and AAV-6 sequences (22). TRS,
terminal resolution site.
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The AAV-1 genome shows more than 80% identity with other known AAV
and contains the characteristic structural features. The
ITRs of
AAV-1 are predicted to form T-shaped hairpin structures
(Fig.
3). The right and left ITRs of AAV-1
are identical and virtually
the same as the right ITR of AAV-6,
except for 1 nucleotide in
the A and A' sequences and the last
nucleotide in the D sequence.
The AAV-2 Rep binding motif
(GCTCGCTCGCTCGCTG) found in the AAV-2
preintegration
region in human chromosome 19 is well conserved
in AAV-1.
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FIG. 3.
Potential secondary structure of AAV-1 ITR.
Nucleotide differences in the corresponding sequences of AAV-2 and
AAV-6 relative to AAV-1 are underlined. AAV-2 sequence
differences are lowercase, underlined, and italicized, whereas
AAV-6 sequence differences are lowercase and underlined.
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Although the overall features of AAV terminal repeats are conserved
among the different serotypes, their lengths differ slightly.
The
terminal repeats of AAV-1 are 143 nucleotides long, while
those of
AAV-2, AAV-3, and AAV-4 are 145 or 146 nucleotides long.
The loop region of the AAV-1 ITR mostly resembles that of the
AAV-4 ITR in that it also uses TCT instead of the TTT found in
the
AAV-2 and AAV-3 ITR. We ruled out the possibility of a
sequencing
error at this site by using restriction enzyme digestion of
the
replicative intermediate, since these 3 nucleotides are part of
the
SacI site
(GAGCTC).
The p5 promoter region of AAV-1 shows some divergence from
homologous regions of other AAV serotypes but maintains critical
regulatory elements; the repeated YY1 sites are present throughout
all
known AAV serotypes, including AAV-1. In AAV-4, 56 additional
nucleotides are inserted between YY1 and the E-box/USF site, while
in
AAV-1, 26 additional nucleotides are inserted before the E-box/USF
site. The p19 promoter, the p40 promoter, and poly(A) can also
be
identified in the AAV-1 genome by homology to those in known
AAV
serotypes, which are also highly conserved (
3).
AAV-6 is a hybrid of AAV-1 and AAV-2.
The sequence
of the AAV-1 coding region is virtually identical to that of
AAV-6 from position 452 to the end of the coding region (99%
homology). The first 508 nucleotides of AAV-6 were shown to
be identical to those of AAV-2 (22).
AAV-6 is organized in the following manner: AAV-2
left ITR, AAV-2 p5 promoter, AAV-1 coding region, and AAV-1
right ITR. This organization suggests that AAV-6 is a naturally
occurring hybrid of AAV-1 and AAV-2 (Fig.
4).
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FIG. 4.
Hypothesis for the creation of AAV-6 from homologous
recombination between AAV-1 and AAV-2. The first line
represents the AAV-2 genome, with Rep and Cap sequences demarcated
by hatched arrows. The second line represents the AAV-1 genome,
with the Rep and Cap sequences depicted as solid arrows. The hypothesis
suggests homologous recombination between AAV-2 and AAV-1 at
the region of the box containing wavy lines; this region contains a
highly homologous sequence of the Rep gene shared by AAV-1 and
AAV-2. PA, poly(A). A more detailed illustration of the common
region of these three viruses is shown at the bottom. Arrows indicate
nucleotides that differ.
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Recombinant vector based on AAV-1.
Our initial goal was to
create a recombinant vector which uses the Cap proteins of AAV-1
together with a vector genome comprised of AAV-2 ITR. We wanted to
take advantage of the well-characterized and favorable biology of
AAV-2 ITR while developing a recombinant virus whose capsid
proteins are derived entirely from simian-based AAV-1. This vector,
referred to here as the AAV-1 vector, should be serologically
distinct from AAV-2.
To create the desired recombinant vector, we developed plasmids that
could transcomplement vector production with AAV-1 Cap.
The first
plasmid is a homolog of the psub201 vector of AAV-2
in which the
ITRs were deleted from pAAV-1, leaving behind Rep
and Cap expressed
from endogenous promoters (pAV1H). A recombinant
vector was produced by
transfecting into 293 cells three plasmids:
an AAV-2-based vector
(i.e., AAV-2 ITR with a reporter gene),
pAV1H, and a plasmid
containing adenovirus type 5 helper functions.
Lysates were purified on
CsCl gradients, yielding low titers of
the recombinant vector
(i.e., 5 × 10
10 genomes/50 15-cm
2 plates).
The AAV helper vector was revised in two ways to improve yield. First,
the AAV-1 Rep gene in pAV1H was substituted for the
corresponding
gene in AAV-2. Second, the p5 promoter was relocated
behind the Rep
and Cap open reading frames to reduce the expression
of the p78 and p68
forms; this modification has been used to enhance
the titers of
AAV-2 vectors (15,300). The resulting plasmid (p5E182/1)
produced
10- to 20-fold higher quantities of the vector than pAV1H
(i.e.,
10
12 genomes/50 15-cm
2 plates).
The performance of AAV-1 vectors was evaluated with immunodeficient
mice (i.e., RAG-1 KO) injected intramuscularly or in the
portal
circulation to target the liver (Fig.
5).
Direct comparisons
were made to equivalent quantities of AAV-2
vectors containing
the same vector genomes encoding secreted proteins
easily measured
in blood (i.e., murine erythropoietin and human
1-antitrypsin).
Early experiments indicated similar in vivo
performances of AAV-1
vectors produced with pAV1H and p5E18(2/1)
(data not shown). All
subsequent studies used AAV-1 vectors
derived from p5E18(2/1)
because of the increased yield.
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FIG. 5.
In vivo activities of AAV-1 and AAV-2 vectors.
Recombinant stocks of virus were generated by transfection and purified
through cesium chloride gradients. (A) AAV-1 and AAV-2 vectors
expressing murine erythropoietin (Epo) from a cytomegalovirus-enhanced
-actin promoter (CB). Equivalent stocks of these two vectors were
injected into muscle (5 × 1010 genomes) or liver via
the portal circulation (1 × 1011 genomes), and the
animals (four groups) were analyzed on day 30 for the presence of
erythropoietin in blood. Data for the groups were combined. Error bars
show standard errors. (B) An identical experiment was performed with
AAV-1 and AAV-2 vectors expressing human 1-antitrypsin
(1AT) from the same promoter. Serum harvested from four groups of
animals 30 days after gene transfer was analyzed for the presence of
1-antitrypsin by an ELISA. Data are reported as in panel A.
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AAV-2 vectors consistently produced 10- to 50-fold more serum
erythropoietin or
1-antitrypsin when injected into liver compared
to
muscle. This result was very different from that for AAV-1
vectors,
with which muscle expression was equivalent to or greater
than liver
expression. In fact, AAV-1 outperformed AAV-2 in muscle
when
equivalent titers based on genomes were
administered.
NAB to AAV-1 and AAV-2 in nonhuman and human primates.
Simple and quantitative assays for NAB to AAV-1 and AAV-2 were
developed with recombinant vectors. A total of 33 rhesus monkeys and 77 normal human subjects were screened. Figure
6 summarizes the results. A reciprocal
dilution of NAB equal to 20 is the baseline (i.e., no detectable NAB).
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FIG. 6.
NAB to AAV-1 and AAV-2 in nonhuman and human
primates. Sera were analyzed for neutralizing activity against
rAAV-1 and rAAV-2 as described in Materials and Methods. The
reciprocal dilution of the NAB was plotted for AAV-2 versus
AAV-1. Sera were titrated in sequential twofold dilutions. A titer
of 1:20 represents the background (i.e., no detectable NAB). The number
next to each datum point represents the total number of animals or
humans with a particular NAB profile. In panel A, 33 adolescent rhesus
monkeys were analyzed. In panel B, 77 normal human volunteers were
evaluated.
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Analysis of NAB in rhesus monkeys showed that 61% of animals tested
positive for AAV-1; a minority (24%) had NAB to AAV-2.
Over
one-third of animals had antibodies to AAV-1 but not AAV-2
(i.e., were monospecific for AAV-1), whereas no animals were
positive
for AAV-2 without reacting to AAV-1. These data
support the hypothesis
that AAV-1 is endemic in rhesus monkeys. The
presence of true
AAV-2 infections in this group of nonhuman
primates is less clear,
since we cannot rule out cross-neutralizing
activity of an AAV-1
response to AAV-2. It is interesting that
there is a linear relationship
between AAV-2 NAB and AAV-1 NAB
in animals that had
both.
Analysis of sera from normal human subjects was remarkable for a
relative lack of NAB to either virus, with 71% of subjects
scoring
negative for both AAV-1 and AAV-2. Only 1 of 77 individuals
was
positive for AAV-1 and not AAV-2 (the titer in this person
was
only twofold higher than the baseline), whereas 6 individuals
(8%)
were positive for AAV-2 but not AAV-1. Overall, more
patients
had NAB to AAV-2 (27%) than to AAV-1 (20%),
and the overall titers
against AAV-2 appeared to be higher. These
data support the hypothesis
that AAV-2 infections are present in
the human population, although
independent primary infections with
AAV-1 cannot be ruled out.
The lack of monospecific antibodies to
AAV-1 argues against this
virus infecting humans to any
appreciable
degree.
Applications of AAV-1 vectors in animal models of gene
therapy.
AAV-1 could have a role in human gene therapy in
situations in which AAV-2 is rendered ineffective because of
preexisting NAB, such as in patients with a prior history of AAV-2
infection or those who received gene therapy with an AAV-2 vector.
This hypothesis presumes that there is little cross-neutralization in
vivo between AAV-1 and AAV-2. Experiments were designed to test
this hypothesis in the context of AAV-mediated gene transfer to
murine liver and muscle. The basic paradigm is to inject AAV-1 or
AAV-2 expressing human 1-antitrypsin and to follow up with a
second vector of the same or different serotype and expressing murine
erythropoietin. The efficiency of gene transfer is quantitatively measured by ELISA analysis of erythropoietin and 1-antitrypsin in blood.
An analysis of vector readministration in muscle is presented in Fig.
7. Administration of AAV-2 vectors
led to high levels
of AAV-2 NAB that blocked the readministration
of AAV-2 in vivo
(group 1). The NAB in these animals did not
neutralize AAV-1 in
vitro, although there was a modest diminution
(i.e., fivefold)
in the transduction of an AAV-1 vector in vivo
(group 5) to a
level below that achieved in naive animals (group 4).
Similarly,
AAV-1 vectors resulted in high levels of AAV-1 NAB
that blocked
AAV-1 readministration in vivo (group 2) but did not
interfere
with AAV-2 vector transduction in vivo (group 6),
compared to
the results for naive animals that received AAV-2 alone
(group
3).
View larger version (26K):
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[in a new window]
|
FIG. 7.
Readministration of AAV vectors in muscle. C57BL/6 mice
were evaluated for AAV-mediated gene transfer following
introduction into naive animals as well as 30 days following the first
vector administration. The different experimental groups are shown
below the graphs. In each case, vector 1 expressed 1-antitrypsin
(1AT) from a cytomegalovirus-enhanced -actin promoter, while
vector 2 expressed murine erythropoietin (EPO) from the same promoter.
Group 1, AAV-2 followed by AAV-2; group 2, AAV-1 followed
by AAV-1; group 3, phosphate-buffered saline (PBS) followed by
AAV-2; group 4, PBS followed by AAV-1; group 5, AAV-2
followed by AAV-1; group 6, AAV-1 followed by AAV-2. (A)
Serum 1-antitrypsin 30 days after vector 1 administration. (B) Serum
erythropoietin (Epo) measured by an ELISA 30 days after vector 2 administration. (C) Reciprocal dilution (diln) of NAB to AAV-1 at
day 30. (D) Reciprocal dilution of NAB to AAV-2 at day 30.
|
|
Similar studies performed with a model of liver-directed gene transfer,
with vectors being injected into the portal circulation
via the spleen,
gave mixed results (Fig.
8). Previous
exposure
to AAV-1 does not interfere with AAV-2 gene transfer.
However,
the opposite is not true. Initial treatment with AAV-2
elicited
high levels of NAB to AAV-2 and low levels of NAB to
AAV-1 sufficient
to suppress the gene transfer of AAV-1
20-fold.
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 8.
Readministration of AAV vectors in the liver. These
experiments are identical to those described in the legend to Fig. 7,
except that animals received vectors in the portal circulation to
target the liver.
|
|
| |
DISCUSSION |
AAV vectors have been based primarily on serotype 2, a
human-derived parvovirus (19, 23). The early availability of
an infectious clone of AAV-2 stimulated work on the development of replication-defective vectors. The utility of AAV-2-based vectors for achieving long-term, stable, and safe gene transfer has been demonstrated with small-animal models; a number of studies with large
animals, including humans, are either planned or under way (9, 10,
21, 27). The tropism of replication-defective AAV-2 for
efficient in vivo gene therapy is narrow.
The goal of this study was to isolate an infectious clone of AAV-1,
which would be sequenced and used to develop a recombinant vector. The
hypothesis was that AAV-1 vectors would have distinct biological
profiles with advantages over AAV-2 vectors in some applications.
Furthermore, AAV-1 and AAV-2 vectors should not cross-neutralize, providing important advantages when used in the
context of preexisting immunity due to a natural infection or prior
gene therapy treatment.
An infectious clone of AAV-1 was generated from a replication
intermediate. Sequencing of the AAV-1 genome confirmed the presence of the structural components that characterize the AAV family. There is
approximately 80% homology in nucleotide sequence between AAV-1
and AAV-2. Delineating the entire sequence of AAV-1 has clarified a potential etiology of AAV-6, which was previously isolated as a contaminant in a human adenovirus preparation. AAV-6 appears to be a hybrid of AAV-1 and AAV-2 formed by homologous recombination between highly conserved regions spanning 452 to 552 nucleotides. There has been minimal divergence from the precursor genomes since this homologous event occurred. The implications of this
observation for gene therapy are unclear. The homologous sequences at
the site of potential recombination are located at the 5' end of the
Rep gene, which is deleted in the AAV vectors. Recombination of this
kind may lead to additional diversity within the AAV family, although
it is not clear if AAV-6 resulted from recombination in vitro or
occurred in vivo during naturally acquired infections.
The initial strategy for exploiting the biology of AAV-1 for
vectors was to create actual chimeras in which the vector genome was
formed from AAV-2 ITRs, whereas the Rep and Cap sequences used to
generate the virions were derived from AAV-1. Our goal was to
retain the favorable biology of AAV-2 in terms of efficient and
stable transduction while modifying the entry pathways and potentially
diverting humoral immune responses (8, 31). Analysis of
these vectors with murine models of liver and lung gene therapy demonstrated some important differences. When normalized for equivalent doses of administered vector genomes, AAV-2 performs better in liver than in muscle, whereas the opposite is true of AAV-1. This finding presumably is due to differences in the efficiency of entry,
although postentry events that differ between the two vectors cannot be
ruled out.
Another important advantage of alternative serotypes is to avoid
neutralization due to preexisting humoral immunity. The existence of
monospecific NAB to AAV-1 in nonhuman primates and to AAV-2 in
humans supports the same serospecificity of these AAV. It should be
noted, however, that seropositivity to AAV-1 and AAV-2, which is not neutralizing, is detected at higher frequencies with Western assays or ELISAs (data not shown). Administration of the vectors into
murine muscle demonstrated the predicted result, in that previous
treatment with AAV-2 blocks the subsequent administration of
AAV-2 while having only a modest impact on the efficiency of AAV-1 treatment. The opposite is also true, in that AAV-1
administered to muscle blocks a second treatment with AAV-1 but
does not interfere with gene transfer mediated by AAV-2. The murine
model of AAV gene transfer to liver yielded mixed results. Clearly,
AAV-2 and AAV-1 completely blocked readministration of the same
serotype. Initial treatment with AAV-1 did not affect a follow-up
administration of AAV-2, although the opposite was not true.
Specifically, initial treatment with AAV-2 diminished retreatment
with AAV-1 approximately 20-fold, a value which will likely be
significant in therapeutic applications. We conclude that AAV-2
generates cross-neutralizing antibodies to AAV-1 that have an
impact on AAV-1-mediated gene transfer. This effect is more
significant in the context of intravascular administration. There is no
evidence that antibodies to AAV-1 cross-neutralize
AAV-2-mediated gene transfer to muscle or liver.
One should consider these results in the context of human applications
of AAV vectors. The majority of humans do not have NAB to either
AAV-1 or AAV-2. In fact, the absence of monospecific antibodies
to AAV-1 in humans, together with the fact that it was isolated
from monkeys, argues against it being a human virus. What is the
relevance of neutralizing activity to AAV-1 in 20% of humans? Does
this value reflect primary infection or cross-neutralization in the
context of infection with wild-type AAV-2? Will this neutralizing activity have an impact on the efficiency of AAV-1 uptake? The data
presented here suggest that the AAV-1 vector would be the preferred
initial vector for muscle-directed gene therapy. It is more efficient
than AAV-2 and does not preclude follow-up treatment with
AAV-2. The situation for the liver is less clear. The AAV-1 vector is less efficient in the context of naive animals, although initial treatment with AAV-2 partially inhibits a second
administration of AAV-1.
The impact of humoral immune responses to AAV vectors on human
applications of gene therapy is an important consideration for chronic
diseases. It appears that AAV expression will persist for a prolonged
period of time in most target organs. What is less clear is how long
the humoral immune response will persist. The use of vectors based on
different serotypes should allow at least two treatments. A requirement
for more frequent readministration of vectors over short time periods
may require other strategies which blunt the initial humoral immune
response (12, 16).
| |
ACKNOWLEDGMENTS |
We thank the Vector, Cell Morphology and Immunology Cores of the
Institute for Human Gene Therapy and Wei Cao, Marcia Houston-Leslie, Rosalind Barr, Ruth Qian, George Qian, and Sarah Ehlen-Haecker for
technical support.
This work was supported by grants from the NIH (P30 DK47757-06 and P01
HD32649-04), the Muscular Dystrophy Association, and Genovo, Inc., a
company that J. M. Wilson founded and has equity in.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 204 Wistar
Institute, 3601 Spruce St., Philadelphia, PA 19104-4268. Phone: (215)
898-3000. Fax: (215) 898-6588. E-mail:
wilsonjm{at}mail.med.upenn.edu.
| |
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Journal of Virology, May 1999, p. 3994-4003, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
