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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2263-2270
In Vivo Marking of Rhesus Monkey Lymphocytes by Adeno-Associated Viral
Vectors: Direct Comparison With Retroviral Vectors
By
Yutaka Hanazono,
Kevin E. Brown,
Atsushi Handa,
Mark E. Metzger,
Dominik Heim,
Gary J. Kurtzman,
Robert E. Donahue, and
Cynthia E. Dunbar
From the Hematology Branch, National Heart Lung and Blood Institute,
National Institutes of Health, Bethesda, MD; and Avigen Inc, Alameda,
CA.
 |
ABSTRACT |
We have compared adeno-associated virus (AAV)-based and
retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model.
To avoid the complication of immune rejection of lymphocytes transduced
with xenogeneic genes in tracking studies primarily designed to
investigate transduction efficiency and in vivo kinetics, the vectors
were designed without expressed genes. All vectors contained
identically mutated  -galactosidase gene (-gal) and neomycin resistance gene (neo) DNA sequences separated by
different length polylinkers, allowing simple differentiation by
polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood
lymphocytes from 4 rhesus monkeys were transduced with either AAV or
retroviral vectors. The in vitro transduction efficiency (mean vector
copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction,
Southern blot analysis of AAV-transduced lymphocytes showed
double-stranded and head-to-tail concatemer forms but failed to show
integration of the AAV vector. AAV and retroviral aliquots were
reinfused concurrently into each animal. Although the retrovirally
marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
LYMPHOCYTES HAVE several features that
are attractive as targets for gene therapy; ease of ex vivo
manipulation, acceptable transduction efficiencies with retroviral
vectors, and long-term in vivo life span. Therefore, many investigators
have been studying lymphocytes as potential targets for gene-therapy
applications, including correction of severe combined immunodeficiency
syndromes, targeted anti-cancer/anti-human immunodeficiency
virus (HIV) therapy, or as factories for soluble
factors.1,2 However, human in vivo trials with retroviral
vectors have resulted in very low expression levels from retrovirally
introduced genes in circulating lymphocytes, precluding clinical
efficacy for most applications.3 Interpretation of results
has been complicated by the stimluation of immune responses against
xenogeneic genes included in the vectors, such as the bacterial
neomycin resistance gene (neo) or the herpes thymidine kinase
gene.4,5 Lymphocytes expressing at high levels may have
been immunologically selected against in vivo, or shut down of
expression from proviral control elements may have
occurred.6
Adeno-associated viruses (AAVs) are small, nonenveloped,
single-stranded DNA viruses of the Parvoviridae
family.7 Much current interest in AAVs stems from their
potential use as vectors for gene therapy. To date, 5 primate AAVs have
been distinguished serologically by their antigenically distinct capsid
proteins, but AAV-2 has been predominantly used for the vector
platform.8,9 The AAV-2 viral genome is composed of 4,679 nucleotides, flanked at both ends by 145 nucleotides of palindromic
inverted terminal repeats (ITRs).
Several characteristics of AAV-based vectors suggest that they might be
useful for gene transfer to T lymphocytes. AAVs infect a wide variety
of cell types, including lymphocytes,10 and infection of
humans or other animals with AAVs has not been associated with any
disease or pathogenicity. AAV vectors will transduce both dividing and
nondividing cells in culture.11 High-level, long-term expression in myocytes, neurons, and hepatocytes in vivo has been achieved, even without chromosomal integration, implying expression from a stable episomal form of AAV vectors.12-14
Replication-defective AAV vectors have been shown to integrate, but
inefficiently and randomly,15,16 although wild-type AAV
integrates more efficiently into a specific locus on human chromosome
19, a process dependent on intact rep gene
function.17,18
Although successful AAV vector transduction of human T lymphocytes
accompanied by transgene expression has been documented in
vitro,19-21 in vivo analysis of AAV-transduced lymphocytes
has not been performed. Using the rhesus macaque, we have compared the
in vivo fate of autologous lymphocytes transduced with either AAV or
standard retroviral vectors. Prior studies of rhesus and human
T-lymphocyte retroviral transductions have been reported and clinical
trials using these cells completed; thus, retroviral vectors are an
appropriate standard for comparison.22,23 In these initial
studies, vectors that do not produce xenogeneic gene products were used
to avoid complicating the analysis of the persistence of transduced
cells by active immune rejection.4,5
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MATERIALS AND METHODS |
Plasmid construction.
We constructed 2 AAV-2 and 1 retroviral nonexpression vector
(Fig 1). The first ATGs of the
-gal sequences in pCMV (Clontech, Palo Alto, CA) and the
neo sequences in pG1Na (Genetic Therapy Inc, Gaithersburg, MD)
were mutated into CTGs by standard polymerase chain reaction (PCR)
mutagenesis. The mutated neo gene cassette from pG1Na was
introduced into the 3' end of the mutated -gal gene in
pCMV (pCMVneo). Polylinkers (PLII and PLIII) derived from pSE420
(Invitrogen, San Diego, CA) were introduced between the mutated
-gal and neo genes in pCMVneo. The
-gal-polylinker-neo fragments were then subcloned
into pBluescript (Stratagene, La Jolla, CA) and XbaI linkers
were generated at the both ends of the
-gal-polylinker-neo fragments. The resulting
-gal-polylinker-neo cassettes (4.3 kb) were inserted
into XbaI sites of pSub201 (a gift of Dr Jude
Samulski, University of North Carolina, Chapel Hill, NC9)
(pAAVPLO and pAAVPLIII) and into the SnaBI and HindIII sites of pG1 (Genetic Therapy Inc) (pG1PLII).
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| Fig 1.
Vector construction. All 3 nonexpression vectors contain
identical -gal and neo sequences separated by
different-length polylinkers. KpnI releases the retroviral
sequence at the length of 6.0 kb. XbaI cuts the AAV vector
sequence twice and NotI cuts once. These restriction enzymes
were used for Southern blotting as shown in Figs 4 and 5.
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Vector production and purification.
The nonexpression AAV vectors, AAVPLIII and AAVPLO, were produced in an
adenovirus-free system and CsCl-purified as previously reported.19 The titers of AAV vector stocks used in these
studies were estimated to be 1012 to 1013
genome copies/mL by Southern blot.
The nonexpression retroviral vector G1PLII producer cell line was
generated by first transfecting the retroviral plasmid into BOSC23
ecotropic packaging cells using calcium phosphate precipitation, and
harvesting supernatants 48 to 60 hours
later.24 These supernatants were then used to infect PA317
amphotropic packaging cells using a standard protocol.25
Stable producer clones were then isolated by limiting dilution. Fresh
supernatants from 80% to 90% confluent producer clones were collected
and used for rhesus lymphocyte transductions. The titers of these
G1PLII supernatants were 5 × 105/mL according to
viral RNA slot blot and Southern blotting of target HeLa
cells.26
Lymphocyte transduction and infusions.
Four rhesus monkeys were enrolled in the study, and all animals were
housed and handled according to guidelines set forth by the Committee
on Care and Use of Laboratory Animals of the Institute of Laboratory
Animal Resources, National Research Council (Bethesda, MD, Department
of Health and Human Services [DHHS] Publication No. NIH
85-23) and the protocol was approved by the Animal Care
and Use Committee of the National Heart, Lung, and Blood Institute.
Fifty milliliters of heparinized peripheral blood was separated on a
density gradient (LSM; ICN Biomedical, Aurora, OH) to obtain
mononuclear cells. These cells were incubated in AIM-V medium (Life
Technology, Gaithersburg, MD) in the presence of 500 IU/mL recombinant
human interleukin-2 (IL-2) (R&D, Minneapolis, MN) and FN18, a
monoclonal antibody to rhesus CD3 (Biosource International, Camarillo,
CA) for 3 days. The cells were then divided into 2 equal aliquots. One
sample was transduced with AAVPLO or III once on day 3 by adding the
purified AAV vector to culture medium in the presence of 500 IU/mL
human IL-2, and was incubated for another 7 days. Lymphocytes from
rhesus 94E068 were transduced with 2,000 viral genomic copies (gc) of
AAVPLO per cell, those from RQ1307 with 20,000 gc AAVPLO per cell,
those from RQ1303 with 20,000 gc AAVPLIII per cell, and those from
RQ854 with 200,000 gc AAVPLO per cell. The other aliquot was transduced
with the retroviral vector G1PLII 3 times over 7 days according to the
published method.22 Briefly, cells were first incubated in
phosphate-free media for 6 hours, and then resuspended in the
retroviral G1PLII supernatant at a multiplicity of infection
(MOI) of 1 to 5, along with 4 µg/mL protamine sulfate
(Sigma, St Louis, MO) and 500 IU/mL human IL-2 , followed by
centrifugation at 1,000g at 32°C for 1 hour and incubation
at 32°C overnight. This optimized transduction was performed twice,
and a standard transduction was performed once, without phosphate
depletion, centrifugation, or low-temperature transduction. Finally,
both aliquots were reinfused simultaneously into each monkey after a
total of 10 days of culture and transduction.
PCR analysis.
DNA was isolated from peripheral blood mononuclear cells using the
QIAamp Blood Kits (Qiagen, Valencia, CA). Five hundred nanograms of DNA
was used in each reaction. The standards consisted of log dilutions of
DNA extracted from the G1PLII-producer clone (which contains 3 copies
of the vector proviral sequence per cell, documented by Southern blot)
into control normal rhesus peripheral blood mononuclear cell
(PBMNC) genomic DNA. The negative control consisted of
DNA extracted from pretransduced rhesus PBMNCs. PCR primers were used
that span the polylinker in the vectors, allowing differentiation of
the AAV and retroviral vector sequences. Overall reactions were
optimized to yield linear amplifications in the range of the intensity
of the positive controls. The outer primer set was 5'-CGT CAG TAT
CGG CGG AAT TAC AGC-3' and 5'-CAG TCA TAG CCG AAT AGC CTC
T-3'. The inner primer set was 5'-CGC TAC CAT TAC CAG TTG
GTC-3' and 5'-AGA ACC TGC GTG CAA TCC ATC-3'.
Amplification conditions were 95°C for 1 minute, 54°C for 1 minute, and 72°C for 2 minutes with 20 cycles both for both outer
and inner PCR. The outer PCR products were purified by QIAquick PCR
purification kits (Qiagen). The inner PCR was performed in the presence
of 12.5 µCi/mL [ -32P]dCTP. The expected sizes of the
final PCR products were 508 bp, 438 bp, and 355 bp for G1PLII,
AAVPLIII, and AAVPLO, respectively.
PCR for -gal and -actin was also performed. The
primer set for -gal was 5'-CTA CAC CAA CGT AAC CTA TCC
C-3' and 5'-TTC TCC GGC GCG TAA AAA TGC G-3'. The
primer set for -actin was 5'-CAT TGT GAT GGA CTC CGG
AGA CGG-3' and CAT CTC CTG CTC GAA GTC TAG AGC-3'.
Amplification conditions were 95°C for 1 minute, 54°C for 1 minute, and 72°C for 2 minutes in the presence of 12.5 µCi/mL [-32P]dCTP with 30 cycles for -gal and 26 cycles for -actin. The expected sizes of the PCR products
were 249 bp for -gal and 234 bp for -actin. The
final PCR products were separated on 8% polyacrylamide gels. The
quantification of the band intensity was performed using a
Phosphorimager ImageQuant (Molecular Dynamics, Sunnyvale, CA).
Southern blotting.
Ten micrograms of DNA was digested with either KpnI,
XbaI, or NotI restriction enzymes, separated on a 0.8%
agarose gel, and transferred onto Hybond-N+ (Amersham, Cleveland, OH).
The radiolabeled DNA probe was a neo gene-specific sequence
generated by PCR. The forward primer was 5'-TCC ATC ATG GCT GAT
GCA ATG CGG C-3' and reverse primer was 5'-GAT AGA AGG CGA
TGC GCT GCG AAT CG-3'. Radiolabeling of the probes was done using
an oligolabeling kit (Phamacia, Piscataway, NJ).
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RESULTS |
Vector design.
We constructed 2 AAV vectors and 1 retroviral vector (Fig 1), with
mutations of ATGs in neo and -gal sequences to
prevent translation ("nonexpression" vectors). All 3 vectors
contain identical -gal and neo sequences, but with
different-length polylinkers (PLII and PLIII) inserted between the 2 genes to allow identification of the vectors by PCR and direct
comparison of the levels of vector-containing cells in vivo. We
confirmed that significant levels of functional gene products were not
translated by showing a lack of X-gal staining and G418 resistance, as
well as the absence of full-length or truncated proteins on immunoblots
in 3T3 cells transduced with the nonexpression vectors
(Y.H., manuscript in preparation).27
Ex vivo expansion and transduction.
Using these nonexpression vectors, we compared transduction of
autologous rhesus monkey lymphocytes with AAV and retroviral vectors.
Mononuclear cells were obtained from 50 mL peripheral blood and
incubated with IL-2 and anti-CD3 antibody for 3 days. The cells were
then divided into 2 equal aliquots; 1 aliquot was transduced once with
the AAV vector, while the other aliquot was transduced with the
retroviral vector (3 times). Several ratios of AAV vector particles to
target cells (MOI) were used (Table 1).
Expansion during the ex vivo culture period is shown in
Fig 2 and was almost equivalent for the
aliquots transduced with AAV or retrovirus. No toxic effects of either
vector were observed during the ex vivo culture period. By
fluorescence-activated cell-surface analysis, over 98% of cells were T
cells expressing CD2 at the end of expansion (10 days after culture
initiation; data not shown).
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| Fig 2.
Ex vivo expansion of rhesus lymphocytes. The numbers of
lymphocytes decreased for the first 3 days. Thereafter, the cell
numbers increased and the overall expansion is shown. The expansion was
equivalent for both AAV- ( ) and retrovirus-transduced ( )
aliquots.
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After the 10-day ex vivo culture and transduction period, PCR for the
vector (-gal) sequence and internal -actin
control sequence was performed to quantify the in vitro transduction
efficiency (Fig 3A). When 200,000 AAV
genomic copies per lymphocyte were used, very high-level transduction
(on average 3 copies per lymphocyte in RQ854) was seen on analysis at
day 10, just before reinfusion. When 20,000 copies per lymphocyte were
used, copy numbers of 0.17 (RQ1307) and 0.80 (RQ1303) per lymphocyte
resulted. When 2,000 copies per lymphocyte were used (94E068), only
0.015 copies per lymphocyte resulted (Fig 3B and Table 1). The
transduction efficiencies for the retroviral vector were estimated by
PCR to be 0.13 to 0.19, similar to levels previously reported using
this retroviral transduction methodology.22
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| Fig 3.
In vitro transduction efficiencies. (A) Semi-quantitative
PCR for the -gal and actin sequences was performed
at the end of the ex vivo culture period. The -gal is the
vector sequence and the actin is the control sequence. (B) The graph summarizes the in vitro transduction efficiencies
shown as a mean copy number per cell, calculated from the PCR results.
The MOI used was different among monkeys. 94E068 had 2,000, RQ1307 and
RQ1303 had 20,000, and RQ864 had 200,000 AAV genomic copies per
lymphocyte. (), AAV; (), retrovirus.
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Southern blotting was also performed after the ex vivo culture on
retrovirally transduced lymphocytes (Fig
4). All 4 monkeys had the proviral sequence in their lymphocyte genome.
A copy number of 0.14 to 0.22 integrated provirus per lymphocyte was
estimated by Southern blotting, consistent with the PCR results (Table
1). The Southern blot analysis of the AAV-transduced lymphocytes on day
10 just before reinfusion gave very different results
(Fig 5A). In AAV-transduced aliquots from
animals RQ1303 and RQ854, with relatively high-level transduction as
estimated by PCR (0.80 and 3.0 copy number per lymphocyte,
respectively), single-strand DNA (ssDNA) 1.6-kb bands were observed.
These represent the expected size of the single-stranded AAV vector
itself. In RQ854, with the highest in vitro transduction efficiency,
besides the 1.6-kb ssDNA band, a 4.3-kb band was observed after
XbaI digestion (cutting at both ends of the vector genome),
suggesting that some of the vector DNA has become double-stranded and
could be released as viral double-stranded DNA (dsDNA). When
single-cutter NotI was used, a 1.0 kb-band was observed,
consistent with the size from the NotI site to the 3'-end
of the dsDNA vector. Furthermore, one more band of 4.6 kb was observed
in RQ854 when NotI was used. This band can be explained if a
portion of the viral dsDNA has become a head-to-tail concatemer (Fig
5B). Therefore, this Southern blot is consistent with the recent
hypothesis that AAV vector ssDNA first becomes double-stranded and then
forms a stable head-to-tail concatemer episome array before initiating
transcription.13 The Southern blots did not show any
evidence of integration of the vector into the genome by day 10 of
culture (7 days after AAV transduction); however, there is no
convenient assay to rule out the possibility of integration, at
least at a low level. At this point, the cells were reinfused into the
monkeys (see below). It is possible that conversion to the
double-stranded, concatamerized form continued in vivo, but the low
level of modified cells prevented Southern blot analysis of
postinfusion blood samples.
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| Fig 4.
Southern blotting of the retrovirally transduced
lymphocytes after the ex vivo culture (day 10). Ten micrograms of DNA
from transduced lymphocytes was digested with KpnI, which
released the proviral sequence at the length of 6.0 kb (see Fig 1),
separated on an agarose gel, and transferred to a nylon membrane
followed by hybridization with the neo DNA probe. The proviral
sequence in the transduced lymphocyte genome was detected in all 4 monkeys. The positive controls (lanes 6 through 10) were made from
dilutions of the G1PLII-retroviral producer DNA into control rhesus
lymphocyte DNA.
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| Fig 5.
Southern blot analysis for the AAV vector-transduced
rhesus lymphocytes with XbaI and NotI (A). Ten
micrograms of DNA from transduced lymphocytes was digested with
XbaI or NotI, separated on agarose gels, and
transferred to nylon membranes followed by hybridization with the
neo DNA probe. In RQ854, besides the single-strand DNA (ssDNA)
band around 1.6 kb, the double-strand DNA (dsDNA) bands were detected,
which were released by digestion with XbaI (4.3 kb, lane 5) and
with NotI (1.0 kb, lane 10). Another band of 4.6 kb with
NotI (lane 10) is consistent with a head-to-tail concatamer
form of the vector. (B) The hypothesis of AAV vector conversion to a
head-to-tail concatemer. This Southern blot is consistent with the
hypothesis.
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In vivo tracking of transduced lymphocytes.
We reinfused transduced cells into each monkey and followed the in vivo
persistence of marked cells by PCR (Fig 6).
In animal 94E068, receiving cells transduced with low MOI AAV, the AAV
signal disappeared 2 weeks after infusion, whereas the retroviral
signal was still detectable at week 56. In RQ1307 and RQ1303, receiving the intermediate MOI AAV, in vivo marking was equivalently shown for
AAV and retroviral vectors for the first 3 days. However, the AAV
signals decreased more rapidly and disappeared at weeks 8 and 4, respectively, while the retroviral signals persisted. In RQ854
receiving cells transduced with the highest MOI AAV, an in vivo AAV
signal was shown at copy number levels of up to 0.03, several-fold
higher than retroviral marking. However, the intensity
of the AAV marking decreased rapidly, although it was still detectable
at weeks 5 and 6.
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| Fig 6.
In vivo persistence of transduced cells as detected by
PCR. Mononuclear cells from rhesus peripheral blood were obtained at
indicated time points. DNA was extracted and subjected to
semi-quantitative PCR spanning the polylinkers (see Fig 1). The longer
PCR products are derived from the retroviral vector and the shorter
ones are derived from the AAV vectors. The copy number control was made
from log dilution of the G1PLII-retroviral producer clone into the
control rhesus lymphocyte DNA.
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| |
DISCUSSION |
In this study we have shown that both AAV- and retrovirus-based vectors
can be used to transduce primary T lymphocytes, using the rhesus monkey
model. Transduction with retrovirus vectors was performed using the
optimized transduction methods of phosphate depletion, centrifugation,
and low-temperature transduction,22 but similar
optimizations have not been performed for AAV transduction and, hence,
in this study AAV vectors were added on a single occasion to the
culture media, at the same time as the first retroviral transduction.
Despite only a single transduction procedure, we have shown that in
vitro transduction efficiency of lymphocytes using AAV vectors was
greater than retroviral vectors when high-titer AAV was used: a viral
preparation concentrated to allow a ratio of 200,000 genome copies per
target cell achieved, on average, 3.0 AAV genomic copies per transduced cell.
It should be noted that this MOI calculation was based on gc of AAV
vector. Using expression-competent vectors, it is known that only a
small percentage of the overall recombinant AAV particle population (1 in 102-4 particles, depending on the vector preparation) are functional. High-level transduction and sustained gene expression has required very high recombinant AAV gc per target cell, compared with retroviral vectors, where the use of greater than 10 infectious particles per target cell has not resulted in increased transduction efficiencies.28 This requirement for very high numbers of
concentrated AAV vectors might be a drawback for clinical applications,
because generation of these stocks on a large scale is technically
challenging, although improved approaches are being
developed.29-31
It is possible that receptor expression on lymphocytes could be
limiting for AAV transduction. Recently it has been proposed that
heparan sulfate proteoglycan may act as a cellular receptor for
AAV-2,32 with the fibroblast growth factor receptor, FGFR1, or V5 intergrin acting as putative coreceptors.33,34
These molecules are expressed on the cell surface of
lymphocytes,35,36 but the expression levels are low and
this factor may account for the high MOI requirement for AAV
transduction of lymphocytes. Alternatively, our own studies have shown
that these molecules are not required for successful transduction of
cell lines, suggesting that other molecules may act as
receptors42 Regardless, manipulations to increase
AAV-receptor interactions, as has been accomplished via phosphate
depletion for amphotropic retroviral receptors, may help overcome this
possible limitation. Nondividing cells such as neurons, myocytes, and
hepatocytes may be efficiently transduced with AAV vectors, as opposed
to retroviral vectors.12,13 In our study, 2 animals
received cells transduced at the same AAV dose (20,000 gc/cell) but had
different patterns of ex vivo lymphocyte growth. Cells from 1 animal
grew poorly, but transduced at a higher relative level than more
rapidly proliferating cells from the other animal. Likewise, the animal
that had cells transduced at the very high AAV MOI and had the highest
transduction efficiency (3 copies per transduced cell) had very poor ex
vivo growth of the target lymphocytes. More efficient transduction may
be possible by either shortening the ex vivo culture period
(potentially also retaining a greater repertory or improved
functionality of infused T cells) or by adding the AAV vectors on the
first day without prior stimulation.
We have confirmed transgene expression from AAV vectors in both human
and rhesus lymphocytes using an AAV vector expressing the green
fluorescent protein gene (data not shown) Similarly, several other
researchers have reported high expression in vitro of AAV-transduced
genes in lymphocytes.19-21 However, in the current study,
as shown in the Southern blots (Fig 5), most of the vector DNA still
remains as single-strand form at the time of reinfusion into the
animals 7 days after transduction, and only a small fraction of the
vector DNA formed the head-to-tail concatemers. Conversion from a
single-stranded form of AAV vectors to a double-stranded form is
considered to be rate-limiting among steps required for transgene
expression.13,37,38 Radiation and some cytotoxic drugs may
accelerate conversion to expressing forms and, thus, improve AAV
transgene expression.37,38 However, these manipulations are
not appropriate for clinical transductions of primary lymphocytes. The
fact that on the day of reinfusion concatemers were observed suggests
that, in vivo, the conversion process continued, and that expressed
gene products could increase their levels in vivo during the 2-8-week
period AAV-marked cells persisted in the circulation. It is also
possible that integration occurred, although the disappearance of the
AAV vector signal by PCR analysis after 5 to 7 weeks suggests that
integration was not common.
As shown in Fig 6, in vivo retroviral marking persisted longer than AAV
marking, detectable for 1 year after a single infusion to date in
several animals. The lack of xenogeneic expressed and translated
sequences may have contributed to this long persistence. In fact, in
separate experiments, rhesus lymphocytes transduced with the
nonexpression retroviral vector persisted stably in vivo (more than 1 year), whereas lymphocytes transduced with the neo-expressing retroviral vector disappeared or decreased to minimal levels after 5 to
10 weeks.27 This long survival of lymphocytes transduced with the nonexpressed sequence is relevant to the prospects for lymphocyte gene therapy, and emphasizes the need to avoid inclusion of
xenogeneic or allogeneic expressed sequences in vectors. There have
been several reports documenting rejection of retrovirally transduced
lymphocytes expressing xenogeneic genes, even in immunosuppressed HIV-positive patients or allogeneic transplantation
recipients.4,39
The Southern blots for AAV-transduced lymphocytes showed no clear
evidence for integration, although at only 7 days posttransduction this
slow conversion and integration process may have not been completed. It
may have continued in vivo after reinfusion, although AAV-transduced
lymphocytes could not be detected in vivo beyond 6 to 8 weeks. These
observations suggest that the integration frequency of AAV vectors in
primary lymphocyte chromosomes is low. However, AAV transduction
resulted in higher short-term in vivo marking as compared with
retroviral transduction. The disappearance of the AAV-containing cells
could have resulted from episomal loss during cell division.
Alternatively, a preexisisting anti-AAV2 immune response against
residual AAV proteins still present in the transduced lymphocytes could
have contributed to rapid clearance. This seems unlikely, given the
prolonged culture period after exposure to replication-incompetent
vector. Western blotting for anti-AAV2 antibodies has not shown any
evidence for anti-AAV2 humoral immunity in these animals before or
after infusion of transduced lymphocytes (K.E.B., unpublished data,
April 1999). Our results suggest possible clinical
applications of AAV vectors in lymphocyte gene therapy when long-term
vector persistence is not required or desired, for instance in targeted
cancer gene therapy.1,40,41
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FOOTNOTES |
Submitted January 6, 1999; accepted May 27, 1999.
D.H. was supported by grants from the Swiss National Science
Foundation, the Ciba-Geigy Jubilaeumsstiftung, and from Cancer Research
Switzerland KFS 500-8-1997.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Cynthia E. Dunbar, MD, Bldg 10, Room 7C103,
9000 Rockville Pike, Bethesda, MD 20892; e-mail:
dunbarc{at}gwgate.nhlbi.nih.gov.
| |
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