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RESEARCH ARTICLE
Self-complementary recombinant adeno-associated
virus (scAAV) vectors promote efficient transduction
independently of DNA synthesis
DM McCarty1,2, PE Monahan1,3 and RJ Samulski1,4
1
UNC Gene Therapy Center, 2School of Pharmacy, 3Department of Pediatrics, 4Department of Pharmacology University of North
Carolina at Chapel Hill, NC, USA
Adeno-associated virus (AAV) vectors package single- rAAV, with a 5.9:1 particle to transducing unit ratio. This
stranded genomes and require host-cell synthesis of the efficiency is neither greatly increased by co-infection with
complementary strand for transduction. However, when the Ad, nor inhibited by hydroxyurea, demonstrating that trans-
genome is half wild-type size, AAV can package either two duction is independent of DNA synthesis. In vivo, scAAV
copies, or dimeric inverted repeat DNA molecules. Dimeric, expressing erythropoietin resulted in rapid and higher levels
or self-complementary molecules (scAAV) should spon- of hematocrit than a conventional single-stranded vector.
taneously reanneal, alleviating the requirement for host-cell These novel scAAV vectors represent a biochemical inter-
DNA synthesis. We generated and characterized scAAV mediate in rAAV transduction and should provide new
vectors in order to bypass the rate-limiting step of second- insights into the biology of vector transduction. Gene
strand synthesis. In vitro, scAAV vectors were five- to 140- Therapy (2001) 8, 1248–1254.
fold more efficient transducing agents than conventional
1249
anisms to provide a complementary-strand for rAAV
vectors, we have found that we can circumvent the prob-
lem by packaging both strands as a single DNA molecule.
Original studies on wild-type (wt) AAV determined that
defective interfering particles (DI) often carried atypical
genomes comprised of inverted repeats.20 Subsequent
studies have also revealed that rAAV DNA of less than
half the wtAAV genome length can be packaged as a
dimer or a diploid monomer, mimicking a DI particle.21–23
The replication scheme of AAV suggests that the dimeric
DNA molecules would be in the inverted repeat con-
figuration.24,25 These self-complementary molecules
(scAAV) should have the ability to re-fold into double-
stranded DNA (dsDNA) templates for expression with
pseudo-first order kinetics upon uncoating. In this study,
we engineered a number of vector constructs, which
packaged genomes varying in size relative to the wtAAV
genome, and evaluated their ability to transduce in vitro
and in vivo. We observed an increased efficiency of trans-
duction from scAAV vectors over conventional rAAV in
HeLa cells (5- to 140-fold). More importantly, unlike con-
ventional single-stranded AAV vectors, inhibitors of Figure 1 Virion DNA content of rAAV vectors. The drawing illustrates
DNA replication did not affect transduction from scAAV the DNA content of the rAAV vectors used in this study and the predicted
conformation that they adopt upon release from the virions. The trans-
vectors. In addition, scAAV vectors displayed a rapid
genes expressed from the cytomegalovirus immediate–early promoter
onset and a higher level of transgene expression in mouse (CMV) are: green fluorescent protein (GFP),  galactosidase (LacZ),
hepatocytes in vivo. All of these biological attributes sup- mouse erythropoietin (nEpo). Neomycin phosphotransferase (neo) is
port the generation and characterization of a new class expressed from the SV40 early promoter (SV40). The size, in nucleotides
of AAV vectors (delivering duplex DNA) that should (nt) of each packaged DNA molecule is indicated. The self-complementary
significantly contribute to the ongoing development of (scAAV) GFP dimer and mEpo dimer vectors fold into a complete duplex
DNA with one extra copy of the terminal repeat while the GFPneo, LacZ,
parvovirus-based gene delivery systems.
and mEpo vectors require cell-mediated DNA synthesis of the
complementary strand.
Results
Generation of scAAV vector
A rAAV plasmid construct (pTR-CMV-GFP), with a
replicon size of 2299 nucleotides, was used to generate a
viral vector stock (rAAV-GFP) by conventional methods.
The predicted size of the dimeric replicative form of this
vector was 4474 nucleotides (Figure 1), which was 95.6%
of the wt AAV genome length. The viral vectors were
fractionated by isopycnic gradient centrifugation in CsCl
and the vDNA content of each fraction was analyzed on
alkaline agarose gels (Figure 2). PhosphoImager scans
were used to quantify the vDNA-specific bands from
each fraction. Under denaturing conditions, the self-
complementary dimer DNA (Figure 2a, fractions 10–13)
ran at approximately twice the length of the monomeric
genome. The hybridizing material in fractions 2–4 is
unpackaged replicative form DNA that sediments at the
bottom of the gradient. Athough a DNase step was
included in the vector purification (see Methods), the
treatment was not intended to be exhaustive and this
material proved to be DNase sensitive in subsequent
experiments while the material in fractions 10–14 was
Figure 2 Recombinant AAV vectors fractionated on CsCl gradients.
DNase resistant (data not shown). Vectors containing Virion DNA (vDNA) was extracted from CsCl gradient fractionated
mostly dimeric DNA genomes (fractions 10 and 11) were CMV-GFP (a), GFPneo (b), and LacZ (c) rAAV vectors. Alkaline agarose
designated as ‘self-complementary rAAV’ (scAAV). The gels of the vDNA were Southern blotted and hybridized with a CMV-
inverted repeat structure of these molecules was con- GFP DNA fragment. Markers at the left end of panel a were the excised
firmed by restriction enzyme digestion (data not shown). vector sequences from the plasmids used to generate the viral vectors (see
Two additional rAAV vectors (Figure 1) were gener- Results). The number of unit length, ssDNA, vector copies per molecule
are indicated by 1×, 2×, and 4×. The viral vectors used in the experiments
ated and purified in parallel, and analyzed in the same depicted in Figures 3 and 4 were from fractions a-11 or a-10 for scAAV
manner (Figure 2b and c). The first, rAAV-GFPneo, con- CMV-GFP (as indicated in the Figure legends), fraction a-13 or a-14 for
tained a neo gene in addition to the GFP and had a rep- the monomer CMV-GFP, fraction b-13 for GFPneo, and fraction c-12
licating genome length of 3398 nucleotides. This was for LacZ.
Gene Therapy
rAAV double-stranded vector
DM McCarty et al
1250
72.6% of the wtAAV genome size, and was too large to to the dimer fraction. In contrast, the monomeric ssDNA
be packaged as a dimer. The second was a 4898 nucleo- GFPneo and LacZ vectors had particle to transducing
tide rAAV-CMV-LacZ construct, which was slightly unit ratios of 125:1 and 828:1, respectively, comparable to
larger (104.7%) than wtAAV genome size, but within the previously reported efficiencies for these vectors.13,26
limit for efficient packaging.22 The lower density, higher The transducing efficiency of conventional rAAV vec-
mobility hybridizing material in fractions 14 and 15 tors can be greatly enhanced (up to 100-fold) by co-infec-
(Figure 2, panel c) comprised genomes which had under- tion with Ad, or by treatment with DNA damaging
gone deletions and these fractions were not used in agents or other types of cell stress. This enhancement had
subsequent experiments. been associated with the cell-mediated transformation of
the ssDNA genome into active ds-DNA transcription
Transduction with dimeric versus monomeric rAAV templates. Because the dimeric rAAV contains the two
vectors and effects of Ad co-infection complementary strands packaged as a single molecule,
The transducing efficiency of the scAAV-GFP (Figure 2a, we predicted that transduction would be independent of
fraction 11) was compared with the homologous mono- enhancement by adenovirus. This was largely the case
mer (fraction 13), as well as the GFPneo and LacZ vectors when HeLa cells were co-infected with the scAAV vec-
(Figure 2b and c, fractions 13 and 12, respectively), in tors and 5 infectious units per cell of adenovirus
HeLa cells infected at low multiplicity (Figure 3). The (Figure 3). The number of GFP-positive cells in the
particle numbers were calculated from the specific, full- scAAV infected cultures was increased by only 1.6-fold,
length vDNA PhosphorImager signals in each fraction on an effect which could be attributed to the transcriptional
the Southern blot, after correction for monomeric versus effects of adenovirus infection on the activity of the CMV
dimeric DNA copy number. Thus, each scAAV contains promoter as previously reported.27,28 The monomer vec-
two copies of the transgene as a single molecule, in the tor transduction rate was increased 2.4-fold by Ad co-
inverted repeat orientation, while each monomeric infection, while the GFPneo and LacZ vectors were
particle contains one single-stranded copy. induced 6.0-fold and 12.8-fold, respectively.
The scAAV CMV-GFP vector (fraction 11), containing
approximately 90% dimer virus, yielded a 5.9:1 ratio of Transduction with dimeric rAAV vectors in the absence
physical particles to transducing units, thus bearing out of host-cell DNA synthesis
our prediction of high transducing efficiency. Fraction 13, Because the vDNA of the scAAV vectors contained both
from the same gradient, conversely containing approxi- DNA strands on a single molecule, allowing efficient
mately 80–90% monomer virus, had a 24.6:1 particle to renaturation upon uncoating, we predicted that these
transducing unit ratio. This four-fold difference in vectors would obviate the role of host-cell DNA synthesis
efficiency represented a minimum difference when it was in transduction. We compared the scAAV-GFP vector
considered that the dimer contamination in the monomer with the homologous monomer, and the GFPneo vector,
fraction would have a greater impact on its transducing in HeLa cells pretreated with hydroxyurea (HU) 24 h
potential than the monomer component would contribute before infection to inhibit host cell DNA synthesis. Hyd-
roxyurea treatment was continued, uninterrupted, at the
same concentrations following infection and maintained
on the cells for the following 24 h, until GFP transduction
was scored.
Transduction from the scAAV-GFP was stimulated by
up to 1.9-fold in response to increasing concentrations of
HU (Figure 4a). This stimulation, similar in magnitude to
that observed with Ad co-infection, was probably affec-
ted through a combination of transcriptional transactiv-
ation of the CMV promoter brought about by cell stress,
and the accumulation of GFP in the nondividing cells.
In contrast, transduction from the homologous monomer
vector fraction was stimulated at the lowest HU concen-
tration and inhibited at higher concentrations. The
residual transducing activity from the monomer vector at
higher HU concentrations, at a level approximately five-
fold lower than that of the scAAV fraction, is consistent
with the 10–20% contamination of the monomer fractions
with dimer containing particles (Figure 2a). The rAAV-
Figure 3 Transduction efficiency of scAAV versus conventional rAAV GFPneo vector transduction was inhibited greater than
vectors in the absence and presence of co-infecting adenovirus. The
efficiencies of the three CsCl fractionated vectors (Figure 1) were compared
10-fold under the same conditions. Similar results were
in rapidly dividing HeLa cells infected with scAAV-GFP fraction 11 (0.5 obtained by treatment with aphidicolin, a polymerase
particles per cell), monomer CMV-GFP fraction 13 (0.5 particles per cell), ␣/␦ specific inhibitor (Figure 4b). This confirmed our
rAAV-GFPneo fraction 13 (2 particles per cell), or rAAV-LacZ fraction hypothesis that scAAV transduction was independent of
12 (0.5 particles per cell). Transduction was quantified at 24 h after infec- host-cell DNA synthesis.
tion by counting GFP-positive cells using fluorescence microscopy, or by
fixing the cells and X-gal staining. The transducing efficiency was graphed
as the number of physical particles per transducing unit, as determined
Transduction by scAAV in vivo
by the number of cells scoring positive for GFP or LacZ expression. Dark A different reporter was used for the comparison of
grey bars indicate transducing efficiency in the presence of Ad co-infection scAAV and conventional single-stranded rAAV
at 5 p.f.u. per cell. efficiency in vivo. The dimer-producing construct con-
Gene Therapy
rAAV double-stranded vector
DM McCarty et al
1251
Gene Therapy
rAAV double-stranded vector
DM McCarty et al
1252
ome is sufficiently small, the dimeric inverted repeats or effectual using scAAV in preliminary studies. Regard-
themselves can be encapsidated as a self-complementary less of the ability of the target cell to make the rAAV
DNA molecule (scAAV). complementary strand, it is clear that these reagents pro-
In cultured HeLa cells, the scAAV vector was greater vide an alternative AAV delivery system for genes that
than four-fold more efficient than the homologous vector may require rapid onset. More importantly, our data sug-
containing only a monomeric ss-DNA genome. We gest that scAAV vectors achieve overall higher levels of
expect that this difference would be greater if not for the therapeutic product when identical number of particles
approximately 10–20% contamination of monomer frac- are administered. Thus, scAAV vectors will prove most
tions with dimer vectors. Consistent with this interpret- useful where a more timely, robust, or quantitative
ation, the scAAV was 20-fold more efficient than a con- response to vector dose is required, ie in endostatin deliv-
ventional rAAV-GFPneo vector and 140-fold more ery for anti-cancer therapy. This potential for attaining
efficient than a rAAV-LacZ vector, although we cannot critical levels of transgene expression at a minimal dose
rule out the possibility of additional inhibition of the lat- is important considering the level of vector production
ter vectors due to specific cis or trans effects of the differ- required for clinical trials.
ent marker genes. Apart from the potential utility of the scAAV as a vec-
We have further demonstrated the increased efficiency tor, we can also regard these new reagents as a biochemi-
of the scAAV in vivo by transducing mouse hepatocytes. cal intermediate in the rAAV transduction pathway. This
While the CMV promoter is not known to be particularly allows the dissection of events taking place before and
active in liver tissue,28 we chose to target liver in this after DNA synthesis, such as the CMV promoter acti-
experiment because it is feasible to achieve a relatively vation that we observed in the presence of Ad co-infec-
homogeneous distribution of vector particles over a large tion. This kind of effect would otherwise have been
tissue. Infecting mice with scAAVmEpo leads to a faster masked by the enhancement of transduction due to the
response, and a greater rise in hematocrit, than the full- promotion of complementary-strand synthesis. We antici-
length ssDNA vector carrying the same gene. This sup- pate that use of these vectors in specific cell types will
ports our observations in cultured cells and is consistent help determine the impact of promoter choice indepen-
with the view that the dimeric vectors are ready to dently of the efficiency of vector complementary-strand
express the transgene immediately upon uncoating and recruitment. Finally, the scAAV vectors are ideally suited
entry into the nucleus. The higher levels of expression to the transfer of small transgenes, which might include
ultimately achieved might reflect either the inability of blood clotting factor IX and X, erythropoietin, superoxide
many infected cells to form dsDNA from conventional dismutase, lysosomal enzymes, and most antisense RNA
rAAV, or the loss or degradation of ss vDNA before the or ribozyme strategies. For therapies demanding larger
formation of duplex.19 genes, the scAAV may have an important role in pilot
Our indirect assessment of transduction in vivo, studies for target cell permissiveness for AAV vectors.
through increase in hematocrit, provides only a limited Considering that cell cycling per se has not been an accur-
range of response to increasing levels of erythropoitin. It ate predictor of rAAV permissivity,18 the direct compari-
therefore remains possible that the differences that we son of transduction with scAAV versus the homologous
observed between the two vectors represents a minimum monomer may be the best predictor of target cell
difference in the number of transduced hepatocytes. If potential for rAAV DNA synthesis.
indeed it is only complementary-strand synthesis that is
limiting rAAV in these cells, we would expect to achieve
near 100% transduction, based on previous observations Materials and methods
of rAAV delivery to hepatocytes at similar doses.18
Future experiments using heterologous marker genes in Plasmids
liver and other potential target tissues in vivo will provide The rAAV plasmids expressing green fluorescent protein
a more accurate assessment of the potential of the scAAV (GFP) were constructed from the previously described
vectors in cells which appear to be refractory to rAAV pTRBSUF-2 (a gift from Nick Muzyczka, University of Flo-
transduction. rida, Gainesville, FL USA). First, the humanized GFP
As we have demonstrated by pre-treatment of cells coding sequence was replaced with the enhanced GFP
with HU, transduction with the scAAV vector is inde- (eGFP) (Clonetech, Palo Alto, CA, USA) to create the
pendent of host-cell DNA synthesis. The ability to trans- plasmid, pTR-CMV-GFPneo. This plasmid generated the
duce cells in the absence of DNA synthesis represents a rAAV-GFPneo vector. Second, the SalI fragment contain-
fundamental departure in the biology of the scAAV vec- ing the neo coding region and SV40 promoter was deleted
tors from the parent virus, allowing them to function to create pTR-CMV-GFP. The vector from this plasmid
under circumstances where conventional rAAV vectors was referred to as rAAV-GFP in this report.
would normally fail. Certain cell types are extremely inef- The plasmid, p43mEpo, a gift from Barry Byrne
ficient for rAAV transduction, ostensibly due to the (University of Florida, Gainesville, FL, USA) contained
inability to synthesize or recruit a complementary the mouse erythropoietin gene under the control of the
strand.13,14,19 The scAAV will suffer no such limitation CMV promoter and generated a rAAV replicon
and can be used with marker genes to directly determine (rAAVmEpo) of less than half the wtAAV length. A
whether a cell is permissive for rAAV transduction in all longer version of this construct (pmEpo-) was made by
other steps irrespective of DNA synthesis. In light of the inserting the 2.3 kb HindIII fragment from phage into
drastic treatments which have been proposed to promote a ClaI site between the polyadenylation signal and the
rAAV transduction efficiency in vivo (ie gamma radiation, downstream AAV terminal repeat. The rAAV-LacZ vec-
HU, topoisomerase inhibitors)14,29 it will be prudent to tor was generated from pDX11-LacZ, which has been
evaluate whether such lengths would be either necessary described elsewhere.30
Gene Therapy
rAAV double-stranded vector
DM McCarty et al
1253
Viral vectors 4 Samulski RJ, Chang L-S, Shenk T. A recombinant plasmid from
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at 4°C in cesium chloride. VII. Helper requirement for viral deoxyribonucleic acid and
Virion DNA (vDNA) was extracted from 10 l of each ribonucleic acid synthesis. J Virol 1972; 10: 1–8.
fraction by digestion in 50 l reactions containing 11 Alexander IE, Russell DW, Miller AD. DNA-damaging agents
0.4 mg/ml protease K, 1% sarkosyl and 10 mm EDTA at greatly increase the transduction of nondividing cells by adeno-
50°C for 1 h, followed by phenol/choroform extraction. associated virus vectors. J Virol 1994; 68: 8282–8287.
The samples were diluted three-fold with water and pre- 12 Ferrari FK, Samulski T, Shenk T, Samulski RJ. Second-strand
cipitated with ethanol for analysis by alkaline agarose gel synthesis is a rate limiting step for efficient transduction by
electrophoresis and Southern blot hybridization. recombinant adeno-associated virus vectors. J Virol 1996; 70:
27–34.
Cells and infections 13 Fisher KJ et al. Transduction with recombinant adeno-associated
HeLa and HEK 293 cells were grown in DMEM media virus for gene therapy is limited by leading-strand synthesis. J
Virol 1996; 70: 520–532.
containing 10% FBS and penicillin/streptomycin. Viral
14 Alexander IE, Russell DW, Spence AM, Miller AD. Effects of
vector stocks were diluted in media before adding to sub- gamma irradiation on the transduction of dividing and nondi-
confluent cultures and left on the cells until GFP trans- viding cells in brain and muscle of rats by adeno-associated
duction was observed by fluorescence microscopy at 24 h virus vectors. Hum Gene Ther 1996; 7: 841–850.
after infection. 15 Halbert CL et al. Transduction by adeno-associated virus vectors
For expression of erythropoietin from rAAV in mouse in the rabbit airway: efficiency, persistence, and readminis-
livers, 200 l normal saline, containing 2 × 1010 physical tration. J Virol 1997; 71: 5932–5941.
particles of either conventional rAAV or scAAV, was 16 Nakai H, Storm TA, Kay MA. Recruitment of single-stranded
injected directly into the portal veins of 10-week-old recombinant adeno-associated virus vector genomes and inter-
Balb-c ByJ mice (Jackson Laboratory, Bar Harbor, ME, molecular recombination are responsible for stable transduction
USA). Blood samples were collected by retro-orbital of liver in vivo. J Virol 2000; 74: 9451–9463.
phlebotomy at the time of infection and at 7-day intervals 17 Snyder RO et al. Persistent and therapeutic concentrations of
for determination of hematocrit. human factor IX in mice after hepatic gene transfer of recombi-
nant AAV vectors. Nat Genet 1997; 16: 270–276.
18 Miao CH et al. Nonrandom transduction of recombinant adeno-
Acknowledgements associated virus vectors in mouse hepatocytes in vivo: cell cyc-
ling does not influence hepatocyte transduction. J Virol 2000; 74:
We acknowledge the technical assistance of Jennifer Nas- 3793–3803.
pinski with scAAV studies in vivo, and Xiaohuai Zhou 19 Miao CH et al. The kinetics of rAAV integration in the liver. Nat
from the UNC Vector Core for construction of Genet 1998; 19: 13–15.
rAAV/epo/lambda vector. This work was supported in 20 de la Maza LM, Carter BJ. Molecular structure of adeno-associa-
part by NIH grants HL 48347, 51818. Doug McCarty was ted virus variant DNA. J Biol Chem 1980; 255: 3194–3203.
supported in part by NIH grant HL 51818 and PEM 21 Muzyczka N. Use of adeno-associated virus as a general trans-
receives research support from the National Hemophilia duction vector for mammalian cells. Curr Top Microbiol Immunol
Foundation and from NIH HL 03960. 1992; 158: 97–129.
22 Dong JY, Fan PD, Frizzell RA. Quantitative analysis of the pack-
aging capacity of recombinant adeno-associated virus. Hum
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Gene Therapy