Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy S198 AAV VECTORS: VECTOR BIOLOGY and histochemistry.. The effect of 17β-estradiol on
Trang 1Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
S198
AAV VECTORS: VECTOR BIOLOGY
and histochemistry The effect of 17β-estradiol on cell growth, HSPG
and integrins expression, rAAV gene transduction efficiency were
determined The levels of HSPG, integrins ανβ3 and ανβ5 showed
great variation between cell lines Whereas the expression of HSPG
appeared to be essential for and positively correlated with rAAV
transduction efficiency, the integrins ανβ3 and ανβ5 were not
absolutely necessary for rAAV transduction even though their
presence may facilitate transduction 17beta estradiol influences on
some ovarian carcinoma cell line’s proliferation and significantly
increase integrin beta 5 expression on all the three tested ovarian
carcinoma cell lines Integrin-retargeted RGD-modified rAAV showed
much higher gene transfer on ovarian carcinoma cell lines than rAAV
with/without estradiol, which implies RGD-AAV2 may be an
prospective alternative gene delivery vector for ovarian cancer
therapy in vivo.
Primate AAV Vector Serotypes Results in
Widespread Transduction and Gene Activity in the
Mouse Brain
Brian A Karolewski,1 Hennessy Howell,1 John H Wolfe.1
1 Pathobiology, University of Pennsylvania and The Children’s
Hospital of Philadelphia, Philadelphia, PA.
Mucopolysaccharidosis (MPS) VII is a heritable lysosomal
storage disease caused by the deficiency of Beta-glucuronidase
(GUSB) MPS VII is a chronic and progressive multiorgan disorder
with signs of pathology presenting early in life Most therapeutic
approaches for MPS VII have focused on adult treatments Although
these approaches can reverse storage lesions, the central nervous
system (CNS) is more difficult to treat Our lab has previously
shown complementary patterns of transduction between AAV1 and
after neonatal injection into the lateral cerebral ventricles (Passini et
al., 2003; Journal of Virology) Using an AAV2 ITR backbone, we
investigated neonatal gene transfer to the CNS of normal and MPS
VII mice with capsid proteins from AAV7, AAV8, and AAV9 We
injected 1.8 X 10^10 genomes of vector into the lateral cerebral
ventricles (2 µl each) of normal and MPS VII mice at birth (P O.5)
At one month postinjection, each of the AAV serotypes
demonstrated extensive and widespread GUSB activity and mRNA
expression in the brain, but variations in transduction patterns related
to substructures of the brain were observed AAV 2/7 and AAV 2/8
transduced more cells and expressed more GUSB compared to AAV
2/9 Significant amounts of GUSB were present in the ganglion
cells of the retina and spinal cord for each of the tested AAV serotypes
Evaluations of AAV gene transduction patterns and lysosomal
storage correction for the eye, brain and spinal cord are ongoing
These results suggest that AAV7, AAV8, and AAV9 are suitable
vectors for targeting the CNS, and may be useful for CNS gene
therapy
This research was supported by NIH grants T32-RR-07063,
RO1-DK-46637, RO1-NS-38690
Fate of rAAV DNA
Young-Kook Choi,1 Yuanqing Lu,1 Sihong Song.1
1 Department of Pharmaceutics, University of Florida, Powell
Gene Therapy Center, Gainesville, FL.
Previously, we showed that 40-50 % vector DNA and transgene
expression remained in the SCID (DNA-PKcs deficient) mouse liver
after a partial hepatectomy, while less than 10% vector DNA and
transgene expression remained in C57BL/6 (B6) mouse liver These
results demonstrate that DNA-PK inhibits rAAV DNA integration
However, it is not clear whether the integration occurred before or
after partial hepatectomy and whether cell division affected rAAV
integration In the present study, we sought to test the hypothesis that cell division may enhance rAAV DNA integration Immediately after a partial hepatectomy, B6 (n=5) or SCID mice (n=3) were intraportally injected with a rAAV vector (rAAV2-CB-AAT, 3x109
i.u./mouse) As a control, the same vector (3x109 i.u/moue) was also intraportally injected into B6 (n=3) or SCID (n=3) liver without a partial hepatectomy Transgene expressions in all four groups were evaluated weekly by monitoring serum levels of hAAT Serum hAAT levels in both hepatectomized SCID and B6 mice were sustained 3 weeks after injection Six to eight weeks after injection, hAAT levels from hepatectomized SCID liver (dividing model) were nearly 100%
of that from intact SCID liver (non dividing model), while hAAT levels from hepatectomized B6 liver (dividing model) were 25-30%
of that from intact B6 liver (non-dividing model) Southern blot analyses showed that total copies of the vector DNA in dividing and non-dividing SCID models were comparable, while the episomal forms of the vector DNA were significantly lower in the dividing SCID model than in the non-dividing SCID model These results strongly suggested that hepatocyte division (in partial-hepatectomized liver) not only reduced episomal forms of rAAV DNA but also enhanced rAAV DNA integration
Required for Replication of a Minimal Adeno-Associated Virus Type 2 (AAV-2) p5 Promoter Element
Achille François,1 Mickael Guilbaud,1,2 Rafi Awedikian,1 Gilliane Chadeuf,1 Philippe Moullier,1,2 Anna Salvetti.1
1 Laboratoire de Thérapie Génique, INSERM U649, Nantes, France; 2 Etablissement Français du Sang des Pays de la Loire, Nantes, France.
It was previously reported that the p5 promoter region (nt 191 to
350 of wt AAV-2) of the AAV-2 rep gene contained a cis-acting element that behaved as a Rep-dependent origin of replication in the absence of ITR (Nony et al, J Virol 2001; Musatov et al., J Virol 2002) This region was also shown to promote AAV-2 site-specific integration into human chromosome 19 (Philpott et al., PNAS 2002) The present study was conducted to identify the minimal elements
in the p5 region necessary for the Rep-dependent replication The p5 element was analyzed using in vitro nicking assays with purified Rep68 protein to precisely map the terminal resolution site(s) (trs) Deleted versions of the p5 element were then generated and tested for in vitro and in vivo replication in the presence of Rep proteins and adenoviral helper factors These analyses indicated that the minimal p5 element able to replicate into adenovirus-infected cells was constituted by a 55 bp region (D10, nt 250 to 304 of wt AAV-2) The D10 element contained the TATA box, the Rep Binding Site (RBS) and, downstream, the major trs located on the lower strand at the +1 transcription initiation site Surprisingly, deletion
of the TATA box abrogated in vivo replication of the D10 element Our data also indicated that the cellular TATA-binding protein (TBP), that was previously reported to interact with the Rep78 (Hermonat et al., Virology 1998), enhanced Rep binding to the p5 RBS and nicking at the trs site Accordingly, over-expression of the TBP enhanced p5-dependent replication
In conclusion, these studies identified a minimal p5 origin of replication (D10) that shares some homologies with both the minimal ITR and the AAVS1 elements, as they all contain a RBS and a trs However, in contrast to these well-known Rep-dependent origins
of replication, the D10 element requires the TATA box and TBP for efficient in vivo replication
Current studies are focused on the effect of TBP on wt AAV replication and on p5-directed site-specific integration The understanding of the role of this additional cis-acting replication
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AAV VECTORS: VECTOR BIOLOGY
element in the AAV-2 life cycle may have important implications for
the generation of novel rAAV vectors with improved biological
properties
by Adeno-Associated Virus Serotype 6
James M Allen,1,2 Eric E Finn,1 Jeffrey S Chamberlain.1,2
1 Department of Neurology, University of Washington, Seattle, WA;
2 Paul D Wellstone Muscular Dystrophy Cooperative Research
Center, University of Washington, Seattle, WA.
Many different adeno-associated virus (AAV) serotypes are
currently being investigated as potential vectors for gene therapy
applications, with AAV2 being the best characterized One aspect
of AAV2 biology of particular interest is its ability to bind heparin
sulfate proteoglycans, which enables efficient purification via affinity
chromatography The amino acids responsible for heparin binding
have been identified (R585, R588 and A590) and used to confer
heparin binding to AAV serotypes 1, 4 and 5 (Kern et al 2003, J
Virol 77:11072-81 and ASGT abstract 91, 2004) In addition, chimeric
AAV1/AAV2 encapsidated vectors generated in co-transfection
experiments also bound heparin (Hauck et al 2003, Mol Ther
7:419-425)
The AAV2 capsid amino acids R585, R588 and A590 may be
sufficient to enable heparin binding when substituted into AAV 1, 4
and 5 but cannot explain the heparin binding characteristics of AAV
serotypes 3 and 6 Indeed, these heparin binding serotypes share
the identical amino acids as AAV1 at these positions (S585, T588
and P590) The homologous motif R484, R487 and K532 are also
shared by AAV serotypes 1, 2, 3 and 6 yet AAV1 does not bind
heparin
The salt elution profile of heparin column bound AAV2 or AAV6
al 2001, J Virol 75:6615-24) In this study, pair-wise combinations
of the AAV1, AAV2 and AAV6 capsids were used to generate chimeric
AAV vector preparations In agreement with previously published
results, AAV1/2 chimeric vector preparations behave similarly to
AAV2 with regard to heparin binding, eluting at 400 to 500 mM
NaCl Curiously, AAV 1/6 chimeric capsids lost the ability to bind
to heparin columns while AAV2/6 capsids bound heparin columns
but required higher concentrations of NaCl (>700 mM) for elution
We hypothesize that the AAV6 heparin binding activity is distinct
from that described for AAV2 AAV1 capsids, which do not bind
heparin, differ from the AAV6 sequence by only 6 amino acids, none
of which were previously identified as being important for heparin
binding We are substituting each of these amino acids in the AAV 1
sequence to determine whether they can contribute to heparin
binding
Young-Kook Choi,1 Irene Zolotukhin,2 Barry J Byrne,2 Sihong
Song.1,2
1 Departments of Pharmaceutics, University of Florida,
Gainesville, FL; 2 Department of Pediatrics, University of Florida,
Powell Gene Therapy Center, Gainesville, FL.
DNA dependent protein kinase (DNA-PK) is a DNA repair
enzyme with multiple functions It has been shown that it plays an
important role in determining the molecular fate of the rAAV genome
in mouse muscle and liver DNA-PKcs inhibits AAV integration in
vitro and in vivo In the present study, we sought to determine the
effect of DNA-PKcs on recombinant adeno-associated virus (rAAV)
replication We co-infected 293 cells with rAAV vector (UF5) and
recombinant herpes simplex virus (rHSV) helper virus The replicated
forms were detected by southern blot analysis When cells were
treated with a DNA-PKcs inhibitor, wortmannin (WM), rAAV replication is significantly reduced in a dose-dependent manner Similar results were obtained from MO59K (DNA-PKcs positive) cells In order to confirm this observation, we have employed small interference RNA (siRNA) to target DNA-PKcs Transfection of this synthetic siRNA resulted in 70% to 90% reduction of the mRNA and the protein levels of DNA-PKcs Remarkably, this treatment significantly decreased rAAV replication In order to avoid the possible side effects by viral infection on DNA-PK activity, we co-transfected the siRNA treated 293 cells with vector (pUF5) and helper (pDG) plasmids Results from these experiments again showed that targeting of DNA-PKcs decreased rAAV replication by at least two folds compared to the controls Our results demonstrated that inhibition of DNA-PKcs by a DNA-PK inhibitor or siRNA decreased rAAV replication suggesting the important role of this cellular enzyme in AAV replication
Vector Mediated Gene Transfer Is Dependent on Viral Serotype and the Transgene Sequence in Skeletal Muscle
Arkasubhra Ghosh,1 Yongping Yue,1 Dongsheng Duan.1
1 Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO.
The small packaging capacity is one of the major hurdles for adeno-associated virus (AAV)-mediated gene therapy The overlapping approach has been developed recently to expand the AAV packaging capacity (Duan et al, Mol Ther 4:383, 2001; Halbert
et al, Nat Biotechnol.20:697, 2002) In this approach, a large gene is split into two partially overlapping fragments and separately packaged into two AAV vectors, including the upstream (carrying the 5’-end of the gene) and the downstream (carrying the 3’-end of the gene) vectors Transgene expression is achieved after homologous recombination of the overlapping region between the upstream and the downstream vectors in co-infected cells Despite the promising proof-of-principle results in the lung with AAV-6 alkaline phosphatase (AP) overlapping vectors, the transduction efficiency
in skeletal muscle has been very disappointing With AAV-2 LacZ overlapping vectors, the efficiency is only 0.37% of that from an intact AAV-2 LacZ vector This level of expression is far from sufficient to treat muscular dystrophy In this study, we examined two potential rate-limiting factors in the overlapping approach, including AAV serotype and the transgene sequence Previous studies suggest that AAV transduction in muscle is influenced by viral serotype 6 mediates much higher gene expression than
AAV-2 in muscle To test whether AAV-6 can improve overlapping vector-mediated gene transfer in muscle, we delivered a total of 1 x 10e9 viral genome particles of LacZ overlapping vectors (2 or AAV-6; 5 x 10e8 particles of each vector including the upstream and the downstream vectors) to the anterior tibialis (TA) muscle of 6-week-old BL10 mice As a control, we also delivered 5 x 10e8 particles of the intact AAV LacZ vector to the contra-lateral TA muscle Transduction efficiency was quantified at 6 weeks later by scoring the percentage of LacZ positive myofibers Consistent with previous reports, less than 0.03% of myofibers were transduced by AAV-2 overlapping vectors However, AAV-6 overlapping vectors yielded 35.2 ± 5.7% transduction This equals 42.25% of that of the transduction efficiency from an intact AAV-6 LacZ vector To determine whether the transgene sequence effects the transduction efficiency, we compared AAV-6 LacZ and AAV-6 AP overlapping vectors Surprisingly, the transduction efficiency of AP overlapping vectors (80.4 ± 2.8%) reached that of the intact AP vector (84.1 ± 1.9%) In summary, our findings suggest that AAV-6 overlapping vectors represent a promising approach to deliver certain larger therapeutic genes for muscular dystrophy gene therapy