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 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 510 Neonatal Gene Transfer with Non-Human 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 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 511 Effect of Hepatocyte Division on Molecular Fate of rAAV DNA Young-Kook Choi,1 Yuanqing Lu,1 Sihong Song.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 S198 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 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 partialhepatectomized liver) not only reduced episomal forms of rAAV DNA but also enhanced rAAV DNA integration 512 The Cellular TATA-Binding Protein Is Required for Replication of a Minimal AdenoAssociated Virus Type (AAV-2) p5 Promoter Element Achille Franỗois,1 Mickael Guilbaud,1,2 Rafi Awedikian,1 Gilliane Chadeuf,1 Philippe Moullier,1,2 Anna Salvetti.1 Laboratoire de Thérapie Génique, INSERM U649, Nantes, France; 2Etablissement Franỗais 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 Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy 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 513 Novel Characteristics of Heparin Binding by Adeno-Associated Virus Serotype James M Allen,1,2 Eric E Finn,1 Jeffrey S Chamberlain.1,2 Department of Neurology, University of Washington, Seattle, WA; 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, and (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 7:419425) The AAV2 capsid amino acids R585, R588 and A590 may be sufficient to enable heparin binding when substituted into AAV 1, and but cannot explain the heparin binding characteristics of AAV serotypes and 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, and yet AAV1 does not bind heparin The salt elution profile of heparin column bound AAV2 or AAV6 pseudotyped vectors revealed that AAV6 capsids elute at ∼300 mM NaCl while AAV2 capsid elutes at ∼500 mM NaCl (Halbert et 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 not bind heparin, differ from the AAV6 sequence by only 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 sequence to determine whether they can contribute to heparin binding 514 Effect of DNA-PKcs on AAV Replication Young-Kook Choi,1 Irene Zolotukhin,2 Barry J Byrne,2 Sihong Song.1,2 Departments of Pharmaceutics, University of Florida, Gainesville, FL; 2Department 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 Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy 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 cotransfected 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 515 Overlapping Adeno-Associated Viral (AAV) 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 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 AAV-6 mediates much higher gene expression than AAV2 in muscle To test whether AAV-6 can improve overlapping vectormediated gene transfer in muscle, we delivered a total of x 10e9 viral genome particles of LacZ overlapping vectors (AAV-2 or AAV6; x 10e8 particles of each vector including the upstream and the downstream vectors) to the anterior tibialis (TA) muscle of 6-weekold BL10 mice As a control, we also delivered x 10e8 particles of the intact AAV LacZ vector to the contra-lateral TA muscle Transduction efficiency was quantified at 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 S199 ... while AAV2 capsid elutes at ∼500 mM NaCl (Halbert et al 20 01, 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... shared by AAV serotypes 1, 2, and yet AAV1 does not bind heparin The salt elution profile of heparin column bound AAV2 or AAV6 pseudotyped vectors revealed that AAV6 capsids elute at ∼300 mM NaCl... 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 515 Overlapping Adeno- Associated Viral (AAV)