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BIOLOGY OF TRANSDUCTION the binding of cellular proteins to the double stranded D (dsD) sequence of the adeno-associated virus (AAV) inverted terminal repeat (ITRs) by electromobility shift assay We show here that transcription factors belonging to the RFX family, bind specifically and selectively to AAV2 and AAV1 dsD sequences By supershift experiments with antisera against RFX family members and using in vitro translated RFX proteins, we further show that the observed complexes, contain RFX1 homodimer and RFX1/RFX3 heterodimers RFX proteins, which interact with transcriptional enhancers and origins of replication of several mammalian DNA viruses including Hepatitis B, Polyoma and Cytomegalovirus, may therefore be involved in the replication cycle of AAV 105 Use of a Recombinant Adenovirus Expressing the AAV-2 Rep52 Helicase To Enhance Vector Potency from Stable rAAV Producer Cell Lines Chun-Liang Chen, Ruju Chen, Philip R Johnson, K Reed Clark Center for Gene Therapy, Columbus Children’s Research Institute, Columbus Children’s Hospital, Columbus, OH Background To augment yields from stable rAAV producer cell lines, we previously down-modulated Rep78/68 protein levels by replacement of the native p5 rep promoter with the tetracycline responsive element (TRE) Following wild-type Ad5 infection, rAAV/Β-gal producer cells containing this modification yielded 610 times more infectious rAAV than p5 containing lines The modified cell lines also demonstrated an increase in Rep52/40 expression levels Southern blot analysis revealed that purified vector particles derived from these Rep modified cell lines possessed a greater proportion of full-length vector genomes (4 to 8-fold) Moreover, a direct role for Rep52/40 function in this process was suggested by transient transfection experiments with a Rep52/40 expression plasmid, whereby enhanced vector potency was observed in the transfected cells To apply these observations towards a scalable production system, we report the isolation of a replication competent, adenovirus type vector that expresses the Rep52 protein The use of this virus in pilot production experiments is described below Methods The recombinant Ad5 vector (rAd/E3/ TRE-Rep52) was generated by cloning the AAV-2 Rep52/40 coding sequences downstream of the TRE and into the Ad5 E3 shuttle vector pAB26 Recombinant virus was generated following cotransfection of 293 cells with helper plasmid pFG173 and pAB26TRErep52/40 Various rAd and wild-type moi were used to infect an optimal rAAV-2/Β-gal producing cell line termed D6 D6 has a tripartite production plasmid stably integrated into the S1 locus and contains: (i) a rAAV/CMV/Β-gal vector, (ii) p5-rep-cap, and (iii) neomycin resistance gene Infectious rAAV-2/Β-gal yields were quantitated by visual counting of lacZ expressing target C12 cells Results Efficient Rep52 expression was documented by western blot analysis following infection of 293 or HeLa cells with rAd/ Rep52 in the absence of a tet transactivator protein To determine the utility for rAAV-2/Β-gal production, D6 producer cells were infected with rAd/Rep52 at various moi (1, 10, or 100) Compared to wild-type adenovirus infection, the recombinant virus yielded a 3-4 log reduction in infectious vector yield Under these infection conditions, Rep western blot analysis revealed elevated Rep52 levels that correlated with a dramatic reduction in Rep78/68 expression levels A series of temporal coinfection experiments were then performed by varying the order of wild-type and recombinant adenovirus infection Using an optimal coinfection scheme (initial wild-type Ad5 infection, followed 12 hr later by rAd/Rep52), infectious rAAV/Β-gal yields were augmented approximately 8-fold compared to wild-type Ad5 infection alone Moreover, under these permissive infection conditions, Rep78/68 proteins were moderately expressed Conclusions We report the generation of an E3 deleted S42 rAd vector expressing the AAV-2 Rep52 protein and define the optimal infection parameters for the synthesis of highly infectious virus from coinfected producer cell lines These data also suggest that overexpression of Rep52 helicase prior to Rep78/68 protein expression results in inhibition of subsequent Rep78/68 expression 106 Optimal Transductions of Mouse Embryonic Stem Cells with Recombinant AAV Serotypes (1-5) Fusheng Wei,1 Sihong Song,2 Pedro Cruz,3 Yuanqing Lu,2 Jingfang Wei,1 Ge Zhao,2 Terence R Flotte.1 Pediatrics, University of Florida College of Medicine, Gainesville, FL, United States; 2Molecular Genetics & Microbiology & Pharmaceutics, University of Florida College of Medicine, Gainesville, FL, United States; 3Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, United States Pluripotent embryonic stem (ES) cells are derived from normal preimplantation blastocyst embryos In vitro, they can be stably propagated in the undifferentiated state, and retain their ability to develop into all cell types in vivo Pluripotent mouse embryonic stem cells may provide an effective means to analyze gene expression and function during development, and may provide a cell platform for the purpose of gene therapy Adeno-associated virus (AAV) is a non-pathogenic human parvovirus that is capable of establishing persistent infections in a wide range of mammalian cell types Recombinant AAV (rAAV) vectors have been extensively employed to mediate efficient gene transfer in the a wide range of cell types in vivo and in vitro There are at least eight serotypes, AAV to Vectors derived from AAV serotype are currently being evaluated in gene transfer studies To compare how other rAAV serotypes (1,3,4 and 5) perform as gene transfer vectors in embryonic stem cells would be particularly useful given their pluripotent nature In the current study, ES cells were infected with rAAV serotypes to vectors designated to express human alpha-1 antitrypsin (AAT) at an M.O.I of 1,000 The levels of human AAT were measured by enzyme-linked immunoassay (ELISA) at 2,4 and days post-infection The results showed that ES cells transduced with rAAV2, expressed hAAT at levels approximately 9-fold higher than other serotypes tested In another experiment, ES cells were infected with rAAV serotype vector expressing green florescent protein (GFP) and the neo® gene at an M.O.I of 1,000 Cells were selected with G418 By 28 days, all G418-resistant ES cells transduced with the rAAV-GFP expressed GFP as judged by fluorescent microscopy Analysis of Southern blots of transduced clones demonstrated that stable vector integration had occurred To our knowledge, this is the first report of efficient transduction of mouse embryonic cells with rAAV Of particular interest is the possibility that sustained targeted expression may be used in embryonic stem cell based gene therapy 107 Isolation and Characterization of the Heparin Binding Domain of AAV Yu-Hung Kuo,1 Justin F Fraser,1 Michael G Kaplitt.1 Neurological Surgery, New York Hospital, Weill-Cornell Medical Center, New York, NY While the various serotypes of adeno-associated virus (AAV) are believed to have the same physical structure, differences have been seen in viral tropism One possible mechanism for tropism is through specificity of the virus for different receptors The observation that AAV2 and AAV3 are capable of binding to heparin, whereas AAV4 and AAV5 show affinity for sialic acid supports this hypothesis Furthermore, it is likely that receptor affinity is encoded within the primary sequence of the surface capsid proteins Isolation and Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy BIOLOGY OF TRANSDUCTION characterization of the surface epitopes involved in receptor association and selectivity would permit for design of novel AAV forms with restricted or expanded tissue tropism To isolate the heparin-binding site of AAV, we created chimeric AAV isoforms where capsid domains of the heparin binding AAV2 serotype were substituted for equivalent domains in the non-heparin binding serotypes AAV1 and AAV5 By using overlapping PCR to create these chimeric viruses, no exogenous amino acids were introduced Chimeric isoforms between AAV2 and AAV5 demonstrated that heparin binding was located within the VP3 portion of the capsid protein Replacement of the VP1 and VP2 sequence of AAV2 with the equivalent domain of AAV5 resulted in production of functional viral particles capable of infecting HEK293 cells in vitro This AAV5/2 chimera was able to be retained and purified on a heparin column In contrast, replacement of the VP1 and VP2 domain of AAV5 with the same domain from AAV2 produced infectious particles that were not retained by a heparin column.To further delineate the minimal heparin binding domain, chimeric viruses were made between AAV1 and AAV2, which show more homology than AAV2 and AAV5 Substitution of fragments of the C-terminal portion of AAV2 VP3 into AAV1 produced chimeric virus that could be retained and purified on a heparin column The chimera is infectious when tested on HEK293 cells in vitro The heparin binding domain of AAV2 is in the C-terminal portion of the VP3 sequence that is common to all subunits of the viral capsid It includes the clusters of divergence between the amino acid sequences of AAV1 and AAV2 Substitution of this domain of AAV2 with the equivalent peptides of AAV1 ablated heparin binding Further subdivision of this domain did not confer heparin binding, but appears to adversely affect virus function We are currently determining whether this domain is sufficient for conferring heparin binding to other serotypes of AAV which are less homologous than AAV1 and AAV2, as well as identifying the individual amino acids necessary for heparin binding Finally, the effects of heparin association on viral function in the brain in vivo are being delineated 108 A Tropism Modified AAV2-Based Vector System for Gene Therapy of Peritoneal Ovarian Carcinoma Wenfang Shi,2 Gregory Arnold,1 Jeffrey S Bartlett.1,2 Center for Gene Therapy, Children’s Research Institute, Columbus, OH; 2Department of Pediatrics, The Ohio State University, Columbus, OH Ovarian cancer is one of the more common malignancies affecting women in the United States Our goal has been to develop a genebased therapy for ovarian cancer using recombinant adeno-associated virus (AAV) as a vector However, the utility of AAV vectors for ovarian cancer gene therapy is limited by the distribution of heparan sulfate proteoglycan (HSPG), the virus’s primary attachment receptor, on different tumor cell populations In order to achieve HSPG-independent gene delivery, several groups have shown that the tropism of AAV can be expanded by genetically altering the viral capsid However, the parameters of this developing technology have yet to be defined and it has not yet been determined if these modified vectors actually infect cells via these engineered interactions We have analyzed tumor tissue from 40 patients with peritoneal ovarian carcinoma for expression of HSPG and other receptors We found that only about 63% of human tumors express the appropriate HSPG epitope required for AAV infection, whereas greater than 90% express either αvβ3 or αvβ5 integrins We also examined a panel of different ovarian cancer cell lines (PA-1, OV4, OVCAR3, Hey, OV3 and SKOV-3) and similarly found that only half of these cell lines expressed HSPG, yet all expressed either αvβ3 or αvβ5 integrins Not surprisingly, those cell lines that failed to express HSPG were not transduced with AAV2-based vectors Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy We have shown that incorporation of an Arg-Gly-Asp (RGD) – containing peptide at sites within the AAV capsid enables vectors to infect integrin-expressing cells independent of HSPG Mutant AAV vectors displaying this peptide ligand were produced to wildtype titer and shown to specifically target integrin receptors on ovarian cancer cells Furthermore, we show that these modified vectors actually mediate infection via this engineered interaction We report significant increases in gene transfer to previously nonpermissive ovarian cancer cell lines, suggesting that AAV vectors displaying RGD peptides may be of utility for treatment of neoplasms characterized by a deficiency of HSPG expression We next tested these vectors for efficient transduction of ovarian carcinoma in vivo Tumors were established by intraperitoneal injection of SKOV-3 cells in SCID mice Either RGD-AAV2 vector or unmodified AAV2 vector expressing GFP or LacZ marker genes was administered to the peritoneal cavity five days after tumors were established Animals were sacrificed at various times post treatment and gene delivery and expression were evaluated by immunostaining and enzymatic activity Both assays showed that marker gene transfer and expression was nearly two-orders of magnitude greater in RGD-AAV treated animals than in animals treated with unmodified AAV vector Importantly, the modified AAV vectors did not transduce normal mesothelial tissue or other internal organs RGD-modified AAV2 vectors encoding HSV-TK have been developed and tested in vitro and in vivo Incorporation of the RGD tumor-targeting motif in these vectors significantly increased anti-tumor toxicity in vitro and efficacy in vivo These results suggest that RGD-modified AAV vectors may offer significant therapeutic advantages for ovarian cancer gene therapy 109 Characterization of Adeno-Associated Virus Type Minimal Origin of Replication and Packaging Signal Sergei Musatov,1 Hajira Begum,2 Jill Roberts,2 Michael G Kaplitt.1,2 Laboratory of Neurobiology and Behavior, The Rockefeller Uniersity, New York, NY, United States; 2Laboratory of Molecular Neurosurgery, Weill Medical College of Cornell University, New York, NY, United States Adeno-associated virus (AAV) inverted terminal repeats (ITRs) are the only cis-elements required for virus propagation The 20-bp D-sequence of the ITR has been demonstrated to play a critical role in the efficient rescue, replication and encapsidation of the AAV genome While there is little homology between distal parts of the D-sequences of different AAV serotypes, all of them form a nearly perfect palindrome with the juxtaposed A-sequence This palindrome generates a hairpin with a terminal resolution site (trs) in a loop region, an event crucial for Rep-mediated endonuclease activity These observations lend support to the hypothesis that it is the secondary structure of the AD junction, rather than the primary sequence of the D-element, which is important for AAV propagation To address this question, we first generated a series of circular AAV (cAAV) vectors with altered AD junctions Plasmids harboring a single wt AD domain have been shown to replicate exclusively as circular molecules and encapsidate single-stranded progeny genomes Vectors containing a single ITR element (AD domain) with no hairpin can be useful for mutational analysis of the D-sequence, since the mutant genomes can be generated using a simple single-step PCRbased cloning All plasmids retain wt trs but differ in the flanking sequences Southern blot analysis of replication revealed that the plasmid with a GC-rich trs stem did not replicate efficiently, while replication of a construct with an AT-rich junction was readily detectable Most importantly, generation of a single mismatch in the synthetic AT-rich stem increased the efficacy of replication to virtually wild-type levels Furthermore, we observed a direct S43 ... sequences of AAV1 and AAV2 Substitution of this domain of AAV2 with the equivalent peptides of AAV1 ablated heparin binding Further subdivision of this domain did not confer heparin binding, but... isolate the heparin- binding site of AAV, we created chimeric AAV isoforms where capsid domains of the heparin binding AAV2 serotype were substituted for equivalent domains in the non -heparin binding. .. vitro The heparin binding domain of AAV2 is in the C-terminal portion of the VP3 sequence that is common to all subunits of the viral capsid It includes the clusters of divergence between the amino