Transposase Hybrid-Vector System for Somatic Integration

BIOLOGY OF AAV AND VECTOR DEVELOPMENT I formation of a new clade unique to AAV-po4. In vivo performance, biodistribution, seroprevalence and phylogenetic analysis will be presented for all newly isolated porcine AAVs. The use of novel AAVs derived from porcine tissues may contribute to the generation of new gene transfer vectors for gene therapy or vaccine applications acceptable for human use.

99. Structure To Function Analysis of Emerging Chimeric AAV Gene Therapy Vectors

Lauren M. Drouin,1 Wuping Li,2 R. Jude Samulski,2 Lin Yang,3 Xiao Xiao,3 Mavis Agbandje-McKenna.1 1 Biochemistry and Molecular Biology, University of Florida, Gainesville, FL; 2Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC; 3Molecular Pharmaceutics, University of North Carlonia at Chapel Hill, Chapel Hill, NC. In recent years, a plethora of chimeric Adeno-associated virus (AAV) vectors have emerged from either directed evolution, DNA shuffling, or rational mutagenesis of variable surface amino acids of the viral capsid protein. These viruses are remarkable in the sense that they have often become targeted to a specific tissue, and also detargeted from others due to changes in residues that likely play a role in receptor binding. In effect, these chimeric viruses combine all of the best features from their parental viruses to produce an end product that exhibits enhanced transduction, low immunogenicity and is optimized for tissue specificity. Using available crystal structures or homologous 3D models generated for the parental viruses, we are able to annotate the capsid features that confer these desirable phenotypes on emerging vectors towards functional characterization. We have compared the crystal structures for the AAV serotypes determined in our lab (AAV1, AAV4, AAV5, AAV7-AAV9) with that available for AAV2 to identify nine common variable regions on the AAV capsid. 3D models for several emerging chimeric viruses have been generated using parental structures as templates, followed by comparisons to identify novel structural features. Our efforts have consistently identified changes in the nine regions, previously defined as variable between the distinct AAV serotypes, as also being the most susceptible to the sequence “mixing” that is forced in the directed evolution or DNA shuffling approaches for generating tissue specific vectors. This observation is significant in that it is beginning to pinpoint the AAV capsid regions that dictate the capsid-associated phenotypes, such as the differential tissue transduction, reported for the AAV serotypes. This provides a structural platform for fine functional annotation. These studies will be important for the rational design of the next generation of tissue targeted AAV vectors with improved efficacy. Examples will be given of emerging chimeras that have been annotated thus far.

100. Identification of Rep-Associated Factors in HSV-1-Induced AAV-2 Replication Compartments

Armel Nicolas,1 Coline Biollay,2 Lauriane Kuhn,3 Myriam Ferro,3 Nathalie Alazard-Dany,1 Alberto L. Epstein,2 Anna Greco,2 Anna Salvetti.1 1 INSERM U758, Ecole Normale Supérieure de Lyon, Lyon, France; 2CNRS UMR5534, Université de Lyon, Lyon, France; 3 INSERM U880, Commissariat à l’Energie Atomique, Grenoble, France.

Recombinant vectors derived from the Adeno-Associated Virus (rAAV) are among the most promising tools for human gene therapy, yet many aspects of the vectors’ and the wild type virus’ biology remain obscure. In particular, methods for high scale production of vector particles still offer much room for improvement and would highly benefit from advances in our understanding of wild type AAV replication. Adenovirus and Herpes Simplex Virus type 1 (HSV-1) are the best known helper viruses for AAV, and while the helper S40

effect of the former has been well characterised, much remains to be discovered about the cellular and viral factors involved in HSV-1-induced AAV replication. To further explore this issue, we have undergone the purification and identification of factors that interact directly or indirectly with Rep proteins in HSV-1-induced AAV replication compartments. For this study we used a stable cell clone (HeLaAAVtCR) containing a latently integrated AAV genome composed of the AAV-2 ITRs flanking a rep gene fused at its 5’ terminus with two affinity tags and a fluorescent mCherry protein. The viral and cellular factors associated with Rep proteins and/or AAV DNA in infected HeLaAAVtCR cells were purified on two consecutive affinity columns and then identified by LC-MS/MS. This analysis led to the identification of 43 and 65 cellular factors that specifically copurified with Rep complexes in cells co-infected with either a wt or an UL30-deleted HSV-1 strain (HSV∆UL30), respectively, with more than half of them identified by at least two peptides. The differences between the proteins identified after infection with both helper viruses were relatively small. Most of these cellular proteins could be linked to a handful of cellular pathways, of which the foremost were DNA replication, repair, and RNA metabolism. A significant number of ribosomal and cytoskeleton proteins were also found which may reflect Rep proteins’ interactions with the cytoskeleton or the nucleolus’ constituents. In addition, some specific proteins normally localizing to mitochondria were also specifically found associated with Rep complexes. Finally, this analysis also resulted in the identification of 6 and 8 HSV-1 factors in wt HSV and HSV∆UL30 infected cells, respectively, among which the most abundant was ICP8, an essential protein for HSV DNA replication. Interestingly, both constituents of the HSV-1 polymerase complex, UL30 and UL42, were found to specifically co-purify with Rep in cells infected with wt HSV, thus confirming our previous study demonstrating the involvement of these viral factors in AAV DNA replication. Immunofluorescence and co-immuno-precipitation analyses are currently being carried out to validate the association of the most relevant factors with AAV replication centres and their direct interaction with Rep and/or AAV DNA. Further work will aim at assessing the functional role of validated factors in AAV replication.

101. Establishment of an AAV/Transposase Hybrid-Vector System for Somatic Integration

Nadine Muether,1 Nicola M. Wolf,1 Lajos Mates,2 Zsuzsanna Izsvak,2 Zoltan Ivics,2 Anja Ehrhardt.1 1 Department of Virology, Max von Pettenkofer-Institute, Munich, Germany; 2Max Delbruck Center for Molecular Medicine, Berlin, Germany. Recombinant adeno-associated viral (rAAV) vectors remain mostly episomal in transduced cells. However, in rapidly dividing cells, vector genomes and transgene expression are lost over time and therefore also the therapeutic effect. For treatment of genetic diseases originated in cells with a fast cell turnover, a rAAV vector capable of somatic integration in a safe location would be ideal. In this study we created a novel AAV/transposase hybrid-vector with improved genetic elements for stable integration. We explored hyperactive Sleeping Beauty transposase variants HSB5 (Yant et al., Nucleic Acids Research 2007) and the novel mutant SB100 (Mates et al., Nature Genetics, in press) with 10-fold and 100-fold increased activity, respectively. It was demonstrated that transposition only works sufficiently from circular substrates. Thus, in our initial two-vector-system, one rAAV vector delivers transposase and Flp recombinase for circularization and the second transposon donor vector contains the transgene flanked by the respective recombinase recognition sites. To test whether circular substrates are formed, we co-transduced a rAAV2/8 pseudotyped HSB5/Flp encoding vector and either a neomycin or a human coagulation factor IX encoding transposon donor vector into various cell lines. Using a PCR-based Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

BIOLOGY OF AAV AND VECTOR DEVELOPMENT I circularization assay we were able to demonstrate that circular dsDNA substrates from single-stranded input rAAV vector genomes are formed. After the novel hyperactive SB100 with 100-fold increased integration efficiencies became available, we inserted SB100 in our AAV-based two-viral vector system. Colony forming assays revealed that the SB100/Flp delivering vector along with the vector carrying a neomycin resistance cassette leads to 10-fold increased genomic integration in 293 cells compared to the HSB5/Flp vector. In order to avoid having to rely on co-transduction of two AAV-vectors into the same cell and to address the question whether Flp is required at all for the system to work efficiently, we generated a stably SB100 expressing cell line. This cell line was infected with the neomycin encoding donor transposon rAAV2/8 vector at MOI 100. Colony forming assays comparing integration efficiencies in SB100 cells and the respective control cell line without SB100 revealed 5-fold increased integration efficiencies. This demonstrated that transposase mediated integration from the rAAV vector works and that Flp mediated circularization may not be required. We are in process to determine sites of insertion after SB100 mediated integration from the rAAV vector into the host genome by using a modified BD GenomeWalker PCR-based strategy. In addition, we are intending to evaluate our system in vivo using rAAV2/8 pseudotyped vectors. In summary, we believe that our novel SB100-based integrating rAAV vector system harbours great potential for gene therapy by facilitating stable transgene expression in cycling cells. The significantly increased integration activity of SB100 may allow lowering the viral dose required for integration and stabilized transgene expression which in turn may reduce vector-related toxicity.

102. Surface-Tethered AAV Vectors for Enhanced Gene Transfer and Spatial Control of Gene Delivery

Adam S. Davis,1 Jeffrey S. Bartlett.1,2 1 Gene Therapy Center, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Pediatrics, The Ohio State University, Columbus, OH. Overcoming the biological barriers to effective gene transfer is a fundamental goal of biotechnology. Similarly, development of strategies that permit spatial control over gene transfer are recognized as vital for effective translation of these technologies. While, viral vectors have proven the most efficient at mediating gene transduction, most bioengineering efforts have focused of non-viral vector systems due to our ability to chemical and physically adapt and modify these systems toward specific functional aims. It has become widely accepted that adsorption of non-viral vectors to polymer substrates or scaffolds can greatly enhance gene transfer, likely by increasing vector concentrations in the cell microenvironment. DNA modified biomaterials have been widely investigated and are currently at the forefront of most regenerative medicine and tissue engineering paradigms. We propose a novel approach that combines these two strategies in which AAV vectors are tethered to a substrate that supports cell adhesion. The AAV vectors package the DNA for efficient internalization and gene transduction, while the surface tethering functions to define the pattern of gene transduction and maintain an elevated concentration of vector in the cell microenvironment for cells adhered to the substrate. Peptide-modified AAV1-based vectors were modified with biotin groups via metabolic biotinylation using E. coli biotin ligase (BirA). Biotinylation was confirmed by biochemical analysis and had no effect on the gene transfer properties of the modified AAV1 vector particles. Biotinylated AAV vectors encoding reporter genes were incubated on neutravidin (nonglycosylated avidin)-coated surfaces. Vector surface densities were a function of avidin-coating and were minimally effected by the number of biotin groups displayed per AAV vector particle. This allowed incorporation of multiple surface modifications into substrate-bound AAV vector Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

particles. Cells seeded onto the AAV vector-modified surfaces were transduced with efficiencies equal to or greater than those mediated by solution-phase vector particles. Surprisingly, vector particles remained bound to the substrate throughout the infection process. Effect of surface concentration and the number of tethers on gene transduction was determined for different cell types using mosaic AAV1-based vector particles with different amounts of displayed biotin. Transduced cells were observed only in regions in which vectors were tethered, suggesting that the location of transduced cells could be specifically controlled by either surface modification (avidin) or vector particle modification (biotinylation). Therefore patterned expression of multiple transgenes on appropriately modified surfaces can be attained. Surface tethering of AAV vector particles represents a promising approach to both enhance gene transduction of target cells and spatially control gene delivery, which may have applications to a multitude of fields ranging from gene therapy to tissue engineering to functional genomics.

103. A Simplified System for BaculovirusMediated Production of rAAV Particles in Insect Cells

Richard H. Smith, Justin R. Levy, Robert M. Kotin. Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Bethesda, MD. Baculovirus expression vector-mediated production of recombinant adeno-associated virus (rAAV) vectors in insect cells provides a readily scalable alternative to mammalian cell-based rAAV production strategies. The most widely used system of baculovirus/insect cellmediated rAAV production uses three separate baculovirus-AAV constructs to deliver the cis- and trans-acting components needed for rAAV vector production. One recombinant baculovirus encodes the AAV Rep78 and Rep52 proteins, a second recombinant baculovirus encodes the AAV capsid proteins (VP1, -2, and -3), and a third recombinant baculovirus harbors the gene-of-interest flanked by the AAV inverted terminal repeat elements. To increase the efficiency and robustness of the system, we have reduced the number of individual baculovirus constructs needed for rAAV production in insect cells by consolidating rep and cap gene expression to a single recombinant baculovirus. Using the consolidated rep- and capexpressing baculovirus reagent in combination with a recombinant baculovirus bearing an ITR-flanked GFP reporter gene, we obtained an average of 7.2 x 104 (SD = 2.5, n = 6) purified, nuclease-resistant rAAV particles per cell from small-scale Sf9 suspension cultures grown in shaker flasks.

104. Capsid Modification Can Increase AAVMediated Gene Transduction in a ReceptorDependent Manner without Increasing Cellular Attachment or Altering Intracellular Trafficking Pathways

Matthew D. Stachler,1 Jeffrey S. Bartlett.1,2 Gene Therapy Center, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Department of Pediatrics, The Ohio State University, Columbus, OH. 1

Adeno-associated virus (AAV) based vectors have become popular for a variety of gene therapy applications. While the use of different serotypes has expanded the number of cells and tissues amenable to AAV-mediated gene therapy, there are still several cell types that are refractive to AAV-mediated gene transfer. While AAV vectors have been engineered to bind to specific receptors present on these cell types and there have been several reports that this “targeting” approach has lead to increased transduction, little effort has been put forth to describe the actual mechanism of increased transduction. The assumption has been that increased transduction is due to either S41