501. RheoSwitch® Therapeutic System-Inducible Recombinant AAV Vectors for Tightly Regulated Transgene Expression

AAV VECTORS 501. RheoSwitch® Therapeutic SystemInducible Recombinant AAV Vectors for Tightly Regulated Transgene Expression Dawn Karzenowski,1 Zhijian J. Chen,1 David W. Potter,1 Malla Padidam.1 1 Stem Cell Therapeutics, RheoGene, Inc., Norristown, PA. Recent advances in recombinant AAV (rAAV) vector technology have made rAAV a vector of choice for many gene therapy applications. rAAV vectors for delivery of the RheoSwitch® Therapeutic System (RTS) are ideal systems for regulatory control of long-term therapeutic gene expression in vivo. RTS inducible gene expression technology allows the precise timing and dose of the therapeutic protein to be controlled through the systemic administration of a highly specific, pharmacologically inert Activator Drug. The controlled expression of the gene of interest is activated by the RheoSwitch® receptor (a heterodimer of an engineered ecdysone receptor ligand binding domain fused to a GAL4 DNA binding domain (GAL4:EcR) and an RXR protein fused to a VP16 activation domain (VP16:RXR)) only in the presence of Activator Drug. In the present study, we have optimized rAAV-based vectors by using different promoters for the expression of RheoSwitch® receptor and by cloning the inducible transgene and the RheoSwitch® receptor-expressing cassettes in different orientations. We then used a highly active, modified CMV promoter for the expression of RheoSwitch® receptor and cloned the receptor cassette and inducible Gaussia luciferase (GLuc) or GFP cassette in a single rAAV. To evaluate the regulation of GLuc or GFP transgene expression, HEK293 and mouse neural stem cells (NSCs) were transduced with packaged virus. Addition of Activator Drug to transduced HEK293 and NSCs in culture induced transgene expression in a dose dependent manner. For in vivo evaluation, the packaged virus was injected into the quadriceps of mice and subsequently induced with Activator Drug. Both the inducible GLuc and GFP vectors demonstrated little or no basal activity and inducible expression. To reduce the size of the construct further, we tested a format in which a p53 activation domain was fused to the GAL4:EcR domain. This gene was cloned with an inducible secretable alkaline phosphatase (SEAP) in a single rAAV vector. Activator Drug-dependent induction and low basal activity were also observed for this format in vitro and in vivo. Regulatable rAAV vectors based on these designs are ideal for therapeutic gene delivery.

502. Advances in the Application of Combinatorial Technology for the Optimization of Adeno-Associated Virus (AAV) Vectors Luca Perabo,1 Jan Endell,2 Stephan Maersch,1 Michael Hallek,1 Hildegard Buening.1 1 Clinic I - Innere Medizin, University of Cologne, Cologne, Germany; 2Gene Center, University of Munich, Munich, Germany. In vitro evolution protocols have been successfully applied to screen combinatorial AAV libraries in order to obtain mutants that escape neutralization by human sera (Perabo et al. JGM 2006; Maheshri et al. Nat Biotechnol 2006). We have further optimized panning protocols by monitoring selection rounds with Real Time PCR to follow the evolution of the viral pools after each selection round. Application of this technology to new screening experiments allowed in shorter times and at reduced costs the identification of novel capsid variants with higher transduction efficiency and improved ability to circumvent antibody neutralization at the same time. Packaging ability was unaffected by the introduced mutations. Beside providing proof of principle for the advantages of this monitoring procedure, these new variants

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enrich the pool of alternative vectors that can be employed for the treatment of patients with preexisting immunity to AAV-2 and further extend our knowledge of capsid biology. Our results contribute to the development of combinatorial technology for the engineering of viral particles. This emerging approach is gathering interest as alternative to rational design, overcoming the limit posed by our incomplete understanding of the infectious process, and at the same time offering a powerful tool to dissect viral biology by reverse genetics (see also abstract by Goldnau et al).

503. A Novel Herpes Simplex Virus Helper Based Production of Adeno- Associated Virus Vectors for Treatment of Retinal Angiogenesis Guo-jie Ye,1 Chris Drogemuller,1 Darby Thomas,2 Marina Scotti,1 Wen Kang,1 David Knop,2 Sanford Boye,3 Peter Pechan,4 James Peterson,3 William Hauswirth,3 Abraham Scaria,4 Samuel Wadsworth.4 1 Research & Preclinical Development, AGTC, Alachua, FL; 2 Process Development, AGTC, Alachua, FL; 3Department of Opphthalmology, University of Florida, Gainesville, FL; 4Gene Therapy, Genzyme Corporation, Framingham, MA. AGTC’s proprietary rAAV vector production method uses recombinant herpes simplex virus (HSV) as the helper agent for rAAV production. This production method has been demonstrated to be both highly scalable and productive compared to other rAAV production methods such as the transient co-transfection or adenovirus co-infection process. rAAV production by rHSV-1 methodology requires two recombinant HSV-1 vectors, rHSV-1rep2cap2 which expresses the AAV 2 rep and cap genes, and a second rHSV-1 that contains the therapeutic gene of interest flanked by the AAV-2 inverted terminal repeats. Both rHSV vectors are deleted in an essential gene for HSV replication and hence they are propagated only in a complementing cell line. Here we report the construction and characterization of rHSV-TR-shFlt-1 that contains a novel soluble hybrid version of Flt-1, the receptor for vascular endothelial growth factor (VEGF). This novel shFlt-1 is a high-affinity binder of VEGF, a central player in pathologic neovascularization in both wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). To construct rHSV-TR-shFlt-1, a shuttle plasmid containing the expression cassette of sFlt-1 flanked with homologous HSV-1 sequences was first constructed and co-transfected with rHSVd27.1TR-GFP viral DNA into complementing V27 cells. rHSV-TR-shFlt1 was screened against the parental rHSV-1-GFP viruses that express fluorescent GFP, and the homologous recombination was further confirmed by Southern blot analysis. Selected rHSV-TR-shFlt-1 clones were propagated through 10 passages and their ability to produce rAAV2-TR-shFlt-1 vector was examined by real-time PCR. The expression level, size, and in-vitro biological activity of sFlt-1 expressed in rAAV2-TR-shFlt-1 transduced cells were determined by ELISA, immunoblot, and the HUVECs proliferation assay respectively. Stability studies indicated that the rHSV-TR-shFlt-1 helpers maintained their ability to produce rAAV comparable to that of the positive control rHSV-1-GFP through 10 passages. Preliminary data from the oxygen-induced retinopathy (OIR) mouse model suggests that rAAV2-shFlt-1 derived from rHSV1TR-shFlt-1 is able to inhibit retinal neovascularization, and there is no significant difference compared to rAAV2-shFlt-1 vector made by the transfection method. To our knowledge this is the first report of the construction and characterization of a recombinant HSV helper virus to produce a rAAV vector encoding a therapeutic gene for inhibition of ocular angiogenesis. Further preclinical evaluation of the AAV2-shFlt-1 is in the process. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy