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

Abstract

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|[reg]| 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|[reg]| 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|[reg]| receptor and by cloning the inducible transgene and the RheoSwitch|[reg]| receptor-expressing cassettes in different orientations. We then used a highly active, modified CMV promoter for the expression of RheoSwitch|[reg]| 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.