1057. Successful Gene Therapy for Retinal Degenerations by Lentiviral Gene Transfer

Abstract

Our goal is to perform gene therapy by efficient integration into safe positions in the host genome. The integrase enzyme from the phage phiC31 directs site-specific integration into a small subset of native genomic sequences 1. Recent gene therapy studies using phiC31 integrase in mice have demonstrated site-specific integration of transgenes and long-term gene expression in liver, skin, and muscle (e.g., 2). Here, we demonstrate use of the phiC31 integrase as a simple and effective method for non-viral long-term gene transfer in the eye. We developed a method to achieve efficient non-viral delivery of plasmid DNA to rat retinal epithelium (RPE) in vivo. The method involves subretinal injection of plasmid DNA, followed by in situ electroporation (adapted from 3). Delivery of GFP by this method was evaluated in frozen sections using fluorescence microscopy. In subsequent experiments, a luciferase plasmid, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging. Subretinal DNA injection followed by electroporation yielded abundant transgene delivery in rat RPE, as assayed by both GFP and luciferase expression. Expression was strongest 48 hours after delivery. In the absence of integrase, luciferase expression declined to near-background levels within 3-4 weeks after treatment. Co-injection of the integrase plasmid led to long-term stable transgene expression at all time points tested, up to three months post-procedure. Animals receiving integrase showed ~64-fold higher long-term expression than control animals. We conclude that subretinal injection followed by electroporation affords abundant transfer of plasmid DNA in mammalian RPE. Addition of phiC31 integrase confers robust long-term transgene expression, presumably by providing site-specific integration of the gene of interest into the genome of RPE cells. Having established non-viral permanent retinal gene transfer by site-specific genomic integration, we now plan to apply the technology to rodent models of retinal degeneration. Because site-specific integration provides strong, stable gene expression while reducing the probability of insertional mutagenesis, the phiC31 integrase system has great potential as a novel approach to non-viral gene therapy in the eye.