316. Evaluating AAV Hybrid Variants for Improved Tropism after Intravitreal Gene Delivery to the Retina

Abstract

AAV-mediated gene therapy has shown promise for some ocular diseases when injected sub-retinally. Intravitreal AAV administration, while less invasive, is far less efficient due to the inner limiting membrane (ILM), which is a particularly profound barrier in the primate retina. Recently AAV2.7m8, which has a 10 amino acid (aa) peptide insertion in the surface exposed loop region of AAV2, was identified using in vivo directed evolution (Dalkara et al., 2013). This variant has been shown to effectively transduce outer retinal cells resulting in extensive gene expression in the non-human primate retina following intravitreal injection.The purpose of our study was to identify AAV variants that could not only penetrate the outer retina from the vitreous, but also have a favorable neutralizing antibody (nAb) titer to allow beneficial AAV-mediated gene expression. We tested whether the 10-aa peptide loop from AAV2.7m8 could be inserted into other capsid backbones, and investigated the tropism and nAb profile of these hybrid capsids. The 10-aa loop was inserted at seven different positions in the receptor binding region of loop IV of capsid 2.5T, which is a chimeric variant of the VP1 region of AAV2 and VP2 and VP3 regions of AAV5 containing a single A581T point mutation (Excoffon et al., 2009). A majority of the 2.5T/7m8 hybrid capsids were successfully packaged and resulted in varying levels of GFP transgene expression following transduction in vitro. Additionally, in vitro transduction was blocked to a lesser extent by nAbs (IVIg) when compared to AAV2, which indicates that the 2.5T/7m8 hybrids may not be impeded by preexisting immunity in human patients. Finally, significantly higher GFP expression was observed with one of the 2.5T/7m8 hybrid variants as compared to the parental 2.5T capsid, following intravitreal administration in rodents.