Novel AAV variants isolated by directed evolution in primate display enhanced retinal transduction following intravitreal injection

Abstract

Purpose : Directed evolution of AAV capsid libraries in mice has identified variants with enhanced transduction of mouse retina following intravitreal (Ivt) injection. In primate retina, improvements were less substantial. We developed a method to generate sortable retinal cells in primate which enabled screening of a highly complex AAV2-based capsid library to identify AAV variants capable of efficient retinal transduction following Ivt injection. In parallel, the same library was screened in mice. After 3 rounds of selection in mice and 2 in macaque, a subset of the most prevalent variants was characterized. The purpose of this study was to evaluate their transduction profiles following Ivt injection in mice and to measure physicochemical parameters previously shown to correlate with increased transduction efficiency by this injection route.

Methods : Capsid variants contained a self-complementary AAV genome carrying the truncated CBA promoter driving mCherry (sc-smCBA-mCherry). Transduction was quantified in vitro using ocular cell lines. Vectors were Ivt injected into Nrl-GFP mice and transduction was evaluated 4 weeks later by fundoscopy and FACS. AAV2wt and AAV2(quadY-F+T-V) were included for comparison. Measurements of capsid stability, as determined by differential scanning fluorimetry (DSF), and affinity for heparan sulfate by heparin column chromatography are underway.

Results : After 3 rounds of screening in mice, two heavily enriched variants, M3-A (32% relative frequency) and M3-B (21%) were chosen for analysis. After two rounds of selection in macaque, the 4 most prevalent variants P2-V1, P2-V2, P2-V3 and P2-V4 were analyzed. Interestingly, P2-V1 is identical to M3-B and P2-V4 is identical to M3-A. All variants displayed substantially improved transduction in vitro compared to AAV2wt. When compared to our most efficient rationally designed capsid, AAV2(quadY-F+T-V), P2-V1, P2-V2 and P2-V3 are all improved, with P2-V3 displaying a >2-fold increase in the number of retinal cells transduced following Ivt injection.

Conclusions : AAV capsids identified by screening in primate and mouse retina show enhanced transduction efficiencies both in vitro and following Ivt injection. An additional round of screening in both primate and mouse has been completed and results are pending.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Ivt-mediated transduction of enhanced AAVs.