Novel AAV Variants Show Superior Gene Delivery in Human Retinal Cell Models

Abstract

Purpose : Gene therapy is a powerful approach for the treatment of inherited and complex retinal disorders. 4D Molecular Therapeutics (4DMT) has applied its Therapeutic Vector Evolution platform to discover novel adeno-associated virus (AAV) variants with superior gene delivery to the human retina following intravitreal delivery using non-human primates (NHP) as a model system. However, due to cross-species differences in AAV gene expression profiles and limitations of animal species to recapitulate human disease phenotypes, we have generated retinal pigment epithelium (RPE) and photoreceptors (PRs) from pluripotent stem cells (PSCs) to evaluate transduction and tropism of our novel retinal AAV vectors in human cell models.

Methods : The Therapeutic Vector Evolution platform involves applying progressively more stringent selective pressures to a genetically diverse library of AAV capsids to select novel variants with improved gene delivery properties compared to the natural AAV serotypes. In order to select variants with improved transduction of the NHP retina, 4DMT’s library of approximately 100 million unique AAV capsid variants was delivered via intravitreal administration.

Results : We identified novel variants after six rounds of selection. To evaluate the selected variants on human RPEs, cultures were differentiated from hPSCs for 30 days, when 90% of all cells in culture expressed mature RPE markers including RPE65 and BEST1, synthesized VEGF and PEDF, and phagocytosed rod outer segments. PRs generated by a multi-step eye cup formation paradigm expressed recoverin, rhodopsin, and S and M/L opsins after 100 days in culture.

Comparative analysis of transduction efficiency and GFP transgene expression by our novel variants in RPEs revealed significantly higher transduction efficiencies versus AAV2 as measured by flow cytometry and western blot. Robust transduction was also observed in the context of PR-directed gene transfer.

Conclusions : This study in human retinal cell models clearly illustrates superior gene delivery of our novel retinal AAV variants discovered through Therapeutic Vector Evolution as compared to the clinically relevant AAV2. These human retinal systems – complemented by in vivo NHP studies – will aid future development of optimized vectors. Moreover, hPSC-dependent disease models can be used to demonstrate functional rescue, thereby facilitating the path from proof-of-concept to clinical application.

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