Directed Evolution of New Viruses for Therapeutic Gene Delivery

Gene therapy has experienced an increasing number of successful human clinical trials – particularly ones using delivery vehicles or vectors based on adeno-associated viruses (AAV) – including trials for hemophilia B, Leber’s congenital amaurosis (LCA2), and spinal muscular atrophy. This progress recently led to the first FDA approval of an AAV-based gene therapy (for LCA2) in December, 2017. Despite this success, vectors in general face a number of barriers and challenges that limit their efficacy, such that the clinical studies to date have required large dosages, invasive routes of administration, and/or specific disease targets that pose less formidable delivery barriers. These shortcomings are not surprising, since the parent viruses upon which vectors are based were not evolved by nature for our convenience to use as human therapeutics. Unfortunately, for most applications, there is insufficient mechanistic knowledge of underlying virus structure-function relationships to empower rational design improvements.

As an alternative, we were the first to develop and have since been implementing directed evolution – the iterative genetic diversification of the viral genome and functional selection for desired properties – to engineer highly optimized, next generation AAV variants for delivery to any cell or tissue target. We have genetically diversified AAV using a broad range of approaches including random point mutagenesis of the cap gene, insertion of random peptide sequences into the AAV capsid, recombination of cap genes from a number of parental serotypes to create random chimeras, and construction of ancestral AAV libraries. The resulting large (~10⁹) libraries were then phenotypically selected for improved function in small and large animal models, yielding AAVs for evasion of neutralizing antibodies, enhanced biodistribution and spread within a target tissue, greatly improved delivery efficiency, and targeted delivery in vitro and in vivo, thereby laying a foundation for translating engineered AAVs into human clinical trials.