Directed Evolution of New Viruses for Therapeutic Gene Delivery

Strong basic and translational efforts in the gene therapy field have culminated in successes in an increasing number of clinical trials involving viral vectors, particularly ones based on adeno-associated virus (AAV). These include trials for monogenic diseases such as hemophilia B, Leber’s congenital amaurosis, Sanfilippo B, and lipoprotein lipase deficiency (for which a gene therapy product was approved in the EU in 2012). AAV is thus capable of safe and therapeutic gene delivery to some cell and tissue targets; however, vectors in general face a number of challenges that limit their efficacy. These include anti-vector neutralizing antibodies, low transduction of some therapeutically relevant cells in vitro or in vivo, difficulty in overcoming cellular and physical barriers within complex tissue structures, and an incapacity for targeted delivery to specific cells. These challenges are not surprising, as nature did not evolve viruses for our convenience to use as human therapeutics, and thus “off the shelf” natural viruses do not meet a range of clinical needs. In most situations there is insufficient mechanistic knowledge of underlying virus structure-function relationships to empower rational design to improve such vectors; however, directed evolution has been emerging as a strategy to engineer novel viral variants that meet specific biomedical needs. We were the first to develop and have since been implementing directed vector evolution – the iterative genetic diversification of a viral genome and functional selection for desired properties – to address a number of problems with AAV. Genetic diversification has included the random diversification of peptide sequences at defined locations in the capsid, random point mutagenesis of the cap gene, and recombination of cap genes from a number of parental serotypes to create random chimeras. Using a range of in vitro and in vivo selection strategies, we have evolved AAV 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, and the resulting vectors are being clinically translated to meet human therapeutic needs.