Gene therapy has experienced an increasing number of successful human clinical trials, leading to 6 FDA approved products using delivery vectors based on adeno-associated viruses (AAV). These successes were possible due to the identification of specific disease targets for which natural variants of AAV were sufficient. However, vectors face a number of barriers and shortcomings that preclude their extension to most human diseases, including limited delivery efficiency to target cells, pre-existing antibodies against AAVs, suboptimal biodistribution, limited spread within tissues, and/or an inability to target delivery to specific cells. These barriers 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. Furthermore, low efficiencies often necessitate high doses, which challenges manufacturing. Unfortunately, for most applications, there is insufficient mechanistic knowledge of underlying virus structure-function relationships to empower rational design improvements.

As an alternative, for over two decades we have 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 efficient and targeted delivery to any cell or tissue target. Our variants have been effective in both animal models and in numerous human clinical trials to date, and results from both will be discussed. Furthermore, CRISPR based genomewide library screens to improve vector yields from manufacturing cell lines will be discussed.

Learning Objectives: 

1. Summarize the current status and potential of gene therapy.

2. Recognize viral engineering strategies to overcome delivery hurdles.

3. Recognize cellular engineering strategies to improve manufacturing.