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. 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. We have genetically diversified AAV using a broad range of approaches from fully random (e.g. error prone PCR) to computationally guided (e.g. by machine learning). The resulting large (~109) libraries are then functionally selected for substantially enhanced delivery, yielding AAVs capable of highly efficient therapeutic gene delivery. Our variants have been effective in both animal models and in 6 human clinical trials to date, and results from both will be discussed.

Learning Objectives:

• Describe the process of directed evolution for optimizing AAV vectors used in gene therapy.
• Differentiate between random and computationally guided approaches for AAV genetic diversification.
• Discuss the clinical implications of using evolved AAV variants in gene therapy based on results from animal models and human trials.