Discovering the Next Generation AAV Vector- Capsid Engineering & Expression Cassette Optimization

At REGENXBIO, a clinical-stage gene therapy company, Dr. Liu’s team works on optimizing AAV vectors for gene therapy through capsid engineering strategies and improved expression cassettes. First, Dr. Liu provided an overview of the timeline for AAV discovery and initial use as a vector for gene therapy. Additionally, he introduced critical features of the AAV genome, structure, and transduction pathway, making this viral vector useful for engineering gene therapies.

Dr. Liu and colleagues work to improve the AAV capsid and transgene cassette to maximize the full potential of AAV gene therapy. Mutations are introduced targeting the capsid’s variable loops to engineer capsid variants. These mutations may encode as many as ten amino acids, resulting in the expression of new peptides on the capsid’s surface. Capsid engineering approaches aim at improving manufacturability and enable the development of AAV variants with new affinities for specific tissues. For transgene cassette optimization, the promoter, transgene, intron sequences, and inverted terminal repeats may be modified to improve the expression of genes of interest.

Dr. Liu’s team relies on structure-based rational design approaches to introduce mutations on the AAV’s surface variable loops for capsid engineering. Libraries containing millions of AAV variants may be developed through random peptide insertions or error-prone PCR. The workflow for AAV variant selection is performed in vivo through AAV library injections into animal models, tissue harvesting, and viral recovery for additional rounds of selection. This process, referred to as directed evolution, takes advantage of natural selection to identify AAV variants with desirable properties. Lastly, Dr. Liu shared how these strategies have been implemented in developing an AAV8 gene therapy, RGX-202, for Duchene muscular dystrophy, currently in the preclinical stage.