(623c) Deep Mutational Scanning of the AAV rep Gene

Adeno-associated virus (AAV) is a commonly used viral vector for in vivo gene delivery. However, there are factors limiting its use on a large scale, including low titer, DNA impurities, and empty capsids. Most efforts to resolve these issues have focused on the AAV cap gene, which encodes its three capsid proteins. We have focused our efforts on the AAV rep gene, which encodes four protein isoforms responsible for DNA replication, packaging, and RNA transcription regulation. We have performed deep mutational scanning to generate a library of rep variants comprised of every possible single amino acid substitution. Deep sequencing of the library before and after transfection into mammalian cells has revealed the effect of each mutation on the ability to form capsids with the correctly packaged DNA. Potentially improved mutants have been identified and characterized based on changes in viral titer, empty to full capsid ratio, mispackaging rates, and toxicity to producer cells.