Factors affecting AAV production: Mutagenesis of AAV2 rep and AAV transgenes

Abstract

Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Previous work in the AAV field focused on capsid engineering to enable targeting of vectors to specific tissues and has resulted in five approved therapies. Here, I turn my attention to the effect of rep and transgene sequence variants on rAAV production. As more rAAV therapies enter the clinic, manufacturing sufficient rAAV quantities to meet patient needs is increasingly difficult. By investigating two relatively understudied components of AAV production, I seek to improve our understanding of the AAV production process and, potentially, identify methods of enhancing it. In Chapter 1, I focus on AAV2 rep, which codes for four proteins that are critical for both AAV genome replication and packaging. I measure the effect of all single codon mutations to AAV2 rep on AAV production and identify numerous novel functional rep variants. This work highlights the importance of the DNA-interacting regions of Rep for AAV production enhancement and provides a useful dataset to enable further Rep engineering. In Chapter 2, I examine the effect of transgene sequence on AAV production. Here, I use synonymous recoding to study the effect of non-coding nucleotide changes to both wild-type and rAAV genomes on production. I demonstrate that nucleotide sequence - absent protein-coding changes - can affect rAAV production. Future work may identify generalizable principles for rAAV transgene design. Through an investigation of rep and transgene variants, this thesis informs our understanding of AAV production and lays the groundwork for improvements in large-scale gene therapy production.