Deep Mutational Scanning of the AAV rep Gene to Assess Effects on DNA Packaging in Expi293F Suspension Culture

ABSTRACT The adeno‐associated virus (AAV) holds great potential as a gene delivery vector for emerging gene therapies. However, AAV's production is currently limited by factors such as low viral titers, inefficient capsid packaging, and producer cell toxicity. We sought to address the issue of low titers through deep mutational scanning of the AAV rep gene, which is involved in AAV replication, transcription regulation, and packaging. After generating a library of all single codon substitutions at approximately 300 sites in the gene, we characterized the mutational effects on packaged virus particles in a high‐throughput replication competition assay in Expi293F cells suspension culture. The resulting values of the enrichment (a metric of packaging efficiency of native cargo) correlated moderately with those of a previous study in HEK293 adherent cells. However, difference in the range and distribution of values between the two studies as well as experimental variability between replicas in both data sets complicated assessment of cell line differences in mutational effects. We determined the ability of select mutants to alter the packaging of a synthetic DNA cargo into the AAV capsid but did not observe statistically significant improvements in either Expi293F or HEK293 cells. Our study adds to the growing evidence that beneficial effects of mutations in rep are highly dependent on the nature of the packaged DNA.