Comprehensive mutagenesis of AAV2 rep

Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large scale gene therapy production. Two plasmid libraries, WT AAV2 and pCMV-Rep78/68, containing all possible single codon mutations to the AAV2 rep gene were cloned. The WT AAV2 library also expresses the AAV2 cap gene while the pCMV-Rep78/68 library only expresses the rep gene. The library formats also differ in the promoter used to drive rep expression: p5 in the WT AAV2 library and CMV in the pCMV-Rep78/68 library. Variants in each library contain unique 20 bp barcodes at the 3' end of rep/cap and ITRs to enable packaging of rep variant sequences into AAV capsids. The plasmid libraries were transfected into HEK293T cells to produce AAV2 capsids. Viral particles were purified and barcodes were amplified and sequenced from both the plasmid and viral pools. The pCMV-Rep78/68 library was also used to investigate the effect of rep mutations on AAV5 and AAV9 capsid production. All transfections were conducted in duplicate.