Key result
Synonymous mutations within the p19 promoter of the AAV Rep gene were enriched by 100 to 10,000 fold in a library selection for genome packaging, though they did not improve packaging of synthetic DNA cargo.
Why the study?
When producing AAV gene therapies, a significant fraction of capsids lack the desired DNA cargo, and how Rep gene mutations affect genome packaging activity was not well understood.
Effect estimate: 10^2 to 10^4 fold increase in abundance
p-value: p=8.96E-10
Synonymous mutations in the AAV Rep gene p19 promoter region are highly enriched during selection for genome packaging, suggesting silent sequence changes can be used to tune AAV packaging efficiency.
When producing Adeno-Associated Virus (AAV) gene therapies, a significant fraction of capsids can lack the desired DNA cargo. In AAV, Rep proteins mediate DNA packaging and virus assembly, suggesting that changes in Rep activity, expression, or DNA binding might affect genome packaging. To understand how mutations in the Rep gene affect activity, we selected a library of Rep mutants for their ability to produce active virions. By sequencing the Rep gene following the purification of viruses that package AAV genomes, we identified Rep mutants having non-synonymous mutations with a range of cellular activities. Surprisingly, synonymous mutations within the p19 promoter were enriched to the greatest extent, increasing in abundance by 102 to 104 fold. When the most highly enriched mutant was used to package a synthetic DNA cargo into the AAV capsid, the packaging efficiency could not be differentiated from native Rep. These findings suggest that these synonymous mutations enhance AAV genome packaging into capsids by affecting Rep-DNA interactions. They also suggest that silent sequence changes in the DNA cargo packaged by Rep can be used to tune packaging DNA packaging efficiency.
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Azim et al. (2025) studied AAV gene therapy manufacturing. Synonymous mutations in AAV Rep gene (p19 promoter) vs. Native Rep (WT) was evaluated on Enrichment of Rep mutants for their ability to produce active virions (10^2 to 10^4 fold increase in abundance, p=8.96E-10). Synonymous mutations within the p19 promoter of the AAV Rep gene were enriched by 100 to 10,000 fold in a library selection for genome packaging, though they did not improve packaging of synthetic DNA cargo.
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