Key result
Synonymous mutations within the p19 promoter of the AAV Rep gene were enriched by 100 to 10,000 fold during library selection, enhancing AAV genome packaging into capsids.
Why the study?
When producing AAV gene therapies, a significant fraction of capsids lack DNA cargo, and how mutations in the Rep gene affect packaging activity was not understood.
Synonymous mutations in the AAV Rep gene p19 promoter are highly enriched during selection but do not significantly improve the packaging efficiency of synthetic DNA cargo compared to native Rep.
Answers are grounded in this paper and cite their sources.
Rep mutant selection may optimize AAV packaging; leaves open validation for clinical gene therapy vectors.
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 10 2 to 10 4 -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. (2024) studied AAV gene therapy manufacturing. Synonymous mutations in AAV Rep gene vs. Native Rep gene was evaluated on AAV genome packaging efficiency (enrichment of active virions) (10^2 to 10^4 fold increase in abundance). Synonymous mutations within the p19 promoter of the AAV Rep gene were enriched by 100 to 10,000 fold during library selection, enhancing AAV genome packaging into capsids.
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