Silberg, JonathanThyer, Ross2025-01-162025-01-162024-122024-10-24December 2https://hdl.handle.net/1911/118200Adeno-associated virus (AAV), a federally approved gene therapy vector that is currently in clinical trials for hundreds of diseases, often presents suboptimal characteristics for therapeutic applications, such as suboptimal tissue specificity, limited cargo size, undesired immune responses, and costly manufacturing. To improve this gene therapy vector, AAV proteins are frequently studied via rational design and combinatorial engineering. While the latter approach increases the sequence space that can be explored, it also presents unique challenges such as genotype-phenotype mismatches, noise arising from mutational errors in cloning, and bias arising in amplicon preparation and sequencing. In AAV, Rep proteins mediate DNA packaging and virus assembly, suggesting that changes in Rep activity, expression, or DNA binding might affect genome packaging. However, these proteins are not as well-understood as the proteins that make up the virus shell. I sought to understand how mutations in the Rep protein affect activity by selecting a library of Rep mutants for their ability to produce virions. To do this, I designed large protein libraries to examine a broad sequence space, I designed a selection strategy that couples genotype-phenotype characteristics of the mutants, and I designed a single-stranded DNA isolation workflow that enabled me to sequence winning variants in deep sequencing. By sequencing the rep gene following the purification of viruses that package AAV genomes, I 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-genome interactions. They also suggest that silent sequence changes in the DNA cargo packaged by Rep can be used to tune packaging DNA packaging efficiency. Additionally, I designed a sequential cloning method for developing barcoded chimeric protein libraries, which enables easier analysis of deep-sequenced these. This cloning strategy has been partially validated. Future work should be done to optimize this cloning method, as it would be applicable for chimeric library design for any protein engineering experiment. Lastly, this work outlines considerations in high-throughput protein engineering experimental design. I hope this section of my thesis enlightens those who wish to begin high-throughput protein experiments and learn from some of the critiques I have of my own work.application/pdfenAdeno-associated virusgene therapyprotein engineeringThesis2025-01-16