Toward a Recombinant Adeno-Associated Virus Origin of Replication Public Deposited

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  • March 22, 2019
  • Hewitt, Frank Curtis.
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
  • Adeno-associated virus (AAV) is widespread throughout the human population. Many of the traits responsible for this ubiquity also make AAV an exceptional gene delivery vehicle. However, the widespread nature of AAV creates the risk that recombinant AAV vectors could be mobilized into unintended tissues or the general population by wild type (wt) AAV replication machinery. Alarmingly, the majority of characterized AAV serotypes are capable of rescuing and replicating the AAV2-based vectors currently used in AAV clinical trials. There are a number of potential methods to prevent AAV vector mobilization. The origin of replication from a less prevalent human AAV serotype or a non-human serotype would decrease, but not eliminate, the risk of mobilization. Ultimately, AAV vectors must utilize an origin of replication incompatible with the Replication (Rep) proteins of any naturally occurring AAV serotype. Unfortunately, the nature of the AAV origin, the inverted terminal repeat (ITR), has been a barrier to understanding the mechanisms of replicative specificity necessary to synthesize a novel origin. By generating a panel of chimeric and mutant ITRs and Rep proteins between two serotypes of AAV, we have mapped two independent DNA-protein interfaces required to generate replicative specificity. In vivo replication assays demonstrated that for AAV2, three residues in the Rep active site are required to make specific contacts with the nicking site of ITR2. AAV5 has a unique interaction between a 12 nucleotide extended Rep binding element and a 49 amino acid region of Rep5 containing two DNA binding interfaces. Structural models display significant differences between serotypes in these regions. Understanding the separation of these elements led to the creation of a recombinant origin of replication with properties independent of either parent serotype. This novel origin stands to prevent AAV vector mobilization and expand our understanding of the basic mechanisms of AAV replication.
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  • ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Genetics and Molecular Biology
  • Samulski, R. Jude

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