Extending the Longevity of AAV-mediated Lung Gene Therapy

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University of Guelph

AAV-mediated gene therapy has progressed rapidly in the past three decades, permitting the use of this vector for the treatment of several diseases. Although the first administration of an AAV vector into a human patient occurred in 1995 to correct cystic fibrosis in the lungs, this organ remains a challenging treatment site and elusive target for gene therapy. Limitations are attributed to the requirement for lung tropic vectors, the defensive barriers of the lungs, immune system considerations, the obstacles in the journey to reach the target cell and be granted entry, and cell turnover. In monogenic disorders such as surfactant protein B deficiency, additional hurdles appear in achieving long term expression due to the developing lung. This disease affects neonates, causing respiratory distress leading to death within a year of birth. Novel vectors such as capsid engineered AAV6.2FF may be able to deliver the gene of interest, but the gene addition delivery of AAV will be maintained in an episomal fashion and eventually wane in expression. Therefore, persistent expression will need to be achieved through either readministration or permanent gene correction. The following chapters feed into each other in progressing this vector forward and extending its persistence in the lungs. In this thesis, we assess the vector performance of AAV6.2FF, analyzing its biodistribution and stability, along with investigating mechanisms to extend expression in the lungs. Here, we administered 1e11 vector genome (vg) of AAV6.2FF with an alkaline phosphatase reporter gene by five routes of administration into C57BL/6 mice and viewed robust and localized expression in the lungs through intranasal instillation. Separately, we examined the thermal stability of this vector and determined that an excipient formulation change to include the non-ionic surfactant chemical PF-68 improved stability under different exposure conditions. In addition, we investigated mechanisms to extend the persistence of AAV6.2FF gene expression in lungs by mitigating the immune response with a stealth design change to the genome that permitted readministration along with demonstrating that permanent gene correction with AAV6.2FF is possible, providing optimistic conclusions that both readministration and gene editing can extend survival in surfactant protein B deficiency.

AAV, Adeno-associated viruses, gene therapy, lung gene therapy, pulmonary, gene editing, readministration, CRISPR-Cas9, biodistribution, PF-68, Viral vectors