Understanding mechanisms of Adeno-associated virus vector transduction through pharmacological and biological approaches

Gene therapy using Adeno-associated virus vectors (rAAV) has garnered much promise recently due to impressive clinical performance for several indications. Indeed, the lack of pathogenicity, broad tissue tropism among several serotypes, and ability to replace greater than 90 percent of the viral genome with a therapeutic transgene makes rAAV an attractive tool to achieve therapeutic benefit. However, recent gene therapy trials have highlighted a particular challenge that has hindered widespread applicability. While administration to local, immunopriviledged sites results in robust, long term expression of therapeutic transgenes; systemic delivery is seemingly restricted to a certain dose, as administration of vectors above this threshold seems to trigger an immune response resulting in decreased transgene expression over time. Thus, a major goal in the field is to identify ways to enhance transduction efficiency of vectors. In this dissertation, two approaches are explored that may provide insight into the future development of more effective vectors. First, a high throughput, small molecule screen was designed and executed to identify and characterize compounds resulting in enhanced transduction. These compounds were validated in vitro and clinically relevant compounds were explored in vivo. Secondly, a mechanism for rAAV2 nuclear entry, a step in subcellular trafficking thought to be a barrier to efficient transduction, was characterized. Efforts in capsid design or pharmacological approaches to overcome cellular barriers to transduction could result in enhanced transduction in a mechanism distinct from those already being targeted. These results provide a foundation for further advancements in pharmacological screens and rational vector design in order to achieve widespread clinical utility of rAAV.Doctor of Philosoph