Evaluation of AAV Vector Constructs to Titrate Transgene Expression (2016)

Undergraduate: Michael Peralta

Faculty Advisor: Steven Gray
Department: Biology

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Adeno-associated virus (AAV) gene therapy vectors represent a promising method of treating central nervous system developmental disorders. Regulatory elements, such as polyadenylation sequences or introns, combined with specific promoters, have been known to affect post-infection viral transgene expression levels. Under- or over-expression of the delivered gene can cause adverse effects in the target cell population, so an important component of therapy design is the engineering of precise expression control while staying within the DNA capacity constraints of the viral capsid. While many regulatory elements have been characterized in isolation, the manner in which they interact to affect expression has not been well characterized. We modified a panel of plasmids incorporating two promoter sequences to incorporate an EGFP reporter gene and one of three different polyadenylation sequences. This resulted in five experimental plasmids, which were transfected into HEK293 and HeLa human cultured cells. The resulting protein expression levels were quantified via flow cytometry and western blotting. We found an unanticipated interaction between a specific combination of the regulatory elements that acted to depress transgene expression. This effect was consistently observed across multiple human cell lines. Future research will focus on packaging these constructs into an AAV9 viral vector for infection of the same cell lines and human fibroblasts.