313. Development of the AAV6-Based Vectors With Selective Tropism and High Transduction Efficiency of Human Cancer Cells

Abstract

Cancer is a leading cause of human death worldwide. Commonly used methods of treatment such as surgery, radiation and chemotherapy tend to be largely successful but are extremely invasive and have serious toxic side effects. Thus, oncolytic viruses represent an attractive alternative to currently used anti-cancer treatments. Among different viral methods for gene delivery, vectors based on a human parvovirus, the adeno-associated virus (AAV) has attracted much attention mainly because of its non-pathogenic nature, and its ability to mediate long-term, sustained therapeutic gene expression.In current studies, AAV serotype 6 was identified to be particularly effective in transduction of several human prostate (PC3), breast (T47D) and liver (Huh7) cancer cell. We also found that transduction efficiency of these vectors can be significantly improved, up to 5-fold, by site-direct mutagenesis of surface exposed residues involved in intracellular trafficking. Over thirty single and multiple tyrosine (Y), serine (S), threonine (T) capsid mutants were developed and AAV6-Y705-731F+T492V was identified the most efficient.However, promiscuity of the AAV vectors makes it difficult to use in vivo. The recent studies on AAV crystal structure have identified specific regions of the capsid which are surface-exposed and tolerant to insertion of the peptides. In the current studies, we developed novel AAV-based vectors for targeting of human cancer cells by incorporating Arg-Gly-Asp (RGD) containing peptide to enable AAV to infect integrin-expressing cells. These AAV6-RGD vectors improve transduction efficiency approximately 3-fold compared with wild-type AAV6 vectors by enhancing viral entry to the cells. We correlated increased transduction efficiency with levels of expression of avβ5 integrin.The combination of RGD-peptide and mutations of surface-exposed residues on a single AAV6 capsid, further increases transduction efficiency up to approximately 10-fold. We next used qPCR analysis to quantitate the vector genomes in cytoplasmic and nuclear fractions of cells infected with the WT and the mutant AAV6 vectors at different time points. We showed that the increase in the transduction efficiency of the AAV6-Y705-731F+T492V vector correlated well with the improvement of both the entry and nuclear translocation of the vectors, compared with AAV6-WT.To conduct animal studies, we also introduced an additional mutation at lysine (K531E) since this was shown to impair the ability of vectors to bind to heparan sulfate proteoglycan and thus decrease the affinity to mice hepatocytes. We used a xenograft animal model in immune-deficient mice to prove the specificity and high transduction efficiency of AAV6-RGD-Y705-731F+T492V-K531E vectors in vivo.In summary, this approach can potentially lead to the development of therapeutic AAV vectors with selective tropism and high transduction efficiency of human cancer cells.