(414b) Directed Evolution of AAV to Target Tissue-Specific Vascular Endothelium

Gene delivery vectors based upon adeno-associated virus (AAV) are highly promising, due to lack of pathogenicity, efficient infection of both dividing and non-dividing cells, and sustained maintenance of the transgene. While vectors based on AAV serotype 2 (AAV2), the best characterized variant, show broad tissue and cell tropism, AAV2 vectors do not efficiently transduce vascular endothelium. The vascular endothelium is associated with many diseases, including pulmonary hypertension and tumor angiogenesis. We hypothesized that the development of AAV vectors capable of efficient gene delivery targeted to vascular endothelium of a specific tissue would have therapeutic benefits for the tissue-associated diseases including cancer. First, tissue-specific AAV2-vectors can reduce potential side effects caused by the delivery of the transgene into unwanted tissues. Second, the amount of vectors required to provide comparable therapeutic effects can be lowered. In order to target AAV2 to vascular endothelium, three different AAV2 plasmid libraries were generated via error-prone PCR, shuffling of cap genes obtained from several serotypes, or insertion of randomized peptides in a defined capsid location. In vivo selection was then performed by tail vein injection of mice with these libraries followed by tissue harvest at 24 hours post-injection. Recovered cap genes of the AAV2 variants were used to construct next round of selection. After three rounds of in vivo selection for lung, the vascular distribution of AAV variants selected was evaluated. Among them, R4L13 variant, showed substantially enhanced localization to lung when compared with wild-type AAV2. R4L13 variant was further evolved via introduction of random mutations in the capsid protein. Another four rounds of in vivo selection for lung were performed. One of the AAV2 variants selected, EP09, showed further improvement in localization to lung compared to R4L13 variant. Recombinant vectors based upon R4L13 and EP09 are being assessed for targeted delivery of the gene expressing luciferase to lung, relative to recombinant vectors with wild-type AAV2 capsid. Similarly AAV libraries are being selected for other tissue-specific vascular endothelium including brain and spinal cord.