抄録 |
AAV vectors are among the most attractive gene delivery vehicles for human gene therapy. Promising therapeutic efficacy of AAV gene therapy has been reported in several clinical trials, including liver-targeted hemophilia B trials. However, further improvements of AAV vectors will be required for making AAV gene therapy a more effective and common practice. Although such improvements would be possible by AAV capsid engineering, this has been hindered by a paucity of knowledge about the AAV capsid structure-function relationships. To overcome this limitation, we have established a novel NGS-based approach that allows streamlined, high-throughput characterization of a wide spectrum of phenotypes of hundreds of different AAV species. This approach enables the collection of a large dataset on AAV capsid amino acid sequence-phenotype relationships by using comprehensive AAV serotype and capsid mutant libraries in which each AAV species is tagged with species-specific DNA barcodes placed within the viral genome. We have applied this approach to cultured cells, wild-type mice, FRG mice with humanized liver, and rhesus macaques, which has allowed us to build a comprehensive, intellectual foundation of engineering novel AAV capsids. To date, we have elucidated a series of previously unidentified, but functionally important capsid amino acids including those responsible for hepatic transduction; and have found that hepatic tropism of certain AAV species is significantly different between rodents and primates. |