PROCESSING VIRUS BARCODES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It has been an ever-tempting big mystery to us how various AAV serotypes and variants exhibit significant dissimilarity in their infectivity, tropism, blood clearance rates, vascular permeability, virus capsid uncoating rates, antigenicity, reactivity to neutralizing antibodies, etc., with the single gene-coded VP protein. Conventional approaches such as capsid mutagenesis and X-ray crystallography have provided some insights, but the obtained results are still puzzling. Based on such a background, we have recently developed a high-throughput reverse genetic approach using the next generation sequencing technologies, named Barcode-Seq. In this approach, we will systematically introduce defined mutations throughout the AAV capsids derived from various serotypes, making an array of barcoded-AAV mutant libraries in which each defined mutation has its unique barcode. Then the libraries will be used to determine the in vitro and in vivo properties of each mutant by taking advantage of the next generation sequencing technologies that allow the identification of over 10-100 millions of viral barcodes in one sequencing run. The concept of systems biology is applied for the data analysis. An immense amount of biological and biostatistical information obtained by this approach will dramatically enhance our understanding of the biology of AAV and help develop new recombinant AAV that is most appropriate for each application.