PROCESSING VIRUS BARCODES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 它一直是一个诱人的大谜团，对我们来说，各种AAV 血清型和变异体在它们的 感染性，嗜性，血液清除率，血管通透性， 病毒衣壳脱落率、抗原性、中和反应性 抗体等，与单一基因编码的VP蛋白。传统型 衣壳诱变和X射线结晶学等方法 提供了一些见解，但得到的结果仍然令人费解。 基于这样的背景，我们最近开发了一种 使用下一代的高通量反向遗传方法 测序技术，命名为条形码-序列。在这种方法中，我们将 系统地在整个甲型肝炎病毒衣壳中引入明确的突变 来自不同的血清型，使一组条形码AAV突变体 库中每个已定义的突变都有其唯一的条形码。然后 这些文库将用于测定体外和体内的 利用下一代的优势获得每个突变体的特性 测序技术，允许识别超过10-100个 在一次测序中有数百万个病毒条形码。这一概念 数据分析采用系统生物学方法。数量巨大的 通过这种方法获得的生物和生物统计信息 将极大地提高我们对AAV生物学和 帮助开发最适合每个人的新的重组AAV 申请。

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.