Synthetic adenovirus libraries for vector optimization

用于载体优化的合成腺病毒文库 腺病毒已被广泛用于解决生物医学研究中的基本问题。这些 小的DNA病毒依赖于许多宿主蛋白质进行自身复制，并且已经成为有用的探针， 探索哺乳动物细胞的基本运作。遗传上易处理的重组腺病毒 已被广泛用作基因递送的载体。理性和无偏见的方法都是 用于增强和改变其功能特性。向量优化的理性方法 由于我们对许多病毒功能的不完全理解， 在技术上受到基因组大小的限制，以及诱变特定基因的固有困难， 兴趣这项建议的重点是开发一个新颖的平台， 腺病毒文库。作为该项目基础的关键创新是最近开发的一种用于 腺病毒基因组的体外组装来自紧凑的模块，可以单独操作，然后 重新组装。在本项目中，将采用综合方法生成高度多样性的焦点区域 在人腺病毒5的基因组中，最广泛使用的载体血清型。所得DNA 子文库将被组装成完整的病毒基因组，每个组装体被分配一个独特的条形码 设计用于促进文库表征和单个突变体的追踪。建设和 高含量，局灶性腺病毒文库的表征，及其实用性，将通过一个简单的 筛选逃避中和单克隆抗体识别的突变病毒。的长期目标 该项目旨在开发一种可共享和可持续开发的资源，以满足不断变化的需求 跨学科的科学家。

Synthetic adenovirus libraries for vector optimization Adenoviruses have been extensively utilized to address fundamental questions in biomedical research. These small DNA viruses rely on numerous host proteins for their own replication, and have been useful probes to explore the fundamental workings of the mammalian cell. Genetically tractable, recombinant adenoviruses have become widely used as vectors for gene delivery. Both rational and unbiased methods have been employed to enhance and modify their functional characteristics. Rational approaches to vector optimization are constrained by our incomplete understanding of many viral functions, while unbiased methods are technically limited by the size of the genome and the inherent difficulty of mutagenizing specific genes of interest. This proposal is focused on the development of a novel platform for the creation of customized adenovirus libraries. The critical innovation that underlies this project is a recently developed system for the in vitro assembly of adenovirus genomes from compact modules that can be individually manipulated and then reassembled. In this project, synthetic methods will be employed to generate focal regions of high diversity across the genome of human adenovirus 5, the most widely-employed vector serotype. The resulting DNA sub-libraries will be assembled into complete viral genomes, with each assembly assigned a unique barcode designed to facilitate library characterization and the tracking of individual mutants. The construction and characterization of high-content, focal adenoviral libraries, and their utility, will be demonstrated by a simple screen for mutant viruses that evade recognition by a neutralizing monoclonal antibody. The long term goal of this project is to develop a resource that can be shared and continually developed to meet the evolving needs of scientists across many disciplines.