Synthetic adenovirus libraries for vector optimization

用于载体优化的合成腺病毒文库

• 批准号: 10188568
• 负责人: FRED BUNZ
• 金额: $ 36.84万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188568
• 关键词: Address; Adenovirus Vector; Adenoviruses; Adopted; Bar Codes; Biomedical Research; Capsid Proteins; Cell physiology; Cellular Stress; Characteristics; Complex; Cosmids; Custom; DNA; DNA Library; DNA Viruses; DNA biosynthesis; Development; Discipline; Epitopes; Frequencies; Gene Delivery; Generations; Genes; Genetic Screening; Genetic Transcription; Genetic Variation; Genome; Genomic Library; Goals; Human; In Vitro; Individual; Infection; Knowledge; Libraries; Mammalian Cell; Methods; Mutagenesis; Mutation; Nature; Normal Cell; Open Reading Frames; Plasmids; Process; Production; Property; Proteins; RNA Processing; Reaction; Resources; Scientist; Serotyping; Signal Pathway; System; Techniques; Technology; Tissues; Toxic effect; Transgenes; Tropism; Tube; Variant; Viral; Viral Genome; Viral Physiology; Virus; base; biological adaptation to stress; cell type; combinatorial; design; field study; homologous recombination; improved; innovation; interest; mutant; neutralizing monoclonal antibodies; next generation; novel; recombinant adenovirus; synthetic biology; synthetic construct; tool; trait; transgene delivery; vector; whole genome

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.

用于矢量优化的合成腺病毒库 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 小型DNA病毒依靠许多宿主蛋白进行自身复制，并且是有用的探针 探索哺乳动物细胞的基本工作。可基因拖动的重组腺病毒 已被广泛用作基因输送的向量。理性和公正的方法都是 用于增强和修改其功能特征。矢量优化的理性方法 受我们对许多病毒功能的不完全理解的限制，而公正的方法是 在技​​术上受到基因组大小的限制以及诱变的特定基因的固有难度 兴趣。该提案的重点是开发一个新颖的平台，以创建定制的 腺病毒库。基于该项目的关键创新是一个最近开发的系统 来自紧凑型模块的腺病毒基因组的体外组装，可以单独操纵，然后 重新组装。在这个项目中，将采用合成方法来产生高多样性的焦点区域 跨越人类腺病毒5的基因组，是最广泛的载体血清型。产生的DNA 子图片将组装成完整的病毒基因组，每个组装分配了一个唯一的条形码 旨在促进图书馆的表征和单个突变体的跟踪。结构和 高内科，局灶性腺病毒库及其效用的表征将通过简单的 通过中和单克隆抗体逃避识别的突变病毒的筛选。长期目标的 该项目是开发一个可以共享和不断开发的资源，以满足不断发展的需求 许多学科的科学家。