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Synthetic adenovirus libraries for vector optimization

用于载体优化的合成腺病毒文库

基本信息

批准号：
10460635
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
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-20 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10460635
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

项目摘要

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DNA病毒依靠大量宿主蛋白进行自身复制，并且已成为有用的探针探索哺乳动物细胞的基本运作方式。遗传上易处理的重组腺病毒已被广泛用作基因传递的载体。理性方法和无偏见方法都已用于增强和修改其功能特性。矢量优化的理性方法由于我们对许多病毒功能的不完全了解而受到限制，而无偏见的方法则技术上受到基因组大小和诱变特定基因的固有难度的限制兴趣。该提案的重点是开发一个用于创建定制的新颖平台腺病毒文库。该项目的关键创新是最近开发的系统从可以单独操作的紧凑模块中体外组装腺病毒基因组，然后重新组装。在该项目中，将采用合成方法来生成高度多样性的焦点区域跨越人类腺病毒 5 型的基因组，这是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，每个组装体都分配有一个唯一的条形码旨在促进文库表征和单个突变体的追踪。建设及高内涵、焦点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。长期目标是该项目旨在开发一种可以共享和持续开发的资源，以满足不断变化的需求跨多个学科的科学家。