Development of tools for rapid systematic refinement of in vivo gene editing technologies

开发用于快速系统完善体内基因编辑技术的工具

基本信息

批准号：
10740025
负责人：
Nathan James VanDusen
金额：
$ 42.53万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2025-08-14
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10740025
关键词：
CRISPR/Cas technology Cardiac Cardiac Myocytes Cell Proliferation Cells Characteristics Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Custom DNA Repair DNA Repair Gene Dependovirus Development Enabling Factors Ensure Epigenetic Process Gene Expression Gene Transduction Agent Genes Genetic Diseases Genetic Screening Genomic DNA Genomics Hepatocyte Human In Vitro Knock-out Knockout Mice Libraries Measurement Measures Mediating Methodology Methods Mitotic Modification Molecular Mus Mutation Nature Pathway interactions Proteins Regulator Genes Reporting Resources Role Safety Site System Technology Testing Therapeutic Time Tissues Viral Genome adeno-associated viral vector cellular transduction clinical translation genetic variant genome editing genome sequencing improved in vivo insight knockout gene loss of function model organism next generation sequencing novel postmitotic precise genome editing programs repaired screening sound tool tool development

项目摘要

Abstract Genome sequencing efforts are increasingly revealing gene variants that disrupt tissue development and function. Therapies for genetic disorders are currently limited by our inability to make precise and permanent adjustments to dysfunctional genes and associated regulatory programs. However, CRISPR/Cas9- based genome editing is proving to be a powerful gene regulatory tool with tremendous therapeutic potential. One particularly promising approach is the use of adeno-associated virus (AAV) to deliver CRISPR/Cas9 components as well as a template for homology directed repair (HDR; AAV-HDR). In vitro AAV-HDR efficiency can be spectacularly high, with >90% of transduced cells correctly edited in some cases, while in vivo studies have demonstrated more modest, and highly variable, results. To successfully employ AAV-HDR in a therapeutic setting, its efficiency will need to be optimized. In addition, a robust understanding of AAV-HDR mechanisms will be necessary to ensure safety. Unfortunately, efforts to study and improve AAV-HDR have been severely hampered by a lack of tools that allow for high-throughput, systematic analyses. Hypothesis: Development of high-throughput methodologies for measuring in vivo AAV-HDR editing efficiency will enable rapid discovery of the underlying molecular mechanisms and facilitate optimization necessary for clinical translation. This proposal will develop and deploy the tools necessary for rapid, systematic refinement of in vivo AAV-HDR. In Aim 1, using mice as a model organism, we will develop a method for simultaneously measuring AAV-HDR efficiency at many target loci. We will investigate the locus-dependent variability of AAV-HDR efficiency by utilizing the system to analyze the relationship between efficiency and target locus chromatin state in cardiomyocytes. In Aim 2, we will develop a high-throughput method, based on a pooled CRISPR- knockout screen, for assessing the impact of gene perturbations on cardiac AAV-HDR efficiency. We will use the system to gain insights into the molecular mechanism of AAV-HDR, by identifying DNA-repair factors that are necessary for successful gene editing. AAV-HDR can occur at high efficiency within heart muscle cells, although efficiency varies dramatically by target locus. Here we propose development of two systems that will leverage next-generation sequencing to make many parallel measurements of AAV-HDR efficiency. To our knowledge, both systems will be the first of their kind. Our use of these systems will lead to key conceptual advances in understanding the mechanisms underlying AAV-HDR. We anticipate that these technical and conceptual advances will promote development of AAV-HDR based therapies.

抽象的基因组测序工作越来越多地揭示破坏组织发育的基因变异和功能。目前，遗传性疾病的治疗方法因我们无法做出精确而有效的治疗而受到限制。对功能失调的基因和相关调控程序进行永久性调整。然而，CRISPR/Cas9- 基于基因组编辑被证明是一种强大的基因调控工具，具有巨大的治疗潜力。一种特别有前途的方法是使用腺相关病毒 (AAV) 来传递 CRISPR/Cas9 组件以及同源定向修复模板（HDR；AAV-HDR）。体外 AAV-HDR 效率可能非常高，在某些情况下，>90% 的转导细胞被正确编辑，而体内研究已表现出更为温和且高度可变的结果。成功地在 AAV-HDR 中应用治疗环境，其效率需要优化。此外，对 AAV-HDR 有深入的了解需要有机制来确保安全。不幸的是，研究和改进 AAV-HDR 的努力并没有取得进展。由于缺乏可进行高通量、系统分析的工具而受到严重阻碍。假设：开发用于测量体内 AAV-HDR 编辑效率的高通量方法将使得快速发现潜在的分子机制并促进临床所需的优化翻译。该提案将开发和部署快速、系统地改进体内实验所需的工具。 AAV-HDR。在目标 1 中，我们将使用小鼠作为模型生物，开发一种同时测量的方法许多目标位点的 AAV-HDR 效率。我们将研究 AAV-HDR 的位点依赖性变异利用该系统分析效率与目标位点染色质之间的关系心肌细胞中的状态。在目标 2 中，我们将开发一种基于汇集 CRISPR 的高通量方法敲除筛选，用于评估基因扰动对心脏 AAV-HDR 效率的影响。我们将使用该系统通过识别 DNA 修复因子来深入了解 AAV-HDR 的分子机制是成功基因编辑所必需的。 AAV-HDR 可以在心肌细胞内高效发生，尽管效率差异很大通过目标位点。在这里，我们建议开发两个系统，利用下一代测序来对 AAV-HDR 效率进行多次并行测量。据我们所知，这两个系统都将是第一个他们的同类。我们对这些系统的使用将导致理解机制方面的关键概念进步底层 AAV-HDR。我们预计这些技术和概念的进步将促进发展基于 AAV-HDR 的疗法。