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

• 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
• 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

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了破坏组织发展的基因变异 和功能。目前，遗传疾病的疗法受到我们无法确定和 对功能失调的基因和相关调节计划的永久调整。但是，crispr/cas9- 基于基因组编辑被证明是具有巨大治疗潜力的强大基因调节工具。 一种特别有希望的方法是使用腺相关病毒（AAV）提供CRISPR/CAS9 组件以及同源性修复模板（HDR； AAV-HDR）。体外AAV-HDR效率 在某些情况下，可以正确编辑过> 90％的转导细胞，而体内研究正确编辑了> 90％的转导细胞 已经证明了更为适中且高度可变的结果。成功地使用AAV-HDR 治疗环境，其效率将需要优化。此外，对AAV-HDR的强烈理解 确保安全需要机制。不幸的是，研究和改善AAV-HDR的努力已 缺乏允许进行高通量，系统分析的工具严重阻碍。假设： 开发用于测量体内AAV-HDR编辑效率的高通量方法学 快速发现潜在的分子机制，并促进临床所需的优化 翻译。 该建议将开发和部署快速，系统地改进体内所需的工具 aav-hdr。在AIM 1中，使用小鼠作为模型生物，我们将开发一种同时测量的方法 在许多目标基因座上的AAV-HDR效率。我们将研究AAV-HDR的基因座依赖性变异性 效率通过利用系统分析效率与靶基因座染色质之间的关系 心肌细胞中的状态。在AIM 2中，我们将根据合并的CRISPR-开发一种高通量方法 敲除屏幕，用于评估基因扰动对心脏AAV-HDR效率的影响。我们将使用 通过识别DNA修复因子，该系统可以深入了解AAV-HDR的分子机制 对于成功的基因编辑是必需的。 尽管效率差异很大，但AAV-HDR可能以高效率发生在心脏肌肉细胞内 通过目标基因座。在这里，我们建议开发两个将利用下一代测序的系统 对AAV-HDR效率进行许多平行测量。据我们所知，这两个系统将是第一个 他们的那样。我们对这些系统的使用将带来关键的概念进步，以理解机制 基础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。