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.

摘要 基因组测序工作越来越多地揭示了扰乱组织发育的基因变异 和功能。目前遗传性疾病的治疗受到限制，因为我们无法精确和 对功能失调基因和相关调控程序的永久性调整。然而，CRISPR/CAS9- 基于基因组的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。 一种特别有希望的方法是使用腺相关病毒(AAV)来传递CRISPR/Cas9 以及用于同源定向修复的模板(HDR；AAV-HDR)。体外AAV-HDR效率 可能高得惊人，在活体研究中，90%的转导细胞在某些情况下是正确编辑的 已经展示了更温和、更具变数的结果。要成功将AAV-HDR应用于 治疗环境，其效率将需要优化。此外，对AAV-HDR的深入理解 机制将是必要的，以确保安全。不幸的是，研究和改进AAV-HDR的努力已经 由于缺乏能够进行高通量、系统分析的工具，这一工作受到严重阻碍。假设： 开发用于测量体内AAV-HDR编辑效率的高通量方法将使 快速发现潜在的分子机制并促进临床所需的优化 翻译。 这项提议将开发和部署必要的工具，以快速、系统地改进体内 AAV-HDR。在目标1中，我们将以小鼠为模式生物，开发一种同时测量 AAV-HDR在多个靶点的效率。我们将研究AAV-HDR的位置相关变异性 利用该系统分析效率与靶基因座染色质的关系 在心肌细胞中的状态。在目标2中，我们将开发一种基于池CRISPR的高通量方法- 基因敲除筛查，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 是成功进行基因编辑所必需的。 AAV-HDR可以在心肌细胞内高效发生，尽管效率差异很大 按目标轨迹。在这里，我们建议开发两个系统，它们将利用下一代测序来 对AAV-HDR效率进行多次平行测量。据我们所知，这两个系统都将是第一个 他们的同类。我们使用这些系统将导致在理解机制方面取得关键的概念性进展 潜在的AAV-HDR。我们预计，这些技术和概念上的进步将促进发展。 以AAV-HDR为基础的疗法。