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

摘要 基因组测序工作越来越多地揭示了破坏组织发育的基因变异 和功能遗传疾病的治疗目前受到限制，因为我们无法精确和 对功能失调的基因和相关的调节程序进行永久性调整。CRISPR/Cas9- 基于基因组编辑的基因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）来递送CRISPR/Cas9。 在一个实施方案中，所述方法包括将所述多核苷酸组分以及用于同源定向修复（HDR; AAV-HDR）的模板结合到所述多核苷酸组分中。体外AAV-HDR效率 可以非常高，在某些情况下，>90%的转导细胞被正确编辑，而体内研究 已经证明了更温和的，高度可变的，结果。为了成功地将AAV-HDR用于 治疗环境，其效率将需要优化。此外，对AAV-HDR的深入了解 必须有机制来确保安全。不幸的是，研究和改进AAV-HDR的努力已经失败。 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： 用于测量体内AAV-HDR编辑效率的高通量方法学的开发将使得能够 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必要的工具，以快速，系统地完善体内 AAV-HDR。在目标1中，使用小鼠作为模式生物，我们将开发一种同时测量 在许多靶基因座的AAV-HDR效率。我们将研究AAV-HDR的基因座依赖性变异性， 通过利用该系统分析效率与靶位点染色质之间的关系， 在心肌细胞中的状态。在目标2中，我们将开发一种高通量方法，基于合并的CRISPR- 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 该系统通过鉴定DNA修复因子来深入了解AAV-HDR的分子机制， 是成功基因编辑的必要条件。 AAV-HDR可以在心肌细胞内以高效率发生，尽管效率变化很大。 通过目标轨迹。在这里，我们提出了两个系统的发展，将利用下一代测序， 进行AAV-HDR效率的许多并行测量。据我们所知，这两个系统将是第一个 他们的同类我们对这些系统的使用将导致理解这些机制的关键概念的进步 基础AAV-HDR。我们预期这些技术和概念上的进步将促进发展 基于AAV-HDR的治疗。

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.