Genome sequencing for evaluating the efficacy, specificity, and safety of human genome editing

这项提案的目标是开发一种全面的方法来评估效率和 利用全基因组测序技术分析基因组编辑后的人类细胞的特异性。基因组编辑具有巨大的 通过修复遗传缺陷，灭活有害的突变等位基因，以及 增强细胞疗法的功能。尽管基因组编辑技术旨在优化 效率和特异性，目标编辑可能是可变的，编辑的单元格中不想要的突变可能会导致 意想不到的功能后果，包括由于非靶标突变、转基因导致的基因中断 插入或删除、重复或结构重新排列。因此，目前的指导意见草案来自 食品和药物管理局(FDA)建议对基因组编辑的细胞疗法进行评估 靶上和靶外的突变。然而，执行这些分析的现有方法是逻辑上的 复杂，或者使用过时的方法，或者涉及对编辑过程的修改，而不能 应用于将用于患者的细胞药物产品。我们假设全基因组测序 (WGS)是解决FDA基因组编辑细胞基因组分析指南的理想平台 产品，因为它可以检测所有突变类型，并可用于全面评估 制造的“为病人准备的”细胞疗法。在这里，我们建议开发一种全面的WGS分析方法 专门设计用来描述基因组编辑的人类细胞中的突变。在目标1中，我们将修改我们的 最近建立的临床WGS体细胞突变分析(ChromoSeq)来衡量效率和 人类细胞基因组编辑的特异性。我们将使用高覆盖率(&gt；250x)WG配对编辑和 未编辑的控制细胞和联合体细胞变体调用方法，以量化目标编辑效率并检测 转基因整合位点和意想不到的单核苷酸变异、插入/缺失(Indels)以及 染色体重排。然后，我们将使用高置信度的数据集对此WGS方法进行鉴定 在具有CRISPR/Cas9和多重引导RNA池(GRNA)的三个人类细胞系中产生的突变， 将通过iGuide和有针对性的、纠错的深度测序进行鉴定。在目标2中，我们将使用我们的 WGS分析，以确定基因组编辑的人类CAR-T细胞的突变图景。这些将包括5个 用试剂对常见的CAR-T靶标和15种现有的主要人类CAR-T产品进行复制实验 编辑了我们实验室已经产生的一系列与治疗相关的基因。我们将使用 这些数据以生成目标上编辑效率测量的基准数据集，汽车集成站点， 人类CAR-T细胞的意外突变将为未来的临床试验提供有价值的数据。最后， 我们将分析来自体细胞基因组编辑的多达20个额外的基因组编辑细胞产品 财团将进一步确定WGS的性能和效用，以评估 基因组编辑的细胞疗法将使未来的研究性临床研究成为可能。

The goal of this proposal is to develop a comprehensive approach for evaluating the efficiency and specificity of genome-edited human cells using whole-genome sequencing. Genome editing has enormous therapeutic potential by making it possible to restore genetic defects, inactivate deleterious mutant alleles, and augment the function of cellular therapies. Although genome editing technologies are designed for optimal efficiency and specificity, on-target editing can be variable, and unwanted mutations in edited cells can result in unintended functional consequences, including disruption of genes due to off-target mutations, transgene insertions, or deletions, duplications, or structural rearrangements. As a result, current draft guidance from the Food and Drug Administration (FDA) recommends that genome-edited cellular therapies be evaluated for both on- and off-target mutations. However, existing approaches for performing these analyses are logistically complicated and either use antiquated methods or involve modifications to the editing process that cannot be applied to cellular drug products that will be used in patients. We hypothesize that whole-genome sequencing (WGS) is an ideal platform to address FDA guidelines for genomic analysis of genome-edited cellular products because it detects the full spectrum of mutation types and can be used to evaluate fully manufactured ‘patient-ready’ cellular therapies. Here we propose to develop a comprehensive WGS assay specifically designed to characterize mutations in genome-edited human cells. In Aim 1, we will modify our recently developed clinical WGS assay for somatic mutations (ChromoSeq) to measure the efficiency and specificity of genome editing in human cells. We will use high coverage (>250x) WGS of paired edited and unedited control cells and joint somatic variant calling methods to quantify on-target editing efficiency and detect transgene integration sites and unintended single nucleotide variants, insertions/deletions (indels), and chromosomal rearrangements. We will then qualify this WGS approach using a dataset of high confidence mutations generated in three human cell lines with CRISPR/Cas9 and multiplex pools of guide RNAs (gRNA), which will be identified via iGUIDE and targeted, error-corrected deep sequencing. In Aim 2, we will use our WGS assay to define the landscape of mutations in genome-edited human CAR-T cells. These will include 5 replicate experiments with reagents to common CAR-T targets, and 15 existing primary human CAR-T products edited at a range of therapeutically relevant genes that have already been generated in our labs. We will use these data to generate a benchmark dataset of on-target editing efficiency measurements, CAR integration sites, and unintended mutations in human CAR-T cells that will provide valuable data for future clinical trials. Finally, we will analyze up to 20 additional genome-edited cellular products from the Somatic Cell Genome Editing Consortium to further establish the performance and utility of WGS for evaluating the safety and efficacy of genome-edited cellular therapies that will enable future investigational clinical studies.