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

用于评估人类基因组编辑的有效性、特异性和安全性的基因组测序

• 批准号: 10667893
• 负责人: ERIC J DUNCAVAGE
• 金额: $ 46.4万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-20 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667893
• 关键词: Address; Adverse effects; Alleles; Benchmarking; Binding Sites; Biological Assay; CRISPR/Cas technology; Cancer Patient; Cell Therapy; Cell physiology; Cells; Chromosomal Rearrangement; Clinical; Clinical Research; Clinical Trials; Collection; Coupled; DNA; DNA analysis; DNA sequencing; Dangerousness; Data; Data Set; Ensure; Evaluation; Frequencies; Funding; Future; Genes; Genomics; Goals; Guide RNA; Guidelines; Human; Human Cell Line; Human Genome; Joints; Malignant Neoplasms; Measurement; Measures; Methods; Modification; Mutation; Patients; Performance; Pharmaceutical Preparations; Process; Qualifying; Reagent; Recommendation; Safety; Single Nucleotide Polymorphism; Somatic Mutation; Specificity; Technology; Testing; Therapeutic; Transgenes; United States Food and Drug Administration; Variant; chimeric antigen receptor; chimeric antigen receptor T cells; cost effective; deep sequencing; design; efficacy evaluation; experience; experimental study; genome analysis; genome editing; genome sequencing; genome-wide analysis; genomic platform; improved; in silico; insertion/deletion mutation; integration site; manufacture; manufacturing process; mutant; off-target mutation; somatic cell gene editing; targeted nucleases; tumor; whole genome

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 As a result, current draft guidance from the Food and Drug Administration (FDA) recommends that genome-edited cellular therapies be evaluated for both on- and off-target mutations.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.

该提案的目标是制定一个全面的方法，以评估效率和 使用全基因组测序的基因组编辑的人类细胞的特异性。基因组编辑具有巨大的 通过使其有可能恢复遗传缺陷、有害的突变等位基因， 增强细胞疗法的功能。尽管基因组编辑技术是为了最佳目的而设计的 在效率和特异性方面，靶向编辑可以是可变的，并且编辑的细胞中的不需要的突变可以导致 非预期的功能后果，包括由于脱靶突变、转基因 插入或缺失、重复或结构重排。因此， 美国食品和药物管理局（FDA）建议对基因组编辑的细胞疗法进行评估， 在靶和脱靶突变。然而，用于执行这些分析的现有方法是逻辑上的。 复杂，要么使用过时的方法，要么涉及修改编辑过程， 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)是一个理想的平台，以解决FDA的指导方针，基因组编辑的细胞基因组分析， 产品，因为它检测突变类型的全谱，并可用于全面评估 制造“患者准备”的细胞疗法。在这里，我们建议开发一种全面的WGS检测方法 专门设计用于表征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 最近开发的用于体细胞突变的临床WGS测定（ChromoSeq），以测量效率和 人类细胞中基因组编辑的特异性。我们将使用高覆盖率（> 250倍）WGS的配对编辑和 未编辑的对照细胞和联合体细胞变体识别方法，以量化靶向编辑效率和检测 转基因整合位点和非预期的单核苷酸变体、插入/缺失（indel），以及 染色体重排然后，我们将使用高置信度的数据集来验证此WGS方法 在具有CRISPR/Cas9和多重向导RNA（gRNA）库的三种人类细胞系中产生的突变， 将通过iGUIDE和靶向、纠错深度测序进行鉴定。在目标2中，我们将使用 WGS测定以定义基因组编辑的人CAR-T细胞中的突变景观。其中包括5 使用针对常见CAR-T靶标的试剂和15种现有的主要人类CAR-T产品进行重复实验 在我们实验室已经产生的一系列治疗相关基因上进行编辑。我们将使用 这些数据用于生成靶向编辑效率测量，CAR整合位点， 人类CAR-T细胞中的非预期突变，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种额外的基因组编辑细胞产物， 联盟将进一步确定WGS的性能和效用，以评价 基因组编辑的细胞疗法将使未来的临床研究成为可能。