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

基本信息

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
项目类别：
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

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

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项目摘要

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.

该提案的目的是开发一种评估效率和的综合方法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通过恢复遗传缺陷，灭活有害突变等位基因和增强细胞疗法的功能。尽管基因组编辑技术是为最佳设计而设计的效率和特异性，目标编辑可能是可变的，并且编辑的单元格中不需要的突变可能会导致意外的功能后果，包括因靶向突变而导致的基因破坏，转化插入或删除，重复或结构重排。结果，目前的指导草案食品和药物管理局（FDA）建议评估基因组编辑的细胞疗法靶向突变和脱离目标突变。但是，在逻辑上进行这些分析的现有方法是复杂并使用过时的方法或涉及对编辑过程的修改应用于将用于患者的细胞药物。我们假设全基因组测序（WGS）是解决FDA指南的理想平台，用于基因组编辑的细胞基因组分析因为它检测到突变类型的完整范围，因此产品可用于完全评估制造的“准备患者”的细胞疗法。在这里，我们建议开发全面的WGS分析专门设计用于表征基因组编辑的人类细胞中的突变。在AIM 1中，我们将修改我们的最近开发了用于体细胞突变（Chromoseq）的临床WGS测定法，以测量效率和人类细胞中基因组编辑的特异性。我们将使用配对编辑的高覆盖范围（> 250倍）WGS和未经编辑的对照单元和联合体细胞变体呼叫方法，以量化目标编辑效率并检测转基因整合位点和意外的单核苷酸变体，插入/缺失（Indels）和染色体重排。然后，我们将使用高信心的数据集限定这种WGS方法与CRISPR/CAS9的三种人类细胞系中产生的突变和导向RNA（GRNA）的多重池，这将通过iguide识别，并针对错误校正的深层测序。在AIM 2中，我们将使用我们的 WGS测定法定义了基因组编辑的人CAR-T细胞中突变的景观。这些将包括5 用试剂重复实验到常见的CAR-T目标，15种现有的原代人Car-T产品在我们实验室已经生成的一系列治疗基因上进行了编辑。我们将使用这些数据以生成靶向编辑效率测量，汽车集成站点的基准数据集，人类CAR-T细胞中的意外突变将为将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个基因组编辑的细胞产物联盟以进一步建立WGS的绩效和实用性来评估基因组编辑的细胞疗法将使未来的研究临床研究。