Sensitive, unbiased, high-throughput, cellular GUIDE-seq-2 genome-wide activity assay for therapeutic genome editing INDs

用于治疗性基因组编辑 IND 的灵敏、无偏倚、高通量、细胞 GUIDE-seq-2 全基因组活性测定

• 批准号: 10668823
• 负责人: Shengdar Tsai
• 金额: $ 47.91万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668823
• 关键词: Address; Adopted; Adoption; Affect; Biological Assay; Cellular Assay; Chemistry; Collaborations; Computer software; DNA Double Strand Break; DNA Integration; Data; Development; Future; Gene Transduction Agent; Genetic; Genetic Variation; Genome; Genomic DNA; Genomic medicine; Goals; Hepatocyte; High-Throughput Nucleotide Sequencing; Human Activities; Human Genome; Individual; Informatics; Investigational Drugs; Investigational New Drug Application; Lead; Methods; Neurons; Outcome; Patients; Performance; Pharmaceutical Preparations; Phase; Proto-Oncogenes; Protocols documentation; Qualifying; Reproducibility of Results; Safety; Sickle Cell Anemia; Site; Specificity; T-Lymphocyte; Technology; Testing; Therapeutic; Translations; Viral Vector; cancer immunotherapy; cell type; genome editing; genome-wide; human disease; improved; in vivo; lead candidate; leukemia; manufacture; member; novel; nuclease; off-target mutation; patient safety; pre-clinical; preclinical development; prime editor; product development; somatic cell gene editing; therapeutic evaluation; therapeutic genome editing

PROJECT SUMMARY Genome editors have extraordinary potential to become curative, genomic medicines, but require special scrutiny because unintended ‘off-target’ mutations may be permanent and affect long-term patient safety. Of broad concern is that off-target mutations could activate proto-oncogenes, similar to DNA-integrating viral vectors for gene therapy which have caused leukemia in several patients. For increasing numbers of promising therapeutic genome editing strategies, there remains a critical need for optimized and qualified cellular assays to: 1) define the genome-wide activity of targets to inform lead target identification and 2) to functionally characterize the genome-wide activity of editors during the course of investigational new drug (IND) enabling chemistry, manufacturing, and control (CMC) activities. To our knowledge, the most commonly used method to define cellular genome-wide activity of editors for IND applications is GUIDE-seq, a sensitive and unbiased method we previously developed to define the cellular genome-wide activity of genome editing nucleases. As part of our Somatic Cell Genome Editing (SCGE) phase 1 project, we developed GUIDE-seq-2, a new version of GUIDE-seq with improved scalability and accuracy. The objective of this proposal is to optimize, refine, and qualify GUIDE-seq-2 as a fit-for-purpose assay for evaluation of therapeutic genome editors or associated genome edited cellular drug products in regulatory submissions. We propose the following specific aims: 1) Optimize and qualify GUIDE-seq-2 to assess cellular genome-wide activity for IND applications and 2) Apply GUIDE-seq-2 in novel, therapeutically relevant contexts. Together, the expected outcomes of our proposed studies will be an optimized and qualified GUIDE-seq-2 assay that can be directly approlied to establish critical quality attributes of human genome editing products in IND submissions. We envision that the optimized GUIDE- seq-2 protocol we developed and roadmap for assay qualification will become rapidly widely adopted and have a broad positive impact on development and translation of safe and effective genomic medicines.

项目摘要 基因组编辑器具有成为治疗性基因组药物的非凡潜力，但需要特殊的 这是因为非预期的“脱靶”突变可能是永久性的，影响患者的长期安全。的 人们普遍担心脱靶突变会激活原癌基因，类似于DNA整合病毒载体 基因治疗已经导致了几个病人的白血病。对于越来越多的有前途的 尽管存在治疗性基因组编辑策略，但仍然迫切需要优化和合格的细胞测定 目的：1）定义靶标的全基因组活性，以告知前导靶标鉴定，以及2）在功能上 描述研究性新药（IND）启用过程中编辑人员的全基因组活动 化学、制造和控制（CMC）活动。据我们所知，最常用的方法， 定义IND应用程序编辑器的细胞全基因组活动是GUIDE-seq，一个敏感和无偏见的 我们先前开发的方法用于定义基因组编辑核酸酶的细胞全基因组活性。作为 作为我们的体细胞基因组编辑（SCGE）第一阶段项目的一部分，我们开发了GUIDE-seq-2，一个新的版本， 具有改进的可扩展性和准确性的GUIDE-seq。本提案的目标是优化、完善和 将GUIDE-seq-2鉴定为用于评价治疗性基因组编辑或相关基因组编辑的适合目的的测定 基因组编辑的细胞药物产品。我们提出以下具体目标：1） 优化和鉴定GUIDE-seq-2，以评估IND申请的细胞全基因组活性; 2）申请 GUIDE-seq-2在新的，治疗相关的背景下。总之，我们建议的预期成果 研究将是一种优化和合格的GUIDE-seq-2测定法，可直接用于确定关键的 IND申请中人类基因组编辑产品的质量属性。我们设想，优化的指南- 我们开发的seq-2方案和测定鉴定路线图将迅速得到广泛采用， 对安全有效的基因组药物的开发和转化产生广泛的积极影响。