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.

项目概要 基因组编辑具有成为治疗性基因药物的非凡潜力，但需要特殊的 审查，因为意外的“脱靶”突变可能是永久性的，并影响患者的长期安全。 广泛关注的是，脱靶突变可能会激活原癌基因，类似于 DNA 整合病毒载体 对于导致数名患者罹患白血病的基因治疗，有希望的人数不断增加。 治疗性基因组编辑策略，仍然迫切需要优化和合格的细胞检测 目的：1) 定义靶标的全基因组活性，为先导靶标识别提供信息；2) 在功能上 描述新药研究 (IND) 过程中编辑器的全基因组活动特征 据我们所知，最常用的方法是化学、制造和控制 (CMC) 活动。 定义用于 IND 应用的编辑器的细胞全基因组活性是 GUIDE-seq，一种敏感且无偏见的方法 我们之前开发的用于定义基因组编辑核酸酶的细胞全基因组活性的方法。 作为体细胞基因组编辑 (SCGE) 第一阶段项目的一部分，我们开发了新版本 GUIDE-seq-2 GUIDE-seq 的可扩展性和准确性得到提高，该提案的目标是优化、细化和改进。 使 GUIDE-seq-2 符合评估治疗基因组编辑器或相关测定的目的 我们在监管提交中提出了以下具体目标：1) 优化和限定 GUIDE-seq-2 以评估 IND 应用的细胞全基因组活性，并 2) 应用 GUIDE-seq-2 在新颖的、治疗相关的背景下，我们提出的预期结果。 研究将是一种优化且合格的 GUIDE-seq-2 检测，可直接用于建立关键的 我们设想优化的 GUIDE- 中人类基因组编辑产品的质量属性。 我们开发的 seq-2 协议和检测鉴定路线图将迅速得到广泛采用，并已 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。