Ultra-sensitive, unbiased, high-throughput, biochemical CHANGE-seq genome-wide activity and gRNA sequencing assays for therapeutic genome editing INDs

用于治疗性基因组编辑 IND 的超灵敏、无偏倚、高通量、生化 CHANGE-seq 全基因组活性和 gRNA 测序分析

• 批准号: 10668824
• 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: 2023-05-17 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668824
• 关键词: Acceleration; Affect; Biochemical; Bioinformatics; Biological Assay; Cells; Chemistry; Clinic; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Computer software; DNA Integration; Data; Development; Ensure; Eye; Frequencies; Future; Gene Transduction Agent; Genetic Diseases; Genome; Genome Components; Genomic DNA; Genomic medicine; Genomics; Goals; Guide RNA; High-Throughput Nucleotide Sequencing; Human Genome; Inherited; Investigational Drugs; Liver; Methods; Outcome; Patients; Performance; Pharmaceutical Preparations; Proto-Oncogenes; Protocols documentation; Publishing; Qualifying; Reagent; Research Methodology; Retinal Diseases; Risk; Safety; Sickle Cell Anemia; Site; Specificity; Technology; Testing; Therapeutic; Translations; Viral Genes; base editor; cancer immunotherapy; design; first-in-human; gene therapy; genome editing; genome-wide; human disease; improved; in vivo; leukemia; manufacture; member; novel; nuclease; off-target mutation; off-target site; prime editor; safety assessment; therapeutic genome editing; therapeutically effective

PROJECT SUMMARY Genome editors, technologies to modify the genomes of living cells, have extraordinary potential to become safe and effective genomic medicines, direct treatments for the underlying cause of genetic diseases such as sickle cell disease and many others. However, as gene therapy products with novel mechanisms of action, there remains a need for optimized and qualified biochemical IND-enabling assays to assess their safety. We and others have developed sensitive and unbiased research methods for defining the genome-wide activity of editors such as CHANGE-seq and GUIDE-seq. However, they require further optimization and characterization as fit- for-purpose assays to fulfill rigorous regulatory requirements for investigational new drug (IND) submissions. Surprisingly, to our knowledge there are no published methods for high-throughput sequencing characterization of gRNA identity and purity. Thus, there remain urgent unmet needs for publicly available, optimized, and qualified IND-enabling assays to characterize critical genome editing reagents and their associated on- and off- target genome-wide activities. We, therefore, propose the following specific aims: 1) Optimize and qualify CHANGE-seq as IND-enabling biochemical genome-wide activity assay, and 2) Optimize and qualify gRNA sequencing as IND-enabling assay to assess genome editing component identity and purity, and 3) Collaborate to test CHANGE-seq and gRNA sequencing in therapeutic contexts. We anticipate that fulfilling the need for well-characterized assays to identify impurities in critical reagents or characterize key quality attributes of genome editing drug products will have positive impact to accelerate the translation of novel, safe, and effective therapeutic genome editing therapeutic strategies to first-in-human clinical trials.

项目概要 基因组编辑是修改活细胞基因组的技术，具有成为 安全有效的基因组药物，直接治疗遗传病的根本原因，例如 镰状细胞病和许多其他疾病。然而，作为具有新颖作用机制的基因治疗产品， 仍然需要优化和合格的生化 IND 检测方法来评估其安全性。我们和 其他人已经开发出敏感且公正的研究方法来定义编辑的全基因组活动 例如 CHANGE-seq 和 GUIDE-seq。然而，它们需要进一步的优化和表征作为拟合 专用测定，以满足研究性新药 (IND) 提交的严格监管要求。 令人惊讶的是，据我们所知，还没有公开的高通量测序表征方法 gRNA 的身份和纯度。因此，对于公开可用的、优化的和 合格的 IND 检测方法，用于表征关键基因组编辑试剂及其相关的开启和关闭 目标全基因组活动。因此，我们提出以下具体目标：1）优化和限定 CHANGE-seq 作为支持 IND 的生化全基因组活性测定，以及 2) 优化和鉴定 gRNA 测序作为 IND 启用分析来评估基因组编辑成分的身份和纯度，以及 3) 合作 在治疗环境中测试 CHANGE-seq 和 gRNA 测序。我们预计，满足以下需求 特征明确的测定，可识别关键试剂中的杂质或表征关键试剂的关键质量属性 基因组编辑药物产品将对加速新颖、安全、有效的药物转化产生积极影响 治疗性基因组编辑治疗策略到首次人体临床试验。