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

项目总结 基因组编辑，一种修改活细胞基因组的技术，具有非凡的潜力 安全有效的基因药物，直接治疗遗传病的根本原因，如 镰状细胞病和许多其他疾病。然而，作为具有新作用机制的基因治疗产品，有 仍然需要优化和合格的生化检测方法来评估其安全性。我们和 其他人已经开发出敏感和公正的研究方法来定义编辑的全基因组活动 如CHANGE-SEQ和GUIDE-SEQ。然而，它们需要进一步的优化和表征，以适应- 目的分析，以满足研究新药(IND)提交的严格法规要求。 令人惊讶的是，据我们所知，还没有发表高通量测序表征的方法 GRNA的同一性和纯度。因此，仍然存在对公开可用、优化和 合格的IND-Enabling分析来表征关键的基因组编辑试剂及其相关的开关- 以全基因组活动为目标。因此，我们提出了以下具体目标：1)优化和合格 Change-Seq作为IND使能的生化全基因组活性分析，以及2)优化和鉴定gRNA 测序作为IND支持的分析，以评估基因组编辑组件的身份和纯度，以及3)合作 在治疗环境中测试变化序列和gRNA测序。我们预计，满足以下需求 识别关键试剂中的杂质或表征关键质量属性的特征良好的分析 基因组编辑药物产品将对加速新奇、安全、有效的翻译产生积极影响 治疗性基因组编辑治疗策略到首个人类临床试验。

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.