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

项目总结 基因组编辑具有成为治疗性、基因组药物的非凡潜力，但需要特殊的 仔细检查，因为意外的“非靶标”突变可能是永久性的，并影响患者的长期安全。的 人们普遍关注的是，非靶点突变可能激活原癌基因，类似于整合dna的病毒载体。 用于基因治疗，这已经导致了几名患者的白血病。因为越来越多的有希望的 治疗性基因组编辑策略，仍然迫切需要优化和合格的细胞分析 为了：1)定义靶标的全基因组活动，以便为Lead靶标识别提供信息；2)从功能上 描述编辑在研究新药(IND)使能过程中的全基因组活动 化学、制造和控制(CMC)活动。据我们所知，最常用的方法是 为IND应用程序定义细胞基因组范围的编辑活动是指南-SEQ，一个敏感和公正的 我们先前开发的定义基因组编辑核酸酶的全细胞基因组活性的方法。AS 作为我们的体细胞基因组编辑(SCGE)第一阶段项目的一部分，我们开发了新版本GUIDE-SEQ-2 具有改进的可扩展性和准确性的GUIDE-SEQ。该提案的目标是优化、提炼和 将GUIDE-SEQ-2鉴定为一种适用于评估治疗性基因组编辑或相关基因的方法 监管意见书中的基因组编辑细胞药物产品。我们提出了以下具体目标：1) 优化和鉴定GUIDE-SEQ-2以评估IND应用程序的细胞全基因组活性和2)应用程序 GUIDE-SEQ-2在新的、与治疗相关的背景下。总而言之，我们提议的预期结果 研究将是一种优化和合格的指南-seq-2检测，可以直接批准以建立关键的 IND提交的人类基因组编辑产品的质量属性。我们设想优化后的指南- 我们开发的SEQ-2方案和检测鉴定路线图将迅速得到广泛采用，并已 对安全有效的基因药物的开发和翻译产生广泛的积极影响。

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.