Assembly of Novel Gene Editing Particles to Understand Genome Surgery in Patient-Derived Cells

组装新型基因编辑颗粒以了解患者来源细胞中的基因组手术

• 批准号: 9335383
• 负责人: Krishanu Saha
• 金额: $ 36.98万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-19 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335383
• 关键词: Autologous; Biological Process; CRISPR/Cas technology; Cell Therapy; Cells; Custom; DNA Double Strand Break; DNA Repair; Data; Disease; Engineered Gene; Foundations; Future; Gene-Modified; Generations; Genes; Genome; Genomics; Goals; Human; Image Analysis; In Situ; In Vitro; Knowledge; Methods; Mission; Monitor; Mutation; Nonhomologous DNA End Joining; Operative Surgical Procedures; Pathway interactions; Patients; Pharmaceutical Preparations; Prevention; Production; Public Health; Reporter; Research; Stem cells; Techniques; Testing; Therapeutic; Tissues; Transcript; United States National Institutes of Health; Variant; disease diagnosis; gene correction; gene therapy; induced pluripotent stem cell; innovation; novel; nuclease; particle; pre-clinical; precision medicine; programs; public health relevance; repaired; small molecule; stem cell fate; time use; tool; trafficking

There is a fundamental gap in understanding how several components of engineered gene-editing nucleases achieve gene modification in human cells. Continued existence of this gap represents an important problem because, until it is filled, use of genome surgery tools will be limited, as it is not clear why various nucleases fail and why some succeed in producing desired gene edits. The long-term goal is to watch genome surgery in action to understand the bottlenecks in performing genome surgery on human cells in vitro with precisely controlled gene-editing particles, comprised of CRISPR-Cas9 components. Particles will be systematically assembled with various components and delivered in a controlled fashion to patient-derived cells and tissues. Live, in situ high content imaging and analysis within customized cell substrates will monitor genome surgery. These capabilities will explore large sequence variation of CRISPR-Cas9 components along with new assemblies of CRISPR-Cas9 components. The central hypothesis is that new assemblies of CRISPR-Cas9 particles can probe different biological processes of trafficking, DNA-double strand break formation and DNA repair involved in genome surgery. This hypothesis will be tested with respect to generating two types of gene edits involving non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways at several genomic loci within patient-derived stem cells and tissues. An overarching rationale for the proposed research programs is that robust gene editing techniques could enable the production of personalized drugs, cell therapies and gene therapies for future genomic and precision medicine. Guided by strong preliminary data, this hypothesis will be tested by pursuing three research programs: 1) Assemble Cas9 particles to identify biological processes that promote "reporter-less" transcript tagging of stem cell fate in culture; 2) Assemble Cas9 particles to identify biological processes that promote gene correction of diseased mutations in stem cells; and, 3) Assemble Cas9 particles to identify biological processes that promote gene correction of diseased mutations in microtissues. Under the first research program, an already proven platform, to assemble hundreds of unique Cas9 particles and edit patient-derived cells in a multiplexed manner, will be used to monitor the production of small gene edits by NHEJ within stem cell marker genes. Under the second and third research programs, this platform will be applied to gene-correct diseased mutations via HDR in induced pluripotent stem cells and microtissues matured from them. The approach is innovative, in the applicant's opinion, because it departs from the status quo by systematically changing multiple components at a time using novel methods in patient-derived cells. The proposed research is significant, because it is expected to advance and expand understanding of how genome surgery tools can be applied for the generation of advanced therapeutics, ranging from targeted small molecules to autologous cell therapies. Ultimately, such knowledge has the potential to set the foundation for new preclinical platforms in Precision Medicine.

了解工程基因编辑核酸酶的几个组成部分是有根本的差距 实现人类细胞中的基因修饰。持续存在此差距是一个重要的问题 因为，在填充之前，基因组手术工具的使用将受到限制，因为尚不清楚为什么各种核酸酶失败 以及为什么有些人成功地产生了所需的基因编辑。长期目标是观看基因组手术 采取行动了解在体外对人类细胞进行基因组手术的瓶颈，并精确地 受控基因编辑颗粒，由CRISPR-CAS9组件组成。粒子将系统地 与各种组件组装在一起，并以受控的方式传递到患者衍生的细胞和组织。 定制细胞底物中的现场，原位高含量成像和分析将监测基因组手术。 这些功能将探索CRISPR-CAS9组件的大序列变化以及新的 CRISPR-CAS9组件的组件。中心假设是CRISPR-CAS9的新组合 颗粒可以探测运输，DNA双链断裂和DNA的不同生物学过程 涉及基因组手术的维修。该假设将在生成两种类型的基因方面进行检验 在几个 患者衍生的干细胞和组织中的基因组基因局。拟议研究的总体理由 程序是强大的基因编辑技术可以使个性化药物的生产，细胞 用于未来基因组和精度医学的疗法和基因疗法。在强大的初步数据的指导下， 该假设将通过追求三个研究计划来检验：1）组装Cas9颗粒以识别 培养干细胞命运的“无报告”转录标记的生物学过程； 2）组装 Cas9颗粒鉴定促进茎中患病突变基因校正基因的生物过程 细胞； 3）组装Cas9颗粒以鉴定促进基因校正的生物学过程 在微作用中患病突变。根据第一个研究计划，一个已经经过验证的平台，可以组装 数百个独特的CAS9颗粒和以多重方式编辑患者衍生的细胞，将用于 监测NHEJ在干细胞标记基因内的小基因编辑的产生。第二和第三 研究计划，该平台将通过HDR应用于诱导 多能干细胞和微作用从中成熟。这种方法是创新的，在申请人的 意见，因为它一次通过系统地更改多个组件而脱离现状 在患者衍生的细胞中使用新方法。拟议的研究很重要，因为它有望 提高并扩大对如何应用基因组手术工具的了解 晚期治疗剂，从靶向小分子到自体细胞疗法。最终，这样的 知识有可能为精密医学的新临床前平台奠定基础。