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: 10206480
• 负责人: Krishanu Saha
• 金额: $ 41.56万
• 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 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10206480
• 关键词: Award; Biological Process; CRISPR/Cas technology; Cell Cycle Arrest; Cell Proliferation; Cell Therapy; Cells; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Custom; DNA; DNA Double Strand Break; DNA Repair; Development; Disease; Foundations; Future; Gene-Modified; Generations; Genes; Genome; Genomic medicine; Human; Image; In Situ; In Vitro; Knowledge; Methods; Mission; Monitor; Morphogenesis; Mutation; Operative Surgical Procedures; Patients; Polymers; Prevention; Process; Production; Productivity; Public Health; RNA; Research; Techniques; Testing; Therapeutic; Time; Tissue Therapy; Tissues; United States National Institutes of Health; base; disease diagnosis; gene correction; gene therapy; genome editing; improved; induced pluripotent stem cell; innovation; novel; particle; precision drugs; precision medicine; programs; small molecule; stem cell differentiation; stem cells; time use; tool; trafficking

PROJECT SUMMARY/ ABSTRACT. There continues to be a fundamental gap in understanding how CRISPR- based genome editors produce gene modifications in different human cells. A lack of understanding of why various editors fail and why some succeed in creating desired gene edits - while retaining full cell and tissue functionality - limits the use of genome editing tools. By observing genome editing in real-time within patient- derived cells in vitro, I seek to understand the bottlenecks in performing genome editing on human cells with precisely-controlled genome editor particles. Particles will be systematically assembled with various DNA, RNA, and polymeric components and delivered to patient-derived cells and microtissues. In situ high content imaging and analysis within customized cell substrates will monitor genome editing at multiple scales. 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 the genome editing of human cells and tissues, as well as downstream effects on biological processes involving cell cycle arrest and morphogenesis. This hypothesis will be tested within patient-derived stem cells and tissues for both gene disruption and correction. An overarching rationale for the proposed research is that an improved understanding of fundamental biological processes involved with genome editing could enable the development of novel cell therapies and gene therapies for future genomic and precision medicine. Guided by strong productivity in the current early stage R35 award, I will pursue three research programs: 1) Assemble Cas9 particles to identify chromatin structures within human cells that promote gene correction; 2) Assemble Cas9 particles to identify delivery and DNA repair processes that promote gene correction within stem cells; and, 3) Assemble Cas9 particles to identify cell proliferative and tissue morphogenesis processes that promote gene correction of diseased mutations in patient-derived microtissues. Under the first research program, editing will occur at target genes that have variable chromatin structures within induced pluripotent stem cells (iPSCs), differentiated progeny, and with small-molecule treatment. Under the second and third research programs, genome editors will be applied to gene-correct diseased mutations in iPSCs, 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 our understanding of how genome editing tools can be applied for the generation of advanced therapeutics, ranging from targeted small molecules to cell/tissue therapies. Ultimately, such knowledge would solidify the foundation for new translational projects involving genome editing.

项目摘要/摘要。 基于基因组编辑器在不同的人类细胞中产生基因修饰。 各种编辑失败，有些人成功地创建了所需的基因编辑 - 同时保留完整的细胞和组织 通过在患者中实时观察基因组编辑来使用基因组编辑工具。 我试图在体外衍生细胞，以了解在人类细胞上进行基因组编辑的瓶颈 精确控制的基因组编辑器颗粒。 和聚合物成分，并传递到患者衍生的细胞和微观效果。 定制的细胞底物中的分析将在多个尺度上监测基因组编辑。 假设是CRISPR-CAS9颗粒的新组件可以探测不同的生物学过程 人类细胞基因组编辑涉及的运输，DNA双链断裂的形成和DNA修复 和组织，以及对涉及循环停滞和的生物过程的下游影响 形态发生。 破坏和纠正。 基因组编辑涉及的基本生物学过程可以使细胞的发展发展 对未来基因组动物和精确医学的疗法和基因疗法。 当前的早期R35奖，我将追求三个三个CAS9颗粒以识别 促进基因校正的人类细胞中的染色质结构； 促进干细胞内基因校正的递送和DNA修复过程； 鉴定细胞增殖和组织形态发生过程的颗粒促进基因校正的过程 患者衍生的微作用的突变，在第一个研究计划下进行编辑 具有变性干细胞（IPSC）内具有变化的Chrimatin结构的基因，分化 后代，并在第二和第三研究计划下进行小分子处理。 将应用于IPSC中的基因纠正型爆发，而微作用成熟。 在申请人的看来，具有创新性，因为它通过有系统的变化而偏离了现状 一次使用新方法的多个组件在患者衍生的细胞中很重要。 因为期望提高和扩展我们对如何应用基因组编辑工具的理解 为了产生晚期治疗剂，从靶向小分子到细胞/组织手术。 最终，这种知识将巩固涉及基因组编辑的项目项目项目项目。