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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

基本信息

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
项目类别：
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

项目摘要

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。在理解CRISPR-