Editing to Create and Correct Gene Variants

编辑以创建和纠正基因变异

基本信息

批准号：
10256630
负责人：
Alexander Marson
金额：
$ 41.98万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-08 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10256630
关键词：
Address Allogenic Autologous Autologous Transplantation B cell differentiation Base Sequence Benign Blood CD34 gene CRISPR interference CRISPR screen CRISPR/Cas technology Candidate Disease Gene Cataloging Cells Clinical Clinical Medicine Clustered Regularly Interspaced Short Palindromic Repeats Coculture Techniques Code DNA DNA sequencing Defect Deficiency Diseases Development Diagnosis Donor person Elements Essential Genes Etiology Exons Family Fluorescence-Activated Cell Sorting Generations Genes Genetic Genome Genome engineering Genomics Graft Rejection Guide RNA Hematopoietic stem cells Hot Spot Human Human Genetics IL2RG gene Immune Immune System Diseases Immunologic Deficiency Syndromes Immunology Impairment In Vitro Incidence Inherited Knock-out Knowledge Laboratories Lead Libraries Link Lymphoid Maps Methods Molecular Mutate Mutation Nucleotides Pathogenicity Patients Positioning Attribute Regulatory Element Ribonucleoproteins Site T cell differentiation T-Cell Development T-Lymphocyte Technology Testing Therapeutic Translating Untranslated RNA Variant causal variant congenital immunodeficiency course development deep sequencing design disease-causing mutation exome sequencing follow-up frontier gene correction gene discovery gene therapy genetic disorder diagnosis genetic variant genome editing genome sequencing graft vs host disease high throughput technology human cord blood CD34+ cell human pluripotent stem cell immune function improved in vivo multidisciplinary new technology novel novel therapeutics nuclease scalpel therapeutic gene therapeutic genome editing whole genome

项目摘要

Mutations in over 350 genes have been implicated as drivers of primary immunodeficiency (PID), but the genes that are mutated to cause many of these rare, but clinically serious conditions remain unknown, even despite whole exome sequencing. The use of whole genome sequencing promises to reveal coding and non-coding mutations for cases of T lymphocyte deficiency that cannot be solved by whole exome sequencing. However, confidently distinguishing pathogenic PID mutations from the exceedingly large number of benign variants across the entire genome is daunting, due to the rare incidence of each PID, incomplete knowledge of the genes required for T cell development, and our lack sequence-based rules to predict which non-coding variants may be pathogenic. CRISPR-Cas9 genome editing combined with our in vitro T cell differentiation platform offers unprecedented opportunities to test directly how human genetic sequences control immune cell development from hematopoietic stem progenitor cells (HSPCs) and ultimately to arrive at new therapies consisting of rewriting mutations that cause human immune diseases in patient blood-forming cells. Progress in pinpointing each patient’s causal mutation will open the next frontier: precise non-viral correction of endogenous disease-causing mutations for autologous gene therapy in HSPCs, avoiding the necessity to use imperfectly matched allogeneic donor transplants, for which graft rejection and graft vs. host disease are potentially devastating complications. This project will develop high-efficiency, high-throughput CRISPR-based technologies for identification of essential genes T for cell development, rapid functional testing of candidate mutations, and therapeutic genetic correction of a patient’s own HSPCs. We have developed CRISPR-Cas9 as a molecular scalpel to edit specific genome sequences in primary human cells and recently improved this technology for therapeutically-relevant editing in HPSCs. We will further apply CRISPR-based technologies for high-throughput mapping of coding and non-coding mutations in genes related to SCID and other forms of T-cell deficient PID, and we will develop new technologies for therapeutic gene editing in primary human HSPCs. Thus this project’s three central aims address fundamental challenges to achieving cures for PID through gene editing: 1) Discovery of all gene perturbations that could result in T cell deficiency, 2) Rapid identification of causal mutations for PID cases with unsolved genetic basis, and 3) Improvement in technology to introduce efficient and specific gene corrections into primary HPSCs as a forerunner to personalized autologous gene correction to restore immune function.

超过 350 个基因的突变被认为是原发性免疫缺陷 (PID) 的驱动因素，但这些基因突变导致许多这些罕见但临床严重的疾病仍然未知，尽管全外显子组测序的使用有望揭示编码和非编码。然而，无法通过全外显子组测序解决 T 淋巴细胞缺陷病例的突变。自信地将致病性 PID 突变与大量良性变异区分开来整个基因组令人望而生畏，由于每种 PID 的发病率都很罕见，而且对基因的了解不完整 T 细胞发育所需的，并且我们缺乏基于序列的规则来预测哪些非编码变体可能 CRISPR-Cas9 基因组编辑与我们的体外 T 细胞分化平台相结合。直接测试人类基因序列如何控制免疫细胞发育的前所未有的机会从造血干祖细胞（HSPC）中提取，并最终获得包括重写在内的新疗法在患者造血细胞中引起人类免疫疾病的突变在精确定位每种突变方面取得了进展。患者的因果突变将开辟下一个前沿：内源性疾病的精确非病毒校正 HSPC 中自体基因治疗的突变，避免使用不完全匹配的同种异体的必要性供体移植，移植物排斥和移植物抗宿主病是潜在的破坏性并发症。该项目将开发基于 CRISPR 的高效、高通量技术，用于鉴定细胞发育的必需基因 T、候选突变的快速功能测试以及治疗性遗传我们开发了 CRISPR-Cas9 作为分子手术刀来编辑特定的 HSPC。原代人类细胞中的基因组序列，最近改进了该技术以用于治疗相关我们将进一步应用基于 CRISPR 的技术进行编码和高通量作图。与 SCID 和其他形式的 T 细胞缺陷 PID 相关的基因中的非编码突变，我们将开发新的因此，该项目的三个中心目标是在原代人类 HSPC 中进行治疗性基因编辑。通过基因编辑解决 PID 治疗的基本挑战：1) 发现所有基因可能导致 T 细胞缺陷的干扰，2) 快速识别 PID 病例的因果突变未解决的遗传基础，以及3）改进技术以引入有效和特定的基因校正进入原代 HPSC，作为个性化自体基因校正的先驱，以恢复免疫功能。