CRISPR Editing of Primary Human Cells to Model and Correct Primary Immunodeficiency Mutations

对原代人类细胞进行 CRISPR 编辑以建模和纠正原发性免疫缺陷突变

• 批准号: 10433980
• 负责人: David-Huy Nhu Nguyen
• 金额: $ 19.87万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10433980
• 关键词: Address; Affect; Alleles; Animal Model; Autologous; Basic Science; Benign; Biology; CRISPR/Cas technology; CTLA4 gene; Catalogs; Cell Death; Cell Line; Cell Therapy; Cell Transplantation; Cells; Cellular biology; Chemicals; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Complement; Complementary DNA; Counseling; DNA; DNA Modification Process; DNA Repair; DNA Structure; Databases; Development; Diagnosis; Diagnostic; Disease; Doctor of Philosophy; Dose; Drug Delivery Systems; Engineering; Environment; Faculty; Funding; Gene Expression; Genes; Genetic; Genetic Code; Genetic Diseases; Genetic Screening; Genome engineering; Genomics; Goals; Hematopoietic stem cells; Heterozygote; Homo; Human; Hybrids; IL2RA gene; IL2RG gene; Immune; Immune System Diseases; Immune response; Immune system; Immunologic Deficiency Syndromes; Immunology; Individual; Infection; Instruction; JAK3 gene; Knock-in; Knowledge; Lead; Learning; Libraries; Link; Maps; Medical; Mentors; Methods; Modeling; Molecular Diagnosis; Mutate; Mutation; Mutation Analysis; Outcome; Pathologic; Pathology; Patients; Physicians; Population; Prognosis; Proteins; Reporter; Reporter Genes; Research; Research Personnel; Research Training; Scientist; Severe Combined Immunodeficiency; Signal Transduction; Site; Surface; System; T-Lymphocyte; TNFSF5 gene; Techniques; Technology; Testing; Toxic effect; Training; Transformed Cell Line; Translational Research; Variant; Work; antagonist; base; career; career development; causal variant; cell bank; cell growth; cell type; clinical care; clinical sequencing; clinically significant; congenital immunodeficiency; cytotoxic; diagnostic platform; diagnostic tool; disease diagnosis; disease-causing mutation; exome sequencing; fighting; functional genomics; functional outcomes; gene correction; gene therapy; genetic variant; immune function; improved; innovation; large datasets; lectures; member; mutation correction; mutation screening; next generation sequencing; novel; novel diagnostics; novel strategies; novel therapeutics; nuclease; pre-clinical; preclinical development; prototype; receptor; response; skills; small molecule; therapeutic genome editing; tool; transplantation therapy

PROJECT SUMMARY / ABSTRACT The goal of this application is to train Dr. David Nguyen PhD MD, an infectious disease clinician and medical engineer, with the skills necessary to become an independently-funded investigator studying, diagnosing, and eventually curing genetic diseases of the immune system. Primary immunodeficiency (PID) disease is commonly recognized as an inability of the immune system to develop, self-regulate, or fight infection due to a genetic defect in some aspect of the normal immune response. Gene therapy is a possibility to cure PID disease once a patient's mutation can be identified, but there is incomplete knowledge of the genetic underpinnings of PID. PID-causative mutations often remain undiagnosed despite state-of-the-art genomic sequencing efforts because of an inability to separate benign sequence variations from the truly pathologic mutations. This research plan addresses key limitations in the ability to provide a molecular diagnosis and cure for PID patients. Dr. Nguyen will utilize recently developed advanced CRISPR-gene editing tools in innovative ways to study PID-associated mutations and understand how they lead to clinical disease. He will then develop novel strategies for replacing PID mutations in a patient's own hematopoietic stem progenitor cells (HSPCs). He aims to 1) accurately recreate and functionally test PID patient sequencing variants by replacing wild-type alleles in (otherwise healthy) primary human immune cells, 2) utilize high-throughput functional genomics to catalogue all possible mutations in key PID-associated genes that could lead to immune dysfunction, 3) improve site- specific gene correction in primary HSPCS. The proposed a 5-year career development and training plan incorporates didactic lecture-based learning, mentored practical training, and career advising to complement Dr. Nguyen's expertise in ways that are critical to completion of his research and career goals. He will receive instruction in advanced human T cell and HSPC biology, functional genomics methods in particular next-generation sequencing techniques and manipulation of large datasets, and pre-clinical development of cell and gene therapies. He will be training at UCSF, a center for both basic and translational research that provides an excellent environment supporting physician-scientists with local experts in all aspects the proposed research and training goals. He will be closely mentored by Dr. Alexander Marson, a leading expert in using CRISPR gene editing to understand the genetic basis of human immune function, and Dr. Jennifer Puck, a world-leader in the diagnostics, genetics, clinical care, and gene therapy of severe combined immunodeficiency. The long-term goal of this study is to provide Dr. Nguyen with the skills required to become a R01-funded faculty member leading efforts to identify, functionally validate, and correct immunodeficiency patient mutations.

项目摘要/摘要 此应用程序的目标是培训David Nguyen博士医学博士，他是一名传染病临床医生和 医学工程师，具备成为一名独立资助的研究人员所需的技能， 诊断并最终治愈免疫系统的遗传性疾病。原发免疫缺陷(PID) 疾病通常被认为是免疫系统不能发展、自我调节或抵抗感染。 由于正常免疫反应的某些方面存在遗传缺陷。基因疗法是治愈疾病的一种可能性 PID病一旦患者的突变就可以被识别出来，但对其基因的了解还不完全 PID的基础。尽管有最先进的基因组技术，但导致PID的突变通常仍未得到诊断 测序工作，因为无法将良性序列变异与真正病理的序列变异区分开来 突变。这项研究计划解决了提供分子诊断和治愈能力的主要限制 适用于PID患者。Nguyen博士将利用最近开发的先进CRISPR-基因编辑工具在创新 研究与PID相关的突变并了解它们如何导致临床疾病的方法。然后他就会发展成 替换患者自身造血干细胞(HSPC)中的PID突变的新策略。他 旨在1)通过替换野生型等位基因，准确地重建和功能测试PID患者的测序变体 在(其他健康的)原生人类免疫细胞中，2)利用高通量功能基因组学来编目 所有可能导致免疫功能障碍的PID相关关键基因突变，3)改善部位- 原发HSPC的特异性基因矫正。 拟议的五年职业发展和培训计划包括以讲授为基础的教学 学习、有指导的实践培训和职业建议，以补充阮博士的专业知识， 对完成他的研究和职业目标至关重要。他将接受先进的人类T细胞和 HSPC生物学、功能基因组学方法，特别是下一代测序技术和 大型数据集的操作，以及细胞和基因疗法的临床前开发。他将在 加州大学旧金山分校，基础研究和翻译研究中心，提供良好的环境支持 医生-科学家与当地各方面的专家提出了研究和培训目标。他会密切关注 由亚历山大·马森博士指导，他是使用CRISPR基因编辑来理解基因 人类免疫功能的基础，以及珍妮弗·帕克博士，诊断、遗传学、临床护理、 以及严重联合免疫缺陷的基因治疗。这项研究的长期目标是为Dr。 Nguyen拥有成为R01资助的教职员工所需的技能，领导在功能上识别 验证并纠正免疫缺陷患者的突变。