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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要此应用程序的目标是培训 David Nguyen 博士、医学博士、传染病临床医生和医学工程师，具有成为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。原发性免疫缺陷（PID）疾病通常被认为是免疫系统无法发展、自我调节或抵抗感染由于正常免疫反应某些方面的遗传缺陷。基因疗法有可能治愈一旦可以识别患者的突变，即可识别 PID 疾病，但对遗传的了解并不完整 PID 的基础尽管采用最先进的基因组技术，但 PID 致病突变常常仍未得到诊断由于无法将良性序列变异与真正的病理性序列变异区分开而进行的测序工作突变。该研究计划解决了提供分子诊断和治疗能力的关键限制对于 PID 患者。 Nguyen 博士将利用最近开发的先进 CRISPR 基因编辑工具进行创新研究 PID 相关突变并了解它们如何导致临床疾病的方法。然后他会发展替代患者自身造血干祖细胞（HSPC）中 PID 突变的新策略。他旨在 1) 通过替换野生型等位基因来准确地重新创建 PID 患者测序变异并进行功能测试在（其他健康的）原代人类免疫细胞中，2）利用高通量功能基因组学进行编目关键 PID 相关基因中所有可能的突变都可能导致免疫功能障碍，3) 改善位点初级 HSPCS 中的特异性基因校正。拟议的 5 年职业发展和培训计划包含基于教学的讲座学习、指导实践培训和职业建议，以以下方式补充 Nguyen 博士的专业知识：对于完成他的研究和职业目标至关重要。他将接受高级人类 T 细胞方面的指导 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。他将在训练加州大学旧金山分校 (UCSF) 是基础研究和转化研究中心，提供良好的环境支持医师科学家与当地专家在各方面提出了研究和培训目标。他将密切由 Alexander Marson 博士指导，他是使用 CRISPR 基因编辑来了解遗传的领先专家人类免疫功能的基础，以及诊断学、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博士提供帮助。 Nguyen 具备成为 R01 资助的教员所需的技能，领导着识别、功能性的工作验证并纠正免疫缺陷患者的突变。