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：
10040386
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/10040386
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 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 base career career development causal variant cell bank cell growth cell type clinical care clinical sequencing clinically significant congenital immunodeficiency cytotoxic 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 outcome forecast pre-clinical preclinical development prototype receptor response skills small molecule therapeutic genome editing tool

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.

项目摘要 /摘要该应用的目的是培训传染病临床医生David Nguyen Phd MD博士，医学工程师，具有成为独立资助的研究人员学习所需的技能，诊断，并最终治愈免疫系统的遗传疾病。原发性免疫缺陷（PID）疾病通常被认为是免疫系统无法发展，自我调节或抗击感染的一种由于遗传缺陷在正常免疫反应的某些方面。基因治疗是可以治愈的一旦可以确定患者的突变，PID病就有遗传的知识不完整 PID的基础。尽管最先进由于无法将良性序列变化与真正的病理分开，因此进行测序工作突变。该研究计划解决了提供分子诊断和治愈能力的关键局限性适用于PID患者。 Nguyen博士将利用最近开发的高级CRISPR-GENE编辑工具的创新工具研究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突变的新型策略。他目的是1）通过替换野生型等位基因来准确重新创建和在功能上测试PID患者测序变体在（否则健康的）原代人免疫细胞中，2）利用高通量功能基因组来编目可能导致免疫功能障碍的关键PID相关基因中的所有可能突变，3）改善位点 - 初级HSPC中的特定基因校正。提议的为期5年的职业发展和培训计划结合了教学讲座学习，指导实践培训和职业建议，以补充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。他将收到高级人类T细胞的指导， HSPC生物学，功能基因组学方法，特别是下一代测序技术和操纵大型数据集以及细胞和基因疗法的临床前发育。他将在 UCSF，基础研究和转化研究中心，提供了一个极好的环境支持各个方面的当地专家的医师科学家都提出了拟议的研究和培训目标。他会紧密由使用CRISPR基因编辑的主要专家Alexander Marson博士指导人类免疫功能的基础，以及诊断，遗传学，临床护理的世界领导者詹妮弗·帕克（Jennifer Puck）博士和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。 Nguyen具有成为R01资助的教职员工所需的技能的领导努力，以识别功能验证并纠正免疫缺陷患者突变。