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

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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.