Autologous Non-viral CRISPR/Cas Homology-directed Repair for Artemis-deficient Severe Combined Immunodeficiency

PROJECT SUMMARY / ABSTRACT This application for the Mentored Clinical Scientist Research Career Development Award (K08), sponsored by the National Institute of Allergy and Infectious Diseases, describes the five-year career development plan of Dr. Matthew Kan, a pediatric immunologist and early career physician-scientist in the Division of Pediatric Allergy, Immunology, and Bone Marrow Transplant at University of California, San Francisco. Dr. Kan's long-term career goal is to develop genome editing therapies for patients with inborn errors of immunity (IEIs), and in this proposal he specifically focuses on developing a preclinical therapy for Artemis- deficient severe combined immunodeficiency (ART-SCID). The career development goals outlined in this application include developing expertise in non-viral genome editing of human hematopoietic stem cells, preclinical modeling and genetic repair of ART-SCID, in vivo delivery of genome editors, and developing potency assays for genome-edited cell products. The primary mentor for accomplishing these career training goals is Dr. Jennifer Doudna, Li Ka Shing Chancellor's Chair in Biomedical Sciences at UC Berkeley, who is a co-discoverer of CRISPR genome editing. Dr. Jennifer Puck, Professor of Pediatrics, will be co-mentor, and she is a renowned clinical immunologist who is an expert in ART- SCID and has advanced multiple lentiviral gene therapies through clinical trials. The career development plan for Dr. Kan includes individualized mentorship, a career development team that includes other leaders in the field of genome editing and immune diseases, formal coursework, and a research program that builds upon Dr. Kan's strong experience in basic and clinical immunology with training in biochemistry, RNA expression, xenograft mouse models, and lipid delivery of genome editors. The overall objective of the research plan is to develop a non-viral platform technology for genome editing of hematopoietic stem cells that comprehensively addresses all pathogenic mutations in each gene. As over 1/3 of patients with ART-SCID have megabase deletions in DCLRE1C, the affected gene, the native locus cannot be used for “universal” gene repair. The central hypothesis is that targeted integration of a DCLRE1C transgene in the AAVS1 safe harbor locus will restore Artemis in ART-SCID, and the safe harbor will serve as a platform for other IEIs. The specific aims are to 1) develop non-viral techniques of targeted integration to restore Artemis expression in human hematopoietic stem and progenitor cells, initially ex vivo and then in vivo, and 2) develop a novel assay for Artemis activity for both patient diagnosis and potency of gene-edited cell products. This application is relevant to the NIH and the NIAID because Dr. Kan's goal is to develop methods for genome editing that will improve outcomes for a devastating inherited immune disease and the technologies developed will translate to other inborn errors of immunity.