Editing to Create and Correct Gene Variants

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Mutations in over 350 genes have been implicated as drivers of primary immunodeficiency (PID), but the genes that are mutated to cause many of these rare, but clinically serious conditions remain unknown, even despite whole exome sequencing. The use of whole genome sequencing promises to reveal coding and non-coding mutations for cases of T lymphocyte deficiency that cannot be solved by whole exome sequencing. However, confidently distinguishing pathogenic PID mutations from the exceedingly large number of benign variants across the entire genome is daunting, due to the rare incidence of each PID, incomplete knowledge of the genes required for T cell development, and our lack sequence-based rules to predict which non-coding variants may be pathogenic. CRISPR-Cas9 genome editing combined with our in vitro T cell differentiation platform offers unprecedented opportunities to test directly how human genetic sequences control immune cell development from hematopoietic stem progenitor cells (HSPCs) and ultimately to arrive at new therapies consisting of rewriting mutations that cause human immune diseases in patient blood-forming cells. Progress in pinpointing each patient’s causal mutation will open the next frontier: precise non-viral correction of endogenous disease-causing mutations for autologous gene therapy in HSPCs, avoiding the necessity to use imperfectly matched allogeneic donor transplants, for which graft rejection and graft vs. host disease are potentially devastating complications. This project will develop high-efficiency, high-throughput CRISPR-based technologies for identification of essential genes T for cell development, rapid functional testing of candidate mutations, and therapeutic genetic correction of a patient’s own HSPCs. We have developed CRISPR-Cas9 as a molecular scalpel to edit specific genome sequences in primary human cells and recently improved this technology for therapeutically-relevant editing in HPSCs. We will further apply CRISPR-based technologies for high-throughput mapping of coding and non-coding mutations in genes related to SCID and other forms of T-cell deficient PID, and we will develop new technologies for therapeutic gene editing in primary human HSPCs. Thus this project’s three central aims address fundamental challenges to achieving cures for PID through gene editing: 1) Discovery of all gene perturbations that could result in T cell deficiency, 2) Rapid identification of causal mutations for PID cases with unsolved genetic basis, and 3) Improvement in technology to introduce efficient and specific gene corrections into primary HPSCs as a forerunner to personalized autologous gene correction to restore immune function.