Project 3: CRISPR Genome Editing to Understand and Correct STAT3 GOF Immune Dysregulation

Project Summary/Abstract Signal transducer and activator of transcription 3 (STAT3) gain of function (GOF) germline mutations are a monogenic cause of a syndrome of early-onset multiorgan autoimmune and lymphoproliferative disease for which the only current definitive cure is an allogeneic hematopoietic stem cell transplant. Despite the clear

genetic understanding of this syndrome, mechanistic cellular and molecular understanding of this syndrome remains obscure, particularly with regards to the STAT3 GOF effects on T cells, the cell type thought to be responsible for much of the autoimmunity seen in STAT3 GOF patients. This proposal is part of a Program

Project Grant that seeks to gain mechanistic insight into the immunological etiology of this disease incorporating human patient samples, mechanistic mouse studies, T cell culture experiments, and functional genomics approaches. This specific Project within the Program Grant seeks to leverage functional genomics approaches

powered by CRISPR-Cas9 technology to understand how STAT3 GOF variants affect T cell function. CRISPR- Cas9 genome engineering technology is driving a revolution in modern biology. Scientists now possess unprecedented capabilities to test the cellular functions of human genetic sequences and correct mutations that

cause disease directly in primary mouse and human cells. Of relevance to this Project proposal, our lab has pioneered methods and systems to conduct targeted and genome-wide knockout screens in primary T cells from mice and humans. Further, we have developed an efficient method for orthogonal validations using Cas9:single-

guide RNA ribonucleoprotein (Cas9 RNP) electroporation coupled with multiplexed flow cytometric phenotyping. These advances now position us to use functional genomics approaches in mouse and human T cells to reveal novel pathways that control abnormal T cell function in STAT3 GOF syndrome, prioritize novel targets in further

studies for drug development, and improve the design of genetically reprogrammed cell-based therapies. Specifically, Project 3 seeks to implement unbiased CRISPR functional genetic approaches in mouse T cells modeling STAT3 GOF mutations to identify critical gene targets that modulate STAT3-mediated inflammation

(Aim 1a, 1b). Further, we seek to leverage the information from these screens to improve function in human patient T cells (Aim 1c). Finally, Project 3 seeks to use cutting-edge non-viral CRISPR based methods to correct STAT3 GOF mutations in actual patient T cells, as well as to develop a preclinical approach to replace selected

STAT3 exons in primary human hematopoietic stem progenitor cells (HSPCs) (Aim 2). Taken together, these studies will form the basis for a framework to understand how STAT3 GOF variants alter T cell function to drive inflammatory disease as well as open multiple avenues towards immune cell CRISPR gene targeting to treat

autoimmunity.