CRISPR/Cas9 Gene Editing of MIR155HG in Primary Human T Cells to Prevent Acute Graft-Versus-Host Disease

Abstract Introduction: Acute graft-versus-host disease (GVHD) mediated by donor alloreactive T cells is the leading cause of non-relapse mortality in patients post-allogeneic hematopoietic cell transplantation. Here we genetically engineered donor T cells by genomic deletion of microRNA-155 host gene (MIR155HG) to prevent GVHD. Methods: Using CRISPR/Cas9 genome editing, we targeted exon 3 (Ex3) of MIR155HG. Genomic deletion was evaluated by genomic PCR and RNA expression of mature miR-155 by qRT-PCR. In xenogeneic acute GVHD experiments, NSG mice were irradiated and transplanted with miR-155 Ex3 edited or control T cells to assess functionality in vivo. Histological analysis of target tissues (skin and liver) was performed at end of study. Weekly flow cytometric evaluation of human donor T cells in peripheral blood was performed. MOLM-13 leukemic target cells were cultured with miR-155 Ex3 edited or control CD8 +effector T cells for 72 hours and cytotoxic function was assessed by CD107a degranulation, intracellular IFN-γ production and leukemic cell death by flow cytometry. Results: Ex3 deletion was confirmed by genomic PCR resulting in downregulation of mature miR-155 expression. Recipients of Ex3 deleted human donor T cells display lower GVHD clinical scores, lower average histopathological scores and significantly improved survival compared to mice receiving control T cells. Ex3 T cells displayed potent CTL function comparable to control T cells resulting in leukemic cell death. Conclusion: CRISPR/Cas9 deletion of miR-155 in donor T cells protects against development of lethal GVHD while maintaining beneficial anti-leukemic effect. Our studies present genomic editing of donor T cells as a novel strategy to prevent GVHD. Funding for this project was provided through The Ohio State University Leukemia Research Program to PR and RG, NIH R01CA252469 to PR, and NIH T32CA090223 fellowship to KB.