Gene Editing of Hematopoietic Stem Cells Restores T Cell Response in a Familial Hemophagocytic Lymphohistiocytosis Model

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene, which result in impaired processing of cytotoxic vesicles and hence compromised T and NK cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show 30-40% mortality. As a first step to meet this medical need, we developed and tested a curative genome editing strategy in the FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site (cSD) in Unc13d intron 26 and develop the clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). Here, we employed CRISPR-Cas technology to delete the disease-underlying mutation in HSCs, and transplanted the Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Genotyping, phenotyping and CAST-Seq based off-target analyses of cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wildtype cells were comparable. LCMV challenge of the transplanted mice resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, while Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. In sum, our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T cell response in the absence of genotoxicity-associated clonal outgrowth or leukemogenesis. Overall design: Mouse bone marrow HSPCs were treated with two RNP complexes (1:3 molar ratio of Cas9:sgRNA). Genotoxicity was assesed via CAST-Seq.