Gene editing of hematopoietic stem cells restores T-cell response in familial hemophagocytic lymphohistiocytosis

Background: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life -threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T -cell- and natural killer -cell -mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. Objective: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). Methods: We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease -causing mutation in HSCs and transplanted Unc13d- edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker -mediated PCR sequencing (CAST-Seq)-based off -target analyses. Cure from HLH predisposition was assessed by LCMV infection. Results: Hematopoietic 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. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock -edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. Conclusions: 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. (J Allergy Clin Immunol 2024;153:243-55.)