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

Viviane Dettmer-Monaco, Kristoffer Weißert, Sandra Ammann, Gianni Monaco, Lei Lei, Linda Gräßel, Manuel Rhiel, Julia Rositzka, Masako M. Kaufmann, Kerstin Geiger, Geoffroy Andrieux, Jessica Lao, Gudrun Thoulass, Christoph Schell, Melanie Boerries, Anna L. Illert, Tatjana I. Cornu, Stephan Ehl, Peter Aichele, Toni Cathomen

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


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 wild-type 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.

Original languageEnglish
Pages (from-to)243-255.e14
JournalJournal of Allergy and Clinical Immunology
Issue number1
StatePublished - Jan 2024
Externally publishedYes


  • Autologous stem cell transplantation
  • CAST-Seq
  • CRISPR-Cas
  • T-cell repertoire
  • gene therapy
  • genome editing
  • genotoxicity
  • hemophagocytic lymphohistiocytosis
  • hyperinflammation


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