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

Dettmer-Monaco V, Weißert K, Ammann S, Monaco G, Lei L, Gräßel L, Rhiel M, Rositzka J, Kaufmann MM, Geiger K, Andrieux G, Lao J, Thoulass G, Schell C, Boerries M, Illert AL, Cornu TI, Ehl S, Aichele P, Cathomen T
Source: J Allergy Clin Immunol
Publication Date: (2023)
Issue: S0091: 6749(23)00989-2
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Basic Research
Molecular Biology
Cells used in publication:
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
CD8+, human
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

Ribonucleoproteins (RNPs) were preassembled at a 1:3 molar ratio by incubating 18.3 pmol (3 mg) of Cas9 protein (IDT, Coralville, Iowa) with 55 pmol of gRNA (Biolegio, Nijmegen, The Netherlands) (Table E1 in this article’s Online Repository at for 10 minutes at room temperature. Nucleofections were performed with RNPs or 1 mg of PureBoost EGFPmRNA (Cellerna, Baesweiler, Germany) using a 4D-Nucleofector (Lonza, Basel, Switzerland).

Functional T-cell assays
For in vivo evaluation, CD81 T cells were purified from splenocytes (StemCell Technologies, 19853, Vancouver, Canada), activated for 3 days with beads coated with CD3e and CD28 antibodies (Miltenyi Biotec, 130-093-627, Bergisch Gladbach, Germany) in RPMI 1640 medium (Gibco, Life Technologies, Waltham, Mass) supplemented with 10% FCS (PAA, BioPath Stores, Cambridge, UK), Pen/Strep (Sigma-Aldrich, St Louis, Mo), and murine IL-2 (10 U/mL) (Peprotech, Hamburg, Germany) before nucleofection (in P4 with CM-137).

 Transplantation of gene-edited HSCs and LCMV challenge
Lineage-negative HSCs (Miltenyi Biotec, 130-090-858. Bergisch Gladbach, Germany) from femur and tibia-derived bone marrow (BM) were stimulated for 3 hours in StemSpan SFEM II medium (StemCell Technologies, Vancouver, Canada) supplemented with L-glutamine (20 mM) (Gibco, Life Technologies), mSCF, mTPO, and hIGF2 (20 ng/mL each) plus hIGF2 and mIL-3 (10 ng/mL each) (Peprotech. Hamburg, Germany) before nucleofection (in P4 with CA-137).

Jinx reporter cell line
K562 cells (ATCC) were used to create a Jinx reporter cell line using a targeted integration strategy. E1 Briefly, the Unc13d intron 26 sequence present in Jinx mice was inserted into position 146 of plasmid pEGFP-C1 (Clontech) via Gibson assembly E2 to generate pEGFP-intron26-Unc13d that harbors homologous sequences to the AAVS1 region. Integration into AAVS1 was achieved by CRISPR-mediated knock-in (4D-Nucleofector, Lonza, SF FF-120).


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.