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| Status |
Public on Jul 04, 2023 |
| Title |
Gene Editing of Hematopoietic Stem Cells Restores T Cell Response in a Familial Hemophagocytic Lymphohistiocytosis Model |
| Organism |
Mus musculus |
| Experiment type |
Other
|
| Summary |
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.
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| 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.
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| Contributor(s) |
Dettmer-Monaco V, Weißert K, Andrieux G, Rhiel M, Börries M, Aichele P, Cathomen T |
| Citation(s) |
37595758 |
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| Submission date |
Feb 14, 2023 |
| Last update date |
Oct 04, 2023 |
| Contact name |
Geoffroy Andrieux |
| Organization name |
University clinics Freiburg
|
| Street address |
Breisacherstr 153
|
| City |
Freiburg |
| ZIP/Postal code |
79110 |
| Country |
Germany |
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| Platforms (1) |
| GPL21103 |
Illumina HiSeq 4000 (Mus musculus) |
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| Samples (6)
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| GSM7043018 |
Non-treated control CAST-seq sample for samples treated Cas9 nucleases [UNC-UT1] |
| GSM7043019 |
Non-treated control CAST-seq sample for samples treated Cas9 nucleases [UNC-UT2] |
| GSM7043020 |
CAST-seq on sample treated with two RNPs [UNC-u1d4-1] |
| GSM7043021 |
CAST-seq on sample treated with two RNPs [UNC-u1d4-2] |
| GSM7043022 |
CAST-seq on sample treated with two RNPs [UNC-u3d4-1] |
| GSM7043023 |
CAST-seq on sample treated with two RNPs [UNC-u3d4-2] |
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| This SubSeries is part of SuperSeries: |
| GSE225541 |
Gene Editing of Hematopoietic Stem Cells Restores T Cell Response in a Familial Hemophagocytic Lymphohistiocytosis Model |
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| Relations |
| BioProject |
PRJNA934866 |
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