P34
Base editing of hematopoietic stem cells restores immune function in a mouse model of familial hemophagocytic lymphohistiocytosis
L Lei(1,2) J Lao(2,3) M M Kaufmann(1,2) G Thoulass(2,3) S Ammann(2,3) H Xiao(10) M O Rhiel(1,2) V D Monaco(1,2) J Grünewald(6,7) G Andrieux(5,8) L Gräßel(4,5) J Alzubi(1,2) B R Miller(6,7) C Schell(9) M Boerries(5,8) A L Illert(4,5) J K Joung(6,7) T I Cornu(1,2) S Ehl(2,3) M Erlacher(10) P Aichele(2,3) T Cathomen(1,2)
1:Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg; 2:Center for Chronic Immunodeficiency, Medical Center – University of Freiburg; 3:Institute for Immunodeficiency, Medical Center – University of Freiburg; 4:Department of Internal Medicine I, Medical Center - University of Freiburg; 5:German Cancer Consortium (DKTK), Partner Site Freiburg & German Cancer Research Center (DKFZ), Heidelberg; 6:Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA; 7:Department of Pathology, Harvard Medical School, Boston, MA, USA; 8:Institute of Medical Bioinformatics and Systems Medicine, Medical Center – University of Freiburg; 9:Institute of Surgical Pathology, Medical Center – University of Freiburg; 10:Department of Pediatric Hematology and Oncology, Medical Center – University of Freiburg
Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is an immunohematologic disorder caused by hyperactivated T cells and macrophages. Uncontrolled immune activation results from impaired lysis of antigen-presenting cells by T cells and NK cells, and is caused by mutations in the UNC13D locus. Although allogeneic hematopoietic stem cell (HSC) transplantation is curative, the high mortality in FHL patients urges the exploration of novel treatment options. Here, we developed a cytosine base editing (CBE) strategy to disrupt the disease-causing cryptic splice site in Unc13d intron 26 of Jinx mice, a preclinical model of FHL3. Electroporation of CBE-encoding mRNA and guide RNA into Jinx T cells and HSCs resulted in 61-71% of edited Unc13d alleles. Genetic and functional assays confirmed correct splicing of the Unc13d mRNA and restored cytolytic activity of the edited T cells. Furthermore, transplantation of Unc13d-edited HSCs into Jinx mice demonstrated functional restoration of lymphocyte cytotoxicity and protection against FHL. Unexpectedly, CAST-Seq and rhAmpSeq analyses revealed frequent CBE-induced chromosomal translocations and insertion/deletion mutations at on- and off-target sites. Off-target patterns and persistence of chromosomal translocations were however different in T cells and HSCs, proposing cell type-specific effects. Despite the high off-target activity, secondary transplantation of CBE-modified HSCs into 12 mice did not result in graft-related malignant transformation. In conclusion, our data demonstrate successful base editing to reverse the clinical phenotype in a preclinical FHL3 model, but also reveal cell type-specific off-target effects, highlighting that cell type-specific safety studies are vital to properly assess the risk-benefit ratio of these novel technologies.