Byung-Chul Lee ¹, Yifan Zhou ², Erica Bresciani ³, Neval Ozkaya ⁴, Alina Dulau Florea ⁵, Blake Carrington ³, Tae-Hoon Shin ¹, Valentina Baena ⁶, Zulfeqhar A Syed ⁶, So Gun Hong 7, Tao Zhen ⁸, Katherine R Calvo ⁹, Paul P Liu ¹⁰, Cynthia E Dunbar ²

¹Translational Stem Cell Biology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States.
²National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States.
³NHGRI, Bethesda, Maryland, United States.
⁴National Cancer Institute, Bethesda, Maryland, United States.
⁵NIH Clinical Center, Bethesda, Maryland, United States.
⁶Electron Microscopy Core, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States.
⁷NHLBI/NIH, Bethesda, Maryland, United States.
⁸NHGRI/NIH, Bethesda, Maryland, United States.
⁹National Institutes of Health Clinical Center, Bethesda, Maryland, United States.
¹⁰NHGRI, NIH, Bethesda, Maryland, United States.

Corresponding Author: Cynthia E Dunbar

Germline loss-of-function heterozygous mutations in the RUNX1 gene cause Familial Platelet Disorder with associated Myeloid Malignancies (FPDMM), characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant HSPCs in vivo long-term. We generated a rhesus macaque (RM) FPDMM competitive repopulation model using CRISPR/Cas9 NHEJ editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous hematopoietic stem and progenitor cells (HSPCs) and tracked mutation allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared to AAVS1-edited cells. Platelet counts remained below the normal range long-term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long-term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse MDS/AML predisposition and thrombopoietic defects.