A RUNX1-FPDMM rhesus macaque model reproduces the human phenotype and predicts challenges to curative gene therapies

Abstract

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.

Runx1-R188Q germ line mutation induces inflammation and predisposition to hematologic malignancies in mice

“…The functional analysis in transplantation assays demonstrated that Runx1 R188Q/+ HSPCs have higher LT-RC than WT counterparts. Accordingly, a recent study reported the engraftment of RUNX1 -edited HSPCs in rhesus macaques, 49 underscoring the role of Runx1 mutations in preleukemic expansion and highlighting the concerns on the use of potential gene-editing therapies in premalignant FPDHM HSCs. In addition, this analysis reveals that Runx1 R188Q/+ HSPCs have selective expansion in both genetic backgrounds, albeit the differences seen in HSPC compartment, indicating that nonhematopoietic cells have negligible impact on the long-term expansion of Runx1-mutant HSPCs.…”

Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

“…Notwithstanding, the integration of the transgene was stable for at least 45 Tracking studies of corrected HSPCs in Wiscott-Aldrich syndrome or βhemoglobinopathy patients have estimated that as few as 98 corrected HSPCs per 10 6 infused CD34 + cells are able to engraft the bone marrow for long term-production of granulocytes 48 . Given that our experiments were designed using relatively small cell numbers (2.5x10 5 to 1x10 6 CD34 + HSPCs per single nucleofection targeting one candidate GSH site), the results suggest that the scaling-up to clinical preparation numbers (usually >5X10 6 electroporated cells) would increase the bioavailability of therapeutic cells 44,51 . Alternatively, if low cell numbers are involved, the rAAV6 vectors reportedly substantially increase editing efficiencies.…”

“…A recent study on gene therapy-patients suffering Wiscott-Aldrich syndrome or βhemoglobinopathies estimated that as few as 98 corrected HSPCs per 10 6 infused CD34+ cells were able to engraft the bone marrow for long term-production of granulocytes according to the tracking of unique vector insertion sites (IS) 47 . Given that our experiments were designed on a small scale of cell numbers (2.5x10 5 to 1x10 6 CD34+ HSPCs per single nucleofection targeting one candidate GSH site), the results suggest that the scaling-up to clinical preparation numbers (usually >5X10 6 electroporated cells) would increase the bioavailability of therapeutic cells 43,49 .…”