P698: crispr/cas9 gene editing in hematopoietic stem cells to model clonal competition in vivo and in vitro for gata2 deficiency

Background: GATA2 deficiency has been identified as a common hereditary cause of myelodysplastic syndromes (MDS) and acute myeloid leukemia in children. Penetrance and expressivity within affected families is often variable, suggesting that cooperating factors are required to trigger the disease. Somatic mutations in MDS driver genes (i.e. SETBP1, ASXL1) have been identified GATA2-MDS. Despite recent studies identifying germline genetics and acquired mutations in GATA2 deficiency, the understanding of the molecular mechanism that triggers the leukemic progression remains still unknown. Aims: Study functional consequence of GATA2 haploinsufficiency and known somatic mutations (SETBP1 & ASXL1) in vivo. For this aim I will transplant engineered cord blood (CB) CD34+ cells harboring GATA2 mutation alone or in combination with SETBP1/ASXL1 mutations in NSG mice to evaluate the engraftment capacity and the clonal evolution. Methods: Here, we used CRISPR/Cas9 in primary CB CD34+ cells followed by serial transplantation into NSG mice to study the genetic malignant effect of GATA2 (R398W), SETBP1 (gain-of-function) and ASXL1 (loss-of-function) mutations. Specifically, CRISPR/Cas9/rAAV6 were used to knock-in a modified GATA2 exon 6 (exon6-T2A-GFP) carrying the R398W mutation in CB-CD34+ cells alone (referred to as GATA2) or in combination with mutations in SETBP1 and ASXL1 (TRIPLE). Mock-treated CD34+ cells were nucleofected without RNP, rAAV6 or ssODN (Control). 48h post nucleofection, 2x105 unsorted cells were IBMT into irradiated NSG mice. In vitro assays of proliferation, apoptosis, flow cytometry and CFU were used to validate the effect of GATA2 mutation. Results: FACS analysis revealed that, ~12% of cells were GFP+ in GATA2 condition and ~7% in TRIPLE condition, which directly correlates with the percentage of GATA2-R398W mutant cells. 16 weeks after primary transplantation, 19/30 mice showed more than 10% of human engraftment (hCD45) in the bone marrow (BM). Moreover, a multilineage constitution (CD34, CD33 and CD19) was observed in all the conditions. In order to dissect the clonal architecture of concurrent mutations, we performed single-cell DNA-sequencing for GATA2, SETBP1 and ASXL1 in hundreds of hCD45+ engrafted cells derived from CFU. 43% of colonies had targeted editing of at least one gene (GATA2, SETBP1 or ASXL1). Interestingly, in TRIPLE condition 24% colonies, more than one gene was edited. To study the long-term clonal evolution, we performed secondary transplants. We noted that the predominant expanded clones were carrying SETBP1+ASXL1 mutations, while clones with only GATA2 mutation were lost in both GATA2 and TRIPLE conditions. To study the impact of the mutations at transcriptomic level, scRNAseq from primary and secondary hCD45+/CD19- engrafted cells was performed (the analysis is underway). This was validated in vitro showing that cells carrying the GATA2 R398W mutation have an impaired clonogenic capacity and proliferation recapitulating the MDS. Summary/Conclusion: In summary, we developed a new in vivo human model of GATA2 deficiency and GATA2-MDS-specific mutations by CRISPR/Cas9 targeting of CD34+ cells. Our findings strongly suggest that GATA2 R398W mutation is not sufficient to increase the fitness of the mutant cells, suggesting that a co-operation of genetic, epigenetic and stressor factors is necessary to trigger the clonal expansion.Keywords: MDS/AML, GATA-2, Pediatric, Mouse model