Rhesus Macaques As a Preclinical Model Organism for RUNX1-FPD

Germline loss-of-function (LOF) heterozygous mutations in the central hematopoietic transcription factor RUNX1 gene cause the marrow failure/malignancy predisposition syndrome Familial Platelet Disorder with associated Myeloid Malignancies (FPDMM, or FPD). Patients with FPD have defective megakaryocytic development, low platelet counts, and a very high (35-50%) life-long risk of hematological malignancies, particularly MDS/AML. Murine heterozygous gene knockout models do not recapitulate the human phenotype in terms of thrombocytopenia or myeloid leukemia progression. Although gene correction of the RUNX1 mutation in hematopoietic stem and progenitor cells (HSPCs) is being considered as a possible treatment approach, it is unknown whether mutation-corrected HSPCs will have the hoped for advantage over RUNX1 mutant HSCs in vivo, likely necessary to significantly lower leukemia risk. In order to study the relative function of wildtype and RUNX1-mutated HSPCs in vivo in a model with close hematopoietic similarity to humans, we generated a rhesus macaque FPD competitive repopulation model via CRISPR/Cas9 NHEJ editing of the RUNX1 gene versus the AAVS1 safe-harbor control locus. We transplanted mixtures of autologous HSPCs edited at the two loci: 75% RUNX1-edited/25% AAVS1- edited CD34+ HSPCs in animal 1 and 25% RUNX1-edited/75% AAVS1-edited CD34+ HSPCs in animal 2, following conditioning with total body irradiation. Both animals engrafted tri-lineage hematopoiesis promptly following transplantation. However, platelet numbers remained below the normal range long-term in animal 1 receiving a higher ration of RUNX1-edited HSPCS and below counts of macaques receiving HSPCs edited at other loci (Figure 1). Bone marrow morphology at 6 months was normal.

To assess the HSPC function of RUNX1 mutant versus AAV1 control and unedited WT cells we tracked RUNX1 and AAVS1-mutated allele frequencies in blood cells over time via deep sequencing (Figure 2). In the infusion products (IP), allele fractions reflected the desired ratios. In both animals, AAVS1-edited cells dominated compared to RUNX1-edited cells. However, in animal 1, RUNX1-mutated cells expanded over time eventually exceeding the ratio in the IP, and in animal 2, levels of RUNX1 and AAVS1-mutated cells were equivalent long-term. Marrow analyzed at 6 months showed heterozygous RUNX1-mutated CFU at levels concordant with mutation frequencies in the blood, but no homozygous RUNX1 mutated CFU, suggesting homozygous LOF is not compatible with long-term HSPC function.

In conclusion, we have created pre-clinical model for FPD via CRISPR/Cas editing of HSPCs in rhesus macaques. The lack of a competitive advantage for wildtype or control-locus edited HSPCs over RUNX1 heterozygous-mutated HSPCs long-term in our model suggests that gene correction approaches for FPD will be challenging, particularly to reverse the MDS/AML predisposition phenotype.

Figure 1

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Figure 1

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