Targeted correction Of RUNX1 Mutation In The FPD Patient-Specific Induced Pluripotent Stem Cells Restores Megakaryocytic Differentiation

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is a rare autosomal dominant disease caused by RUNX1 mutations. RUNX1 encodes a transcription factor, which is frequently mutated in leukemias. It is also essential in early hematopoiesis and megakaryocytic differentiation. FPD patients have a mild to moderate bleeding disorder, thrombocytopenia, and over 35% of patients will develop AML during their lifetime. Currently there are no suitable animal models for the disease, making it necessary to develop alternative approaches for research studies.

To develop an in vitro model of FPD, we established induced pluripotent stem cell (iPSC) lines using episomal, non-integrating vectors from skin fibroblasts from a family of FPD patients harboring a nonsense mutation in the C-terminal region of the RUNX1 (Y260X). The derived iPSC lines showed typical characteristics of pluripotency such as teratoma formation and expressions of pluripotency markers (Oct4, Sox2, Nanog, and Tra-1-81). In order to correct the mutation in the patient-specific iPSCs by homologous recombination, we designed a pair of RUNX1 zinc finger nucleases (ZFNs) and a targeting vector. We then performed ZFN-mediated gene targeting in one of the FPD iPSC lines and confirmed correction of the mutant RUNX1 allele in at least two iPSC clones by Southern, FISH and sequence analyses. RUNX1 mRNA expression analysis of the FPD iPSC line and the corrected iPSC lines showed expression of only wildtype RUNX1 transcripts in the corrected iPSC lines, but not in the uncorrected lines. Furthermore, we directed hematopoietic differentiation of the FPD iPSCs and the corrected iPSC lines using the spin embryoid body formation method. We found defects in hematopoietic differentiation of FPD iPSCs, particularly towards megakaryopoiesis. Importantly, ZFN-mediated mutation correction rescued the hematopoietic differentiation defects with almost double the number of CD41a⁺/CD42a⁺ megakaryocytes produced from the corrected iPSCs.

Our results collectively demonstrate successful in vitro modeling of FPD using iPSC lines with an innovative approach to correct the RUNX1 mutation by gene targeting. Mutation correction rescues the hematopoietic defects, indicating that hematopoietic stem cell transplantation using patient-specific iPSC lines has great potential to treat the FPD patients in the future.