Prime editing rescues osteogenic function of patient-derived iMSCs carrying the RUNX2 c.516delT mutation

Introduction Cleidocranial dysplasia (CCD) is a skeletal genetic disorder caused by RUNX2 mutation, for which no effective therapy is currently available. Prime editing (PE) enables precise insertion and deletion editing in mammalian cells, offering a promising approach for correcting heterozygous loss-of-function mutations. Methods Here, we applied PE to correct the RUNX2 c.516delT loss-of-function mutation in patient-derived induced mesenchymal stem cells (iMSCs). To improve editing efficiency, we employed mpknot-pegRNA and tevopreQ1-pegRNA designs in combination with PE2, PEmax, and PE7 variants. We then assessed the normalization of RUNX2 protein levels and downstream target activation upon gene correction. Results We achieved an editing efficiency of up to 30.6% in iMSCs. Following PE-mediated repair, the expression of RUNX2 downstream targets CBFβ and SP7 was significantly upregulated, and osteogenic differentiation and bone mineralization were partially rescued in mutant iMSCs. Discussion Collectively, our findings suggest that optimized prime editing systems can efficiently correct this pathogenic RUNX2 mutation in a patient-derived model and partially restore osteogenic function in vitro , providing a proof-of-concept for PE-based therapeutic strategies targeting this specific CCD-associated RUNX2 allele. Although further validation in additional patient samples and different mutation types is required, this study supports the potential of PE-based approaches for correction of loss-of-function frameshift mutations in CCD.