ReprogRAM nanoplatform for direct conversion of fibroblasts to osteoblasts for bone regeneration

mRNA-based direct conversion offers an integration-free route for osteogenic cell-fate reprogramming, yet efficient reprogramming typically requires sustained expression of reprogramming factors. Here, we developed ReprogRAM (Reprogrammable RNA Assemblies for Multi-factor expression), a self-assembled concatemeric mRNA nanoparticle synthesized by rolling-circle transcription and engineered for cap-independent translation. A single delivery of ReprogRAM to human dermal fibroblasts enabled cytosolic expression that persisted over time with minimal cytokine induction. By leveraging this long-acting expression profile, we encoded the osteogenic reprogramming factors Oct4 and Cbfβ and implemented a hybridized delivery strategy to enforce co-expression of both factors. ReprogRAM induced robust osteogenic conversion, as evidenced by increased mineralized matrix deposition, induction of osteoblast markers, and transcriptomic remodeling toward osteoblasts. Finally, transplantation of ReprogRAM-derived reprogrammed osteoblasts (rOBs) embedded in a gelatin scaffold promoted bone repair in a critical-sized mouse calvarial defect model. Together, ReprogRAM provides a long-acting, integration-free mRNA modality for cell-fate engineering and bone regeneration.