Precision RNAi for Fibrodysplasia Ossificans Progressiva: a combinatorial, unimolecular, allele selective approach

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder caused by a dominant mutation in the ACVR1 gene (R206H, 97% of cases), leading to debilitating heterotopic ossification (HO) characterized by abnormal bone growth triggered by inflammatory flare-ups. Here, we report the development of disease-modifying, allele-selective small interfering RNA (siRNA) targeting ACVR1 ^R206H . Allele selectivity is essential as wildtype ACVR1 is crucial for many functions including skeletal homeostasis and development. When conjugated to docosanoic acid (DCA), administration of the fully modified ACVR1 siRNA, either alone or in combination with an siRNA targeting IL1B (a key regulator of inflammation), results in profound reduction of HO using both responsive (post-trauma) and preventative (pre-trauma) intervention strategies in a murine FOP model. Notably, the combination therapy outperforms modulation of either target alone. We also describe the chemical engineering of a new class of lipophilic divalent siRNAs that target both pathways with a single compound, demonstrating superior muscle accumulation and therapeutic efficacy. siRNA treatment inhibits key signaling pathways (e.g. inflammatory, WNT, Notch, Hedgehog, and TGF-β), within muscle-resident fibroadipogenic progenitors (FAPs), leading to a significant reduction in cartilage, bone, and connective tissue formation. This work establishes a foundation for the development of disease-modifying treatments for FOP and offers a platform for targeting other musculoskeletal disorders involving multi-pathway dysregulation.