Development of a Combinatorial siRNA Therapy for the Treatment of Fibrodysplasia Ossificans Progressiva - PROJECT SUMMARY Fibrodysplasia ossificans progressiva (FOP) is an autosomal dominant disorder characterized by spontaneous or trauma-induced accumulation of irreversible ossifications in tendons, ligaments, and skeletal muscle that lead to progressive loss of mobility and early death. Nearly all cases of FOP are caused by a gain-of-function mutation (R206H) in the ACVR1 gene, which results in overactive signaling and heterotopic ossification (HO). The current standard of care for FOP involves pain relief and reducing the risk of trauma. Direct modulation of mutant ACVR1 and related pathways to prevent HO would offer a more powerful way to improve patient quality of life. Small interfering RNAs (siRNAs) enable potent, sequence-specific gene silencing, with six FDA-approved therapeutics. This clinical success is driven by stabilizing chemistries and conjugate-mediated delivery. The Khvorova lab has developed (1) docosanoic acid (DCA) conjugates that support robust delivery to, and silencing in, muscle after subcutaneous administration, and (2) chemically stabilized siRNA that selectively silence the ACVR1R206H mRNA allele. Administering this compound in an FOP mouse model profoundly reduces trauma- induced HO, validating siRNA-mediated ACVRR206H silencing as a therapeutic strategy for FOP. Nevertheless, HO was not fully prevented, suggesting other factors contribute to HO formation. ACVR1R206H results in broadly aberrant inflammatory signaling in FOP. Inflammatory episodes precede bone growth, and elevated levels of pro-inflammatory cytokines, including interleukin-1α and β (IL-1α and IL-1β), are detected at HO sites in patients. IL-1 inhibitors partially suppress HO in an FOP patient, and knocking out the IL- 1 receptor decreased HO volume in FOP mice, suggesting IL-1 is involved in HO formation. However, the precise role of the IL-1 pathway (and possibly other inflammatory pathways) in FOP pathogenesis, and the effects of its modulation in the context of ACVR1R206H regulation, is poorly understood. This project seeks to use siRNAs to study the role of IL-1 signaling in HO, and to develop therapeutic approaches to simultaneously target ACVR1R206H and IL-1 signaling to more efficiently suppress HO. Aim 1 will determine which IL-1 signaling components are most important for HO formation and clarify how the FOP inflammatory signature responds to IL-1 modulation. siRNA targeting IL-1α, IL-1β, or the IL-1 receptor accessory protein will be administered to a muscle injury site in FOP mice, and inflammatory cytokine levels, cell populations, transcriptome changes, and HO formation will be measured. Aim 2 will determine the effects of IL- 1 modulation in the context of ACVR1R206H silencing in FOP mice to identify additional therapeutic targets that lend an additive or synergistic effect in reducing HO. Co-administration of separate siRNA molecules targeting IL-1 signaling and ACVR1R206H (Aim 2a) or unimolecular divalent (i.e., linked) siRNA targeting both (Aim 2b) will be explored. This work will reveal inflammatory mechanisms in HO and FOP, inform FOP therapeutic design, and provide the fellow with training in FOP pathophysiology, immunology, and therapeutic development.
