Abstract
Heterotopic ossification is the most disabling feature of fibrodysplasia ossificans progressiva, an ultra-rare genetic disorder for which there is currently no prevention or treatment. Most patients with this disease harbor a heterozygous activating mutation (c.617 G > A;p.R206H) in ACVR1. Here, we identify recombinant AAV9 as the most effective serotype for transduction of the major cells-of-origin of heterotopic ossification. We use AAV9 delivery for gene replacement by expression of codon-optimized human ACVR1, ACVR1R206H allele-specific silencing by AAV-compatible artificial miRNA and a combination of gene replacement and silencing. In mouse skeletal cells harboring a conditional knock-in allele of human mutant ACVR1 and in patient-derived induced pluripotent stem cells, AAV gene therapy ablated aberrant Activin A signaling and chondrogenic and osteogenic differentiation. In Acvr1(R206H) knock-in mice treated locally in early adulthood or systemically at birth, trauma-induced endochondral bone formation was markedly reduced, while inflammation and fibroproliferative responses remained largely intact in the injured muscle. Remarkably, spontaneous heterotopic ossification also substantially decreased in in Acvr1(R206H) knock-in mice treated systemically at birth or in early adulthood. Collectively, we develop promising gene therapeutics that can prevent disabling heterotopic ossification in mice, supporting clinical translation to patients with fibrodysplasia ossificans progressiva.