Suppression of Heterotopic Ossification in Fibrodysplasia Ossificans Progressiva Using AAV Gene Delivery

Abstract Progressive heterotopic ossification (HO) is the most disabling and defining feature of fibrodysplasia ossificans progressiva (FOP), an ultra-rare genetic disorder. More than 95% of FOP patients harbor a heterozygous activating mutation (c.617G>A;p.R206H) in the Activin A Receptor Type 1 (ACVR1). Currently, there are no definitive preventions or treatments. Gene therapy is thus an attractive treatment strategy. Here, we identify recombinant adeno-associated virus 9 (rAAV9) as the most effective serotype for transduction of the major cells-of-origin of HO in soft tissues. Using AAV-mediated gene delivery, three gene therapy approaches for FOP, including gene replacement by expression of codon-optimized human ACVR1 (ACVR1OPT), ACVR1R206H allele-specific silencing by AAV-compatible artificial miRNA (amiR, amiR-ACVR1R206H), and a combination of gene replacement and silencing (ACVR1OPT;amiR-ACVR1R206H), were developed. AAV gene therapy ablated aberrant Activin A signaling and chondrogenic and osteogenic differentiation of mouse skeletal cells harboring a conditional knock-in allele of human ACVR1R206H (Acvr1(R206H)KI) and human FOP patient-derived induced pluripotent stem cells (iPSCs). Accordingly, trauma-induced HO was markedly reduced in Acvr1(R206H)KI mice treated locally at early adulthood or systemically at birth. These mice displayed a significant decrease in endochondral bone formation in injured muscle while inflammation and fibroproliferation responses to injury remained largely intact. Remarkably, spontaneous HO in juvenile and adult Acvr1(R206H)KI mice was also substantially decreased by AAV gene therapy. Compared to gene replacement or silencing, combination gene therapy was the most potent in preventing both traumatic and spontaneous HO in FOP mice. Collectively, our results identify novel gene therapeutics that can prevent disabling HO in FOP mice and provide evidence to support clinical translation to FOP patients.