Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codonoptimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.

1 Centres of Gene and Cell Therapy and Biomedical Sciences, School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, UK 2 Sorbonne Université, INSERM, Association Institut de Myologie, Centre de Recherche en Myologie, UMRS974, 47 bd de l’Hôpital, 75013 Paris, France 3 Comparative Biomedical Sciences, Royal Veterinary College, London, UK 4 Benitec Biopharma, Hayward, California, USA

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV 'silence' and 'replace' vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the 'silence' and 'replace' cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.