Adeno-associated virus (AAV)-mediated RNA interference to PABPN1 combined with an optimised resistant transgene for rescue of the muscle specific disease oculopharyngeal muscular dystrophy (OPMD)

Background: OPMD is an autosomal dominant, late-onset muscle disorder, caused by a trinucleotide repeat expansion in PABPN1 resulting in an N-terminal expanded polyalanine tract and intracellular inclusions of aggregated expPABPN1. PABPN1 promotes interaction between the poly(A) polymerase and the cleavage and polyadenylation specificity factor and controls the length of mRNA poly(A) tails, mRNA export from the nucleus, and alternative poly(A) site usage. Aims: We ought to design a gene therapy strategy based on RNA interference to silence mutant PABPN1, combined with a codon- modified sequence resistant to degradation using AAV as a vector. Methods: Viral plasmids containing shRNA constructs (pAAV- sh131, pAAV-sh132, pAAV-sh133) and RNA polymerase III promoters were designed in collaboration with BENITEC BIOPHARMA. A viral plasmid expressing a codon-optimised PABPN1 under a muscle specific promoter (pAAV-spc512-OPTPAB) was synthesised by Geneart. Knock-down efficiency was assessed in vitro and in vivo (A17 mice) by western blotting and quantified using ImageJ. Results: pAAV-sh131, pAAV-sh132 and pAAV-sh133 gave 40%, 90% and 95% knock-down of PABPN1 expression, respectively, in vitro. scAAV8 expressing sh133 injected into TA muscles gave 90% knockdown of PABPN1 expression in vivo. Co-transfection studies showed resistance of pAAV-spc512-OPTPAB to sh133 degradation in vitro. Conclusion: PABPN1 can be efficiently knocked-down in vitro and in vivo. Studies are on-going to investigate PABPN1 knock-down and replacement on muscle function in vivo.