Long-Term Systemic Treatment of a Mouse Model Displaying Chronic FSHD-like Pathology with Antisense Therapeutics That Inhibit DUX4 Expression

Silencing the expression of the double homeobox 4 (DUX4) gene offers great potential for the treatment of facioscapulohumeral muscular dystrophy (FSHD). Several research groups have recently reported promising results using systemic antisense therapy in a transgenic small animal model of FSHD, the ACTA1-MCM/FLExDUX4 mouse model. However, the treatment was applied in non-DUX4-induced mice or shortly after DUX4 activation, which resulted in conditions that do not correctly represent the situation in a clinic. Here, we generated progressive FSHD-like pathology in ACTA1-MCM/FLExDUX4 mice and then treated the animals with vivoPMO-PACS4, an antisense compound that efficiently downregulates DUX4. To best mimic the translation of this treatment in clinical settings, the systemic antisense oligonucleotide administration was delayed to 3 weeks after the DUX4 activation so that the pathology was established at the time of the treatment. The chronic administration of vivoPMO-PACS4 for 8 weeks downregulated the DUX4 expression by 60%. Consequently, the treated mice showed an increase by 18% in body-wide muscle mass and 32% in muscle strength, and a reduction in both myofiber central nucleation and muscle fibrosis by up to 29% and 37%, respectively. Our results in a more suitable model of FSHD pathology confirm the efficacy of vivoPMO-PACS4 administration, and highlight the significant benefit provided by the long-term treatment of the disease

Systemic antisense therapeutics inhibiting DUX4 expression ameliorates FSHD-like pathology in an FSHD mouse model

Aberrant expression of the double homeobox 4 (DUX4) gene in skeletal muscle causes muscle deterioration and weakness in Facioscapulohumeral muscular dystrophy (FSHD). Since the presence of a permissive pLAM1 polyadenylation signal is essential for stabilization of DUX4 mRNA and translation of DUX4 protein, disrupting the function of this structure can prevent expression of DUX4. We and others have shown promising results using antisense approaches to reduce DUX4 expression in vitro and in vivo following local intramuscular administration. Here we demonstrate that further development of the antisense chemistries enhances in vitro antisense efficacy. The optimal chemistry was conjugated to a cell-penetrating moiety and was systemically administered into the tamoxifen-inducible Cre-driver FLExDUX4 double-transgenic mouse model of FSHD. After four weekly treatments, mRNA quantities of DUX4 and target genes were reduced by 50% that led to 12% amelioration in muscle atrophy, 52% improvement in in situ muscle strength, 17% reduction in muscle fibrosis and prevention of shift in the myofiber type profile. Systemic DUX4 inhibition also significantly improved the locomotor activity and reduced the fatigue level by 22%. Our data demonstrate that the optimized antisense approach has potential of being further developed as a therapeutic strategy for FSHD

Optimized lentiviral vector for restoration of full-length dystrophin via a cell-mediated approach in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the DMD gene. Restoration of full-length dystrophin protein in skeletal muscle would have therapeutic benefit, but lentivirally-mediated delivery of such a large gene in vivo has been hindered by lack of tissue-specificity, limited transduction and insufficient transgene expression. To address these problems, we developed a lentiviral vector, which contained a muscle-specific promoter and sequence optimized full-length dystrophin, to constrain the dystrophin expression to differentiated myotubes/myofibres and enhance the transgene expression. We further explored the efficiency of restoration of full-length dystrophin in vivo, by grafting DMD myoblasts that had been corrected by this optimized lentiviral vector intramuscularly into an immunodeficient DMD mouse model. We showed that these lentivirally-corrected DMD myoblasts effectively reconstituted full-length dystrophin expression in 93.58±2.17% of the myotubes in vitro. Moreover, dystrophin was restored in 64.4±2.87% of the donor-derived regenerated muscle fibres in vivo, which was able to recruit members of the dystrophin glycoprotein complex at the sarcolemma. This study represents a significant advance over existing cell-mediated gene therapy strategies for DMD that aim to restore full-length dystrophin expression in skeletal muscle