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

Abstract

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.

Microdystrophin Gene Addition Significantly Improves Muscle Functionality and Diaphragm Muscle Histopathology in a Fibrotic Mouse Model of Duchenne Muscular Dystrophy

“…The effect of AAV8-MD1 delivery on muscle fibrosis was analyzed by assessing the expression of collagen VI, periostin, and fibronectin, all considered markers of fibrosis [23][24][25][26]. As shown in Figure 4A, the diaphragms of the Dba2/J control mice already showed a level of fibrosis, as has been reported previously for skeletal muscles of this mouse, but the diaphragms of the D2.mdx mice displayed more collagen VI deposition.…”

“…Histological analysis was performed following a protocol by Lu-Nguyen and colleagues [26]. Collagen VI immunostaining was used to determine the fibrotic area of each sample.…”

A Systemically Administered Unconjugated Antisense Oligonucleotide Targeting DUX4 Improves Muscular Injury and Motor Function in FSHD Model Mice

“…We also studied the number of centrally nucleated myofibers found in gastrocnemius muscles in DUX4-TG mice [ 11 , 15 , 17 , 18 , 29 ]. The nuclei of regenerated mouse myofibers stay central for several months [ 31 ].…”

“…TMX injection causes transient symptoms with a 2-week peak, probably because dying DUX4-expressing muscle cells are compensated by regenerating non-DUX4-expressing muscle cells [ 10 ]. Lu-Nguyen et al [ 17 ] have demonstrated the chronic disease progression model, where DUX4-TG mice were repeatedly treated with 1.5 mg/kg of TMX every other week. However, they did not show how much the disease was exacerbated by such a low dose of TMX compared with TMX-untreated DUX4-TG mice.…”

“…Antisense oligonucleotides that could decrease the causative DUX4 expression have been investigated as a promising treatment strategy [ 13 , 14 ]. Nonetheless, only a few groups have demonstrated a systemic improvement in DUX4-TG mice using ASOs with a ligand that facilitated ASO delivery to skeletal muscles [ 15 , 16 , 17 , 18 ]. Here, we have generated a novel gapmer ASO targeting the human DUX4 gene, MT-DUX4-ASO, with an optimized design and high in vivo activity.…”