AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD
Carlee R. Giesige, Lindsay M. Wallace, Kristin N. Heller, Jocelyn O. Eidahl, Nizar Y. Saad, Allison M. Fowler, Nettie K. Pyne, Mustafa Al-Kharsan, Afrooz Rashnonejad, Gholamhossein Amini Chermahini, Jacqueline S. Domire, Diana Mukweyi, Sara E. Garwick-Coppens, Susan M. Guckes, K. John McLaughlin, Kathrin Meyer, Louise R. Rodino-Klapac, Scott Q. Harper
Carlee R. Giesige, Lindsay M. Wallace, Kristin N. Heller, Jocelyn O. Eidahl, Nizar Y. Saad, Allison M. Fowler, Nettie K. Pyne, Mustafa Al-Kharsan, Afrooz Rashnonejad, Gholamhossein Amini Chermahini, Jacqueline S. Domire, Diana Mukweyi, Sara E. Garwick-Coppens, Susan M. Guckes, K. John McLaughlin, Kathrin Meyer, Louise R. Rodino-Klapac, Scott Q. Harper
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Research Article Muscle biology Therapeutics

AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model — the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.

Authors

Carlee R. Giesige, Lindsay M. Wallace, Kristin N. Heller, Jocelyn O. Eidahl, Nizar Y. Saad, Allison M. Fowler, Nettie K. Pyne, Mustafa Al-Kharsan, Afrooz Rashnonejad, Gholamhossein Amini Chermahini, Jacqueline S. Domire, Diana Mukweyi, Sara E. Garwick-Coppens, Susan M. Guckes, K. John McLaughlin, Kathrin Meyer, Louise R. Rodino-Klapac, Scott Q. Harper

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Figure 2

Histopathology in TIC-DUX4 mice following a medium-dose tamoxifen regimen.

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Histopathology in TIC-DUX4 mice following a medium-dose tamoxifen regime...
(A and B) Left panels show tibialis anterior (TA) and triceps muscles from TIC-DUX4 mice 4 weeks after induction with 5 mg/kg tamoxifen, 3 times per week. Far left images show representative 10× muscle cryosections tiled to display an entire muscle in cross-section. Navy blue coloring was digitally added to each low-power image to easily visualize histopathology, including degenerating myofibers, regenerated myofibers containing central nuclei, and immune infiltration, which are features evident in the adjacent 20× images. The bottom higher-power images show immunofluorescence for DUX4 (V5 epitope, red) or nuclei (DAPI, blue). Widespread damage was present throughout induced TIC-DUX4 muscles. Increased percentage of myofibers with central nuclei and a shift toward smaller bore myofibers in induced TIC-DUX4 muscles support that muscles underwent degeneration and subsequent regeneration during this time course. Tamoxifen-induced TIC-DUX4 mice (TIC-DUX4 I) were significantly different from uninduced TIC-DUX4 (TIC-DUX4 U) and WT animals (2-way ANOVA with Tukey’s multiple comparison test; P < 0.05 for TA and P < 0.01 for triceps). No significant sex differences were measured in induced TIC-DUX4 animals. Mean central nuclei percentages for TA muscle of induced TIC-DUX4 males: 23.4%; uninduced TIC-DUX4 mice, 2.2%; WT, Tam-treated 2.0%; WT untreated, 1.1%. For female TA muscles: induced TIC-DUX4 23.9%; uninduced TIC-DUX4, 2.3%; WT, Tam-treated 2.6%; WT untreated, 1.3%. For triceps, mean central nucleic percentages in induced TIC-DUX4 males: 34.8%; uninduced TIC-DUX4, 4.5%; WT, Tam-treated 1.9%; WT untreated, 1.2%. For induced TIC-DUX4 female triceps: 27.3%; uninduced TIC-DUX4, 4.4%; WT,Tam-treated 2.3%; WT untreated 1.7%. Animals were 8–12 weeks of age during this experiment. For male mice: n = 9 induced and uninduced TIC-DUX4 mice; 4 tamoxifen-treated WT littermates; 4 untreated WT littermates. For females: n = 9 induced and 10 uninduced TIC-DUX4 mice; 4 tamoxifen-treated WT littermates; 4 untreated WT littermates. (C) Representative uninduced triceps muscle of 8- to 12-week-old TIC-DUX4 mice. (D) Representative tiled 10× image showing tamoxifen-treated gas muscle of 8- to 12-week-old WT mice. 20× H&E image (top right) demonstrated that tamoxifen treatment alone has no impact on muscle histology. Scale bars: 500 μm in low-power images; 100 μm at 20×.