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 3

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

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Functional deficits in TIC-DUX4 mice following a medium-dose tamoxifen r...
(A) TIC-DUX4 animals treated with 5 mg/kg tamoxifen, 3× per week for 4 weeks showed significantly reduced overall cage activity and rearing behavior compared with vehicle (untreated) or WT controls. Induced TIC-DUX4 male and female mice had a mean activity of 2,616 and 1,719 mean beam breaks, respectively, compared with 5,999 and 6,173 mean beam breaks in uninduced TIC-DUX4 mice and 4,646 and 4,770 mean beam breaks in WT mice, respectively. Induced mice had a rearing frequency mean of 131.9 (male) and 124.3 (female) beam breaks compared with 659.9 (male) and 580.4 (female) beam breaks in uninduced TIC-DUX4 mice. WT animals had 468.6 (male) and 482.8 (female) mean beam breaks. Error bars show ± SD. I, tamoxifen induced; U, untreated. n = 8 induced male and 5 induced female TIC-DUX4 mice; 12 male and 11 female uninduced TIC-DUX4 mice; and 7 male and 6 female tamoxifen-treated WT littermate controls. For both total activity and rearing, induced TIC-DUX4 animals were significantly different from all control groups (2-way ANOVA with Tukey’s multiple comparison test; P < 0.05). (B) TIC-DUX4 mice treated with 5 mg/kg tamoxifen, 3× per week for 4 weeks developed significant weakness in the tibialis anterior muscles, as measured by absolute and specific force reductions compared with all control groups (P < 0.0001, 2-way ANOVA with Tukey’s multiple comparison test). Animals treated with 10-fold less tamoxifen concentration (0.5 mg/kg) during the same time course showed no measurable deficits. There were no significant differences between sexes, and data presented here are grouped only by treatment and genotype. Error bars represent ± SD. Mean absolute force: TIC-DUX4 mice (5 mg/kg Tam), 363.6 mN; TIC-DUX4 mice (0.5 mg/kg Tam), 1,135 mN; uninduced TIC-DUX4 mice, 1,349 mN; WT (5 mg/kg Tam), 1,231 mN; WT untreated, 1,125 mN. Mean specific force: TIC-DUX4 mice (5 mg/kg Tam), 120 mN/mm2; TIC-DUX4 (0.5 mg/kg Tam), 286.1 mN/mm2; uninduced TIC-DUX4 mice, 274.9 mN/mm2; WT (5 mg/kg Tam), 281.8 mN/mm2; WT untreated, 269.7 mN/mm2. n = 6 (5 mg/kg) induced TIC-DUX4 legs; 9 (0.5mg/kg) induced TIC-DUX4 legs; 7 uninduced TIC-DUX4 legs; 7 (5mg/kg) tamoxifen-treated WT littermate legs; and 14 untreated WT littermate legs. mN = milliNewtons.