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
View: Text | PDF
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

×

Figure 1

Generation of the ROSA26-DUX4 knock-in mouse and DUX4 expression validation in TIC-DUX4 mice.

Options: View larger image (or click on image) Download as PowerPoint
Generation of the ROSA26-DUX4 knock-in mouse and DUX4 expression validat...
(A) Top, WT Rosa26 locus. Asterisk marks pRosa26-DUX4 insertion site. Black rectangles represent Rosa26 5′ and 3′ flanks. Gray rectangles depict Rosa26 genomic DNA sequences not included in the targeting construct. Middle, targeting construct inserted into Rosa26. The 5′ end contains a LoxP-flanked (floxed) neomycin resistance gene (NeoR) driven by the PGK promoter (not shown). Adjacent to the NeoR cassette is 1 DUX4 ORF (exon 1) fused to a V5 epitope sequence, followed by introns (Int1, Int2) and 3′ UTR exons (Ex2 and Ex3) from the last repeat of DUX4 (labeled 3′ UTR/pLAM). Exon 3 derives from the chrom 4q pLAM region and contains the noncanonical 5′-ATTAAA-3′ polyA signal utilized by the last DUX4 copy in FSHD patients. When present, the floxed NeoR cassette inhibits DUX4 expression from the Rosa26 promoter. A bovine growth hormone polyA signal (BGH pA; 5′-AATAAA-3′) was included next to DUX4/pLAM. The HSA-mER-Cre-mER (HSA-MCM) mouse was previously published. HSA, human skeletal actin promoter with β-globin intron (βGlobin-int); mER, mutated estrogen receptor. Arrows indicate PCR primers used to detect WT Rosa26 and the knocked-in transgene in mouse genomic DNA. PCR products sizes are indicated. (B) Southern blot screening of 6 ES cell clones for pRosa26-DUX4 knock-in. DNA was digested with EcoRV and probed with the probe indicated in A. WT ROSA26 alleles produce an 11,517-bp EcoRV band, while correctly targeted clones produced a 4,084-bp band. (C) Configuration of 2 DUX4 transcripts detected in induced TIC-DUX4 mice via nested 3′ RACE RT-PCR. Thirty-five of 36 clones contained correctly spliced products, while 1 clone had a retained intron 1. All transcripts utilized the BGH polyA signal. (D) Western blot of DUX4 expression in TIC-DUX4 mouse tissue. H, high dose tamoxifen; L, low dose tamoxifen; –, untreated. Positive control (+ ctrl) is protein extracted from CMV. DUX4-transfected HEK293 cells. (E) Wfdc3 cDNA, a biomarker of DUX4 in mice, was significantly increased 1,395- and 1,438-fold in high (H) and low (L) Tam– induced TIC-DUX4 muscles compared with untreated (U, sunflower oil) or WT control littermates (–), respectively. Data are measured from qPCR assays (P < 0.01; 1-way ANOVA with Tukey’s multiple comparison test). n = 4 for high Tam dose TIC-DUX4; n = 3 for all other groups.