A Deoxyribonucleic Acid Decoy Trapping DUX4 for the Treatment of Facioscapulohumeral Muscular Dystrophy

Figures

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  Figure 1. Decoys Targeting DUX4 Protein Are Able to Decrease DUX4-Induced Gene Activation In Vitro (A) Design of the different decoys used. The sequence in-between the dashed line is common to all of the decoys except for the random decoy. The DUX4 binding site is in red. The random sequence (generated by the random DNA sequence generator, the percentage of GC was conserved; http://www.faculty.ucr.edu/∼mmaduro/random.htm) is bolded. The decoys are two oligonucleotides that are hybridized together (decoy 3 [D3]) or double-strand DNA synthetized as one single DNA strand (D6–D11 and random), which are ligated before transfection. Chemical modifications are as follows: italic indicates 2′-O-methyl bases; underlined bases carry a phosphorothioate linkage; the hexaethylene glycol linkers are represented by gray brackets; the minimal DUX4 binding sites are in bold; and arrows indicate the junction between the beginning and the end of the oligonucleotide before ligation. (B) FSHD myoblasts were transfected with the different decoys, and the levels of genes downstream of DUX4 were performed, normalized to DUX4 expression. GAPDH was used as a normalizer. The data represent mean ± SEM from at least…

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  Figure 2. Oligonucleotide-Based Decoys Inhibit DUX4-Induced Gene Transcription In Vivo (A and B) Tibiales anterior (TAs) of C57BL/6 mice were electrotransferred with the pCS2 plasmid coding for DUX4 in the presence of the different decoys. Expression levels of both DUX4 and Tm7sf4 mRNAs were analyzed by qPCR since a multiparametric analysis of variance (MANOVA) and a Newman-Keuls post hoc test had revealed a strong correlation between DUX4 and Tm7sf4 (A) (r2 = 0.8948, p = 10e−8). Mice were sacrificed 4 days after injection and total RNA was extracted. The histograms represent Gapdh normalized data (B). At least eight TAs were analyzed per group. Data are presented as the mean ± SEM of three or more independent experiments. ∗p < 0.05, ∗∗p < 0.01, by one-way ANOVA. (C) Decoy 7 carrying a rhodamine (Rho) fluorescence decoy was electrotransferred into the TA of C57BL/6 mice. Four days later, mice were sacrificed and muscles were sectioned (8 μm). The D7-Rho is seen in red, the laminin is labeled in green using a polyclonal rabbit anti-laminin antibody (Z0097, Dako, Les Ulis, France), and nuclei are counterstained in blue using DAPI. In the merged image, examples of decoy accumulation in the…

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  Figure 3. Molecular Stability of the DNA Decoys in the Presence of Nucleases (A–D) Decoys were incubated in the presence of differentiation medium (A), exonuclease III (B), nuclease S1 (C), or FBS for 0, 1, 2, 4, 8, 10, or 18 h (D) and loaded on a 15% acrylamide gel. L, 50-bp ladder. D3–D11 indicate the different decoys. Lengths of the double strands: D3, 82 bp; D6, 34 bp; D7, D8, and D9, 40 bp; D11, 28 bp.

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  Figure 4. AAV-Based Decoys Inhibit DUX4-Induced Gene Transcription In Vivo TAs of C57BL/6 mice were injected with 2.5e10 vg/TA of AAV-GFP or AAV-decoy (35 μL). 14 days later, the pCS2 plasmid was electrotransferred into the muscles. Mice were sacrificed 4 days later and total RNA was extracted and expression levels of DUX4 and Tm7sf4, wfdc3, and Duxbl were analyzed by qPCR. The boxplots were normalized using Gapdh. Data are presented as the mean ± SEM of three or more independent experiments (n ≥ 8 TAs/group). ∗p < 0.05, by a t test.