Oligonucleotide Therapies for Facioscapulohumeral Muscular Dystrophy: Current Preclinical Landscape

Figures

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  Figure 1. (A) A schematic representation of the D4Z4 region in healthy individuals and FSHD patients. The D4Z4 macrosatellite tandem repeat array is found in the subtelomeric region 4q35. Each gray triangle indicates a 3.3 kb D4Z4 repeat unit, within each of which a DUX4 retrogene is contained. The 1st and 2nd DUX4 exons (blue boxes) occur in each full D4Z4 unit. A partial D4Z4 (gray trapezoid) occurs after the most distal complete unit, followed by the 3rd DUX4 exon. Healthy individuals have 11–100+ repeats and full epigenetic repression (purple line). FSHD patients have fewer repeats and compromised epigenetic repression (purple dotted line) arising from one of two genetic changes indicated in red text. (B) A schematic representation of aberrant DUX4 expression from the most distal complete D4Z4 repeat within the FSHD-permissive 4qA haplotype. The 1st and 2nd DUX4 exons are within each D4Z4 unit. The 3rd DUX4 exon and PAS site are found directly downstream of the most distal D4Z4 unit. Compromised repression results in low-level DUX4 protein expression existing within the skeletal muscle, perturbing downstream gene expression to cause the FSHD pathology (oxidative stress, apoptosis,…

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  Figure 2. An overview of oligonucleotide therapies for FSHD and where they inhibit DUX4 expression (gene, mRNA, or protein) to ameliorate FSHD symptoms. (1) sgRNA-dCas9-TRD targets the promoter or coding region of the DUX4 gene, resulting in transcriptional repression. (2) U7-snRNA alters the specificity of a small nuclear ribonucleoprotein complex (snRNP) to inhibit DUX4 pre-mRNA maturation. (3) Antisense oligonucleotides bind to DUX4 mRNA, causing steric blocking (and downstream effects) or RNase H-mediated degradation, depending on the AO type. (4) Various RNA molecules (miRNA, siRNA, etc.) degrade DUX4 mRNA through the RNA interference pathway. (5) Decoy dsDNA molecules have DUX4-binding motifs that trap the DUX4 protein in a binding sink, inhibiting the transactivation of downstream DUX4 targets. (6) DNA aptamers bind to the DUX4 protein, inhibiting DUX4 activity through steric inhibition.

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  Figure 3. The mechanism of action for (A) AO and (B) RNAi therapies. (A) Antisense oligonucleotides (AOs) can degrade target mRNA transcripts by recruiting RNase H, or by inducing steric blocking. AO-mediated steric blocking of a target mRNA transcript can inhibit proper translation, splicing, and/or stability. Further downstream degradation pathways can be initiated on steric-blocked mRNA transcripts. (B) RNAi-based therapies (siRNAs, miRNAs, etc.) degrade target mRNA transcripts using the RNA interference (RNAi) pathway. RNAi can be induced by miRNAs or siRNAs complementary to an mRNA transcript. DICER processes the precursor molecules to produce miRNA or siRNAs, which then are loaded into the Argonaute (AGO) protein of the RNA-induced silencing complex (RISC). Using the guide strand, the RISC targets a complementary mRNA transcript and induces translational inhibition, sequestration, and/or mRNA degradation (miRNA-RISC) or simply mRNA degradation (siRNA-RISC).

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  Figure 4. An overview of the oligonucleotide target sites on DUX4 mRNA. Orange (antisense oligonucleotides) and purple (RNAi) lines indicate oligo target sites. Partially overlapping target sites are simply represented as a continuous line. All attempted target sites are included, even oligos that showed poor indications in corresponding studies. The figure is not to scale and approximate oligo sites/sizes are shown. (Top) A schematic representation of the DUX4 gene (downwards arrow indicates start codon; blue, open reading frame; boxes, exons; lines, introns; red line, polyadenylation signal). The distal D4Z4 unit, partial D4Z4 unit, and pLAM region are indicated by double-sided arrows. Note that the following groups used some or all of the same oligonucleotides (indicated by *): Vanderplanck et al. (2011), Ansseau et al. (2017), and Derenne et al. (2020) [25,29,30]; Marsollier et al. (2016) and Chen et al. (2016) [27,28]; Marsollier et al. (2016) and Falzarano et al. (2021) [27,34]; Lu-Nguyen et al. (2021), Lu-Nguyen et al. (2022a), and Lu-Nguyen et al. (2022b) [35,36,37]; Wallace et al. (2012) and Wallace et al. (2018) [39,40].