Oligonucleotide Therapies for Facioscapulohumeral Muscular Dystrophy: Current Preclinical Landscape

1. Introduction

1.1. FSHD Overview

Facioscapulohumeral muscular dystrophy (FSHD, MIM: 158900 & 158901) is an autosomal dominant myopathy with a global incidence of approximately 1 in 8000–22000, making it one of the most common forms of muscular dystrophy worldwide [1,2,3]. FSHD is characterized by progressive muscle weakness and atrophy that develops in an asymmetric fashion, primarily affecting muscles of the face, shoulder girdle, and upper arms. Additional muscle groups can be affected with age, such as the ankle dorsiflexors and proximal leg muscles, resulting in obligate wheelchair use for approximately 20% of patients [1,4]. Some FSHD patients also experience extramuscular symptoms such as hearing loss, retinal vasculopathy, and/or cardiac conduction defects. FSHD is highly variable in terms of disease onset and severity [4]. There are no curative treatments available for FSHD, with current interventions limited to managing symptoms [5].

FSHD is a genetic condition with two distinct types. Patients can be classified as having either FSHD1 or FSHD2 depending on the genetic mechanism that results in the de-repression of the D4Z4 macrosatellite repeat array, located in the subtelomeric region 4q35 (Figure 1A). Healthy individuals have 11-100 3.3-kb D4Z4 repeat units. FSHD1, affecting 95% of patients, is caused by array contraction to ≤10 repeats [6]. FSHD2, affecting 5% of patients, is caused by mutation in genes involved in epigenetic methylation of the D4Z4 array (E.g., SMCHD1, DNMT3B, and LRIF1) [7,8,9]. Curiously, FSHD2 patients also tend to have fewer D4Z4 repeats than healthy individuals (12-16), demonstrating the complexity of this condition and how the distinction between FSHD1 and FSHD2 may not be as straightforward as initially thought. Regardless, the shared outcome in both types of FSHD is the loss of repressive methylation in the D4Z4 array. Therefore, clinical presentation is identical between FSHD1 and FSHD2 [4].

1.2. DUX4 Is the Central Cause of FSHD

FSHD is caused by the aberrant expression of double homeobox 4 (DUX4) protein in adult skeletal muscle, arising from the loss of repressive methylation in the D4Z4 array. The DUX4 gene encodes a transcription factor that is normally involved in zygotic genome activation during the 4-cell stage of early embryonic development [10,11]. Afterwards, DUX4 is epigenetically silenced in all adult tissues apart from limited expression of unrelated DUX4 isoforms in the testis and thymus [12,13]. Alternative splicing is known to occur for the DUX4 transcript, however, only mis-expression of the full-length isoform in muscle tissue is relevant to FSHD. Any mention hereafter of DUX4 mRNA is referring only to this full-length, pathogenic isoform.

Narrowing in on the genetic region from which FSHD arises, we return to the D4Z4 repeat array and the DUX4 gene therein. Within each 3.3-kb D4Z4 repeat unit is a retrogene containing exons 1 and 2 of the DUX4 open reading frame. A partial D4Z4 unit occurs after the most distal full unit, followed by the 3rd DUX4 exon (Figure 1B) [14]. Aberrant DUX4 expression occurs off this most distal D4Z4 unit in the FSHD-permissive 4qA haplotype. FSHD can only manifest in one of two major 4q allele variants: 4qA and 4qB. Unlike the non-permissive 4qB haplotype, the 4qA haplotype contains the pLAM region with a polyadenylation site (PAS), allowing transcription of a stable DUX4 mRNA when epigenetic repression is compromised in the D4Z4 array [1,14].

Following transcription of the DUX4 gene, stochastic, low-level DUX4 protein occurs in myofiber nuclei of the skeletal muscle. Inappropriate DUX4 protein in adult skeletal muscle is highly toxic, driving gene programs that result in oxidative stress, dysregulated transcript quality control, protein aggregation, inflammation, apoptosis, impaired myogenesis, and muscle atrophy (Figure 1B) [14,15,16]. DUX4 protein directly activates various genes including TRIM43, ZSCAN4, MBD3L2, WFDC3, PRAMEF1, RFPL2, and KHDC1 [17,18]. Disruption of these signaling pathways by reactivated DUX4 produces the FSHD pathology that we see in patients, manifesting primarily in the muscle tissue. In this review, we discuss targeted therapies aimed at removing or inhibiting this mis-expressed DUX4 protein, summarize the preclinical results obtained so far, and discuss further considerations for these treatment approaches.

2. Oligonucleotide Therapies Targeting DUX4

FSHD is a condition arising solely from the aberrant reactivation of a dormant gene: DUX4. Therefore, therapies that directly target aberrant DUX4 expression present a compelling treatment option. This review focuses on preclinical FSHD therapies that use a sequence-specific approach for targeting DUX4. This includes oligonucleotides with sequence complementarity to either the DUX4 gene, DUX4 mRNA, or DUX4 protein. This complementarity is used to inhibit DUX4 somewhere along its gene>mRNA>protein expression axis, thereby preventing DUX4 transactivation and the resulting FSHD pathology (Figure 2). Notably, the scope of this review is limited to only non-gene editing approaches that target DUX4.

The targeted oligonucleotide therapies discussed in this review are divided into three categories: Antisense Oligonucleotides (AOs), RNA interference (RNAi), and Other. These therapies have shown promising preclinical results in cellular and murine models of FSHD, primarily in their ability to lower DUX4 mRNA levels, reduce DUX4-target gene expression, and alleviate FSHD symptoms (Table 1, Table 2 and Table 3).

2.1. Antisense Oligonucleotides (AOs)

Antisense oligonucleotides (AOs) are synthetic, single-stranded nucleic acids that target a complementary mRNA molecule, dictated by Watson-Crick base pairing, to initiate post-transcriptional gene silencing. AOs were first identified in 1978 by Zamecnik and Stephenson, who found that complementary oligonucleotides inhibited translation of Rous sarcoma virus mRNA [19]. AOs bind to a target mRNA in a sequence-dependent manner and prevent its translation, thereby reducing the amount of target protein [20].

AOs are known to utilize various chemistries, a fact that makes them distinct from other oligonucleotide therapies. Modern AO drugs have chemical modifications to improve pharmacological properties like tolerability, target affinity, nuclease resistance, and intracellular uptake [21,22]. Commonly used AO chemistries involve modifying the phosphate backbone (PS, phosphorothioate; PMO, phosphorodiamidate morpholino oligomer) or ribose sugar (2’OMe, 2’-O-methyl; 2′-MOE, 2′-O-methoxyethyl; LNA, locked nucleic acid) [21]. In addition, peptide and fatty acid conjugate modifications have been used to facilitate delivery of DUX4-targeting AOs [23,24]. AOs can also be synthesized as gapmers: a chimeric molecule comprised of a central DNA region and a flanking region of modified RNA [25].

The formation of an AO-mRNA duplex results in (1) RNase-H mediated degradation of the target mRNA, or (2) steric blocking of the target mRNA (Figure 3A) [20,22,26]. Only gapmer AOs recruit RNase H to cleave target mRNA. Gapmer AOs produce a DNA:RNA substrate when bound to mRNA, recognizable by RNase H [27,28]. Steric blocking AOs inhibit proper mRNA translation, splicing and/or stability in an RNase H-independent manner [20,22,26]. Additionally, these steric blocking AOs can initiate further downstream degradation pathways, such as nonsense-mediated decay and no-go decay [29,30].

As summarised in Table 1, numerous preclinical studies have demonstrated that AO therapies can effectively reduce the amount of DUX4 mRNA and DUX4 target gene expression both in vitro and in vivo [23,24,31,32,33,34,35,36,37,38,39,40,41,42]. Other measures were also used to evaluate AO treatment efficacy, such as DUX4 protein levels, muscle fibre health, and murine functional performance. Most groups used primary or immortalized myoblasts/myocytes (often differentiated into myotubes) as an in vitro FSHD model, and FLExDUX4 mice as an in vivo FSHD model [43,44,45]. Earlier studies used local (intramuscular) injection for in vivo AO treatment, while studies after 2021 evaluated systemic (intraperitoneal, subcutaneous) injection routes. Systemic injection, being more clinically viable, managed to yield a similarly efficient DUX4 knockdown compared to local injection. Regarding the DUX4 target site, the >10 studies produced since 2011 tend to target exons 2 and 3, often with an emphasis on the polyadenylations site (PAS), pre-mRNA cleavage sites, and/or splice sites therein (Figure 4).

2.2. RNA Interference (RNAi)

Like AOs, RNAi-based oligonucleotides act at the RNA level, binding to a target mRNA according to antisense sequence complementarity to initiate post-transcriptional gene silencing. Where these classifications differ is that RNAi-based oligonucleotides initiate the RNA interference (RNAi) pathway to knockdown target mRNAs. First defined by Fire and Mello in 1998, RNAi is a conserved, biological mechanism by which double-stranded RNA triggers the loss of homologous mRNA [46]. RNAi can be induced by miRNAs or siRNAs complementary to a mRNA transcript. DICER endonucleases cleave precursor molecules (pre-miRNA or dsRNA) to produce mature microRNA (miRNA) or small-interfering RNAs (siRNA), which then get loaded into the Argonaute (AGO) protein of the RNA-induced silencing complex (RISC). Using the guide strand, RISC targets a complementary mRNA transcript and induces translational inhibition, sequestration, and/or mRNA degradation (miRNA-RISC) or simply mRNA degradation (siRNA-RISC) (Figure 3B) [46,47].

The two types of RNAi-based oligonucleotide therapies for FSHD are miRNAs (natural or artificial) and siRNAs. Since 2011, several studies have shown that these RNAi-based oligos can knockdown DUX4 mRNA and reduce DUX4 transactivation, in addition to improving other markers of FSHD symptom reversal (E.g., DUX4 protein levels, muscle fibre health, murine functional performance, etc.) (Table 2) [31,32,48,49,50,51,52,53,54,55,56,57]. Notably, the amount of published work is less for RNAi-based approaches compared to AOs. These studies commonly use primary or immortalized myoblasts/myocytes (often differentiated into myotubes) as an in vitro FSHD model, and AAV-DUX4 mice as an in vivo FSHD model [43,44,58]. FLExDUX4 mice were also used as an in vivo model for testing RNAi therapies, but to a lesser extent [45]. Most studies used local (intramuscular) injection to evaluate preclinical efficacy in vivo, except for two groups with partially released findings using systemic (intravenous) injection [51,55]. All current preclinical studies opted for adeno-associated virus (AAV)-mediated delivery of siRNAs or miRNAs in vivo, except for the partially released findings from Avidity Biosciences, Inc. and Dyne Therapeutics, Inc., who each describe a proprietary anti-mTfR1 mAb conjugate for delivery [54,55,56,57]. Lastly, as summarized in Figure 4, these miRNAs and siRNAs target DUX4 mRNA at all 3 of its exons, especially exon 1. Other sites like upstream D4Z4 regions, intronic regions, and pre-mRNA cleavage sites have also been tested.

2.3. Other

Other non-gene editing, preclinical oligonucleotide therapies have also been investigated as potential treatments for FSHD (Table 3). Unlike AOs and RNAi therapies which target DUX4 mRNA, these oligos tend to target DUX4 expression at the gene or protein level (Figure 2). These approaches present further compelling options for treating FSHD, in addition to the antisense approaches previously discussed.

2.3.1. CRISPR/dCas9 Transcriptional Repression

Multiple research groups have explored CRISPR/dCas9-mediated transcriptional repression of the DUX4 gene as a targeted therapy for FSHD. This is a form of CRISPR inhibition (CRISPRi) which uses the sequence-specificity of the sgRNA-Cas9 complex to target the DUX4 promoter, but with a catalytically inactive, ‘dead’ Cas9 (dCas9) fused to a transcriptional repressor domain (TRD) [59]. This allows for specific re-silencing of the D4Z4 region, reducing DUX4 expression and the resulting FSHD pathology. While various TRDs have been used, most studies opted for the Krüppel-associated box (KRAB) domain. Evaluation of this treatment approach has been largely done in primary FSHD myoblasts, myocytes, or myotubes, with the only in vivo testing performed by Himeda et al. (2021) using AAV-delivery and local (intramuscular) injection in FLExDUX4 mice [60]. These studies have all reported reduction in DUX4 mRNA following treatment, as well as reduction in DUX4 target gene expression and/or increased H3K9 tri-methylation at the D4Z4 array [60,61,62,63,64]. Notably, rather than directly repressing the DUX4 gene, Himeda et al. (2018) used the CRISPR/dCas9-KRAB system to repress epigenetic activators of DUX4 (ASH1L, BRD2, KDM4C, SMARCA5). Similar reduction of DUX4 mRNA was observed for this approach [62].

Abstracts proposing other non-gene editing, CRISPR-based approaches have been recently published. Results are preliminary and not fully released, however. One group is developing CRISPR/Cas13-mediated cleavage of DUX4 mRNA, reporting effective DUX4 knockdown in vivo [65]. Another group suggests using CRISPR/Cas13-ADAR (adenosine deaminase acting on RNA)-mediated editing of DUX4 mRNA to create a C>U nonsense mutation [66]. No definitive results have been published at this time.

2.3.2. DNA Aptamers

Aptamers are single-stranded oligonucleotides that can bind to a specific protein or protein family thanks to their secondary and tertiary folding structure. The unique 3D conformation of an aptamer allows target interaction like that of an antigen and antibody [67]. Therefore, aptamers can be used to specifically target a protein of interest, and in the case of FSHD, bind to and inhibit DUX4. Klingler et al. (2020) designed DNA aptamers with high affinity to DUX4 protein [68]. While not evaluated, these DNA aptamers could be used to treat FSHD by sterically inhibiting DUX4 protein in skeletal muscle.

2.3.3. dsDNA Decoy Trapping

Mariot et al. (2020) demonstrated a unique approach to prevent DUX4 transactivation known as decoy trapping [69]. Decoy trapping uses double-stranded DNA fragments whose sequence corresponds to DUX4 binding motifs, akin to the DNA regions that DUX4 normally binds to as a transcription factor. By saturating the cellular environment with dsDNA decoy binding sites, DUX4 protein is trapped in a binding sink and unable to activate its normal target genes. Mariot et al. (2020) found that dsDNA treatment was able to reduce expression or downstream DUX4 target genes in vitro and in vivo [69].

2.3.4. U7-snRNA pre-mRNA Inhibition

Rashnonejad et al. (2021) describe a strategy to inhibit DUX4 mRNA expression using U7-small nuclear RNA (snRNA) antisense expression cassettes [70]. U7-snRNA is a part of the small nuclear ribonucleoprotein complex (snRNP), which is involved in 3’ end processing of histone pre-mRNAs in the nucleus. This therapeutic approach uses modified U7-snRNA with antisense sequence specificity to DUX4, capable of inhibiting pre-mRNA production or maturation. Rashnonejad et al. (2021) showed that these U7-snRNA expression cassettes, delivered by AAV, effectively reduced DUX4 mRNA, DUX4 protein, and DUX4 target gene expression in immortalized FSHD myotubes [70].

3. Further Considerations

Antisense therapies for FSHD, meaning AOs or RNAi drugs that target DUX4 mRNA, are the furthest along in preclinical development compared to other oligonucleotide approaches. Many AO and RNAi therapies have shown promising indications both in vitro and in vivo, as discussed previously. Therefore, discussion of certain advantages and disadvantages of DUX4-targeting oligonucleotide therapies will focus on antisense approaches only.

3.1. Advantages of Antisense Approaches

Antisense therapies are ideal for monogenic diseases that can be attributed to a single root cause. In the case of FSHD, this is aberrant DUX4 expression in adult skeletal muscle. DUX4 is an especially ideal therapeutic target for antisense therapies because it is practically absent in all adult tissues under normal, healthy circumstances, making concerns of unwanted DUX4 knockdown elsewhere in the body largely insignificant [12,13]. This, however, is not something that should be entirely ignored when evaluating candidate therapies for clinical trials. Overall, antisense therapies are highly specific and potent molecules with a relatively simple mechanism of action, often taking advantage of conserved cellular processes [20].

Compared to gene-editing approaches that prevent DUX4 expression, antisense therapies involve no changes to genomic DNA, acting only at the RNA level [20,26]. This makes them more acceptable from a regulatory standpoint, unburdened by the moral concern surrounding CRISPR/Cas9 editing of the human genome, even if for therapeutic purposes. For this reason, it may be fair to suggest that antisense therapies are a more clinically viable form of targeted, genetic therapy for FSHD.

3.2. Disadvantages of Antisense Approaches

Efficient delivery to muscle tissue is a considerable challenge for antisense therapies, often hindering the clinical utility of oligonucleotides that otherwise demonstrate good preclinical efficacy. AOs and RNAi oligonucleotides are relatively large nucleic acids that tend to be negatively charged and hydrophilic [21]. Molecules with such properties do not readily pass through the plasma membrane. Furthermore, upon systemic injection, these molecules must avoid nuclease degradation, mononuclear phagocyte system entrapment, protein entrapment, and high renal clearance [71,72,73]. If these can be overcome, there remains the issue of inefficient cellular uptake, as these oligonucleotides are also prone to endosomal entrapment within the cell [71,72,73]. All this means that only a small percentage of injected drug becomes bioavailable to provide therapeutic benefit to a patient. However, various strategies to improve delivery are currently being explored for AOs and RNAi oligos, including chemical modification, delivery conjugates, and carrier molecules [71].

Persistence of therapeutic effect is another notable disadvantage of antisense therapies. Given that antisense therapies act on the mRNA, a transient and replenishable molecule, regular lifelong administrations may be necessary to offer long-term reversal of FSHD symptoms for patients. This problem is not shared by other proposed genetic therapies that would permanently inactivate the toxic DUX4 gene (E.g., CRISPR/Cas9 editing) such that multiple treatments are not needed.

3.3. Early-Stage Clinical Trials for Select FSHD Therapies

Two RNAi-based oligonucleotide therapies for FSHD are currently recruiting for Phase 1/2 clinical trials to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy in adult patients. First, ARO-DUX4, developed by Arrowhead Pharmaceuticals, is a DUX4-specific siRNA using an unspecified and proprietary delivery method (Phase 1/2 NCT06131983) [74,75]. Second, AOC 1020, developed by Avidity Biosciences, is a DUX4-specific siRNA using a proprietary anti-mTfR1 mAb delivery conjugate (Phase 1/2 FORTITUDE™ NCT0574792) [76,77]. Both therapies have previously demonstrated preclinical efficacy in cellular and murine models of FSHD [51,52,54,55].

4. Conclusions

Since DUX4 was identified as the central cause of FSHD, numerous targeted oligonucleotide therapies have been proposed, many of which have shown promising results in preclinical stages. However, despite DUX4 presenting itself as an ideal therapeutic target, there are still considerable challenges that may prevent these therapies from reaching clinical use and benefiting patients. Additionally, more progress towards fully characterizing FSHD is needed, as it remains an incredibly complicated condition with many unanswered questions. Further research into the molecular underpinnings of FSHD may offer additional therapeutic targets amenable to oligonucleotide therapies. Similarly, it is important to continue investigating the normal physiological role of DUX4 in the testis and thymus, as this is not fully understood and could impact decisions made when targeting DUX4 in skeletal muscle, possibly in terms of off-target effects. Another consideration would be the potential synergistic effect of combining multiple therapies together, particularly those that target DUX4 expression via different modes of action. This has not yet been attempted for FSHD. Overall, with a strong pipeline of candidate oligos from many different research groups, and two siRNA drugs entering early clinical trials, the future appears hopeful for a targeted treatment option for patients with FSHD.