Therapeutic Strategy and Clinical Path of Facioscapulohumeral Muscular Dystrophy: Review of the Current Literature

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant genetic disease, which is caused by the mistaken expression of double homeobox protein 4 protein 4 (DUX4) in skeletal muscle. Patients with FSHD are usually accompanied by degenerative changes in the face, shoulders, and upper muscles, gradually accumulating in the lower limb muscles. The severity of patients is quite different, and most patients end up using wheelchairs and losing their self-care ability. At present, the exploration of treatment strategies for FSHD has shifted from relieving symptoms to gene therapy, which brings hope to the future of patients, but the current gene therapy is only in the clinical trial stage. Here, we conducted a comprehensive search of the relevant literature using the keywords FSHD, DUX4, and gene therapy methods including ASOs, CRISPR, and RNAi in the PubMed and Web of Science databases. We discussed the current advancements in treatment strategies for FSHD, as well as ongoing preclinical and clinical trials related to FSHD. Additionally, we evaluated the advantages and limitations of various gene therapy approaches targeting DUX4 aimed at correcting the underlying genetic defect.

1. Introduction

1.1. Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy found after Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) [1]. It affects about one in every 8000 people and is an autosomal dominant hereditary disease [2,3]. FSHD usually involves skeletal muscles of the face, shoulders, and upper limbs first, followed by muscles of lower limbs, and is characterized by progressive asymmetric muscular atrophy. In addition to muscle symptoms, some patients have lung diseases, hearing impairment, and retinopathy. The onset age and severity of FSHD vary greatly among different individuals; symptoms of FSHD usually appear in the second or third decade after birth, and the disease progresses slowly. Studies have shown that about 20% of mutation carriers are asymptomatic, while the final outcome of 20% of patients need wheelchair support, and the risk of fatigue and chronic pain is also increased. At present, there is no effective treatment in clinic, which mainly focuses on relieving the symptoms of the disease to improve the life qualities of patients.

1.2. Molecular Mechanism of FSHD

The genetic mechanisms that contribute to the onset of FSHD are extremely complicated, but they are characterized in common by the misexpression of the DUX4 gene in skeletal muscle, which consequently causes the muscle disease. Patients are classified as FSHD1 and FSHD2 based on different genetic mechanisms; approximately 95% of patients are categorized as FSHD 1. In patients with FSHD1 (Figure 1), it is caused by contraction of a distal array of microsatellite repeats on chromosome 4q35; in normal individuals, these regions usually consist of 11-100 3.3 kb-sized D4Z4 repetitive units containing two exons and a double homeobox protein 4 protein 4 (DUX4) gene throughout the open reading frame (ORF), which is typically hypermethylated in healthy individuals, while the D4Z4 repeats shrinks to 10 or fewer units and methylation is disrupted in FSHD1 [4]. FSHD2, about 5% of the FSHD, shrinkage of the D4Z4 array is not the primary factor; the reduction of this array does not even occur (Figure 1). The pathogenic causes of FSHD2 are more related to gene mutations encoding D4Z4 array methylation proteins; for example, most FSHD2 patients carry mutations in SMCHD1, which mainly encodes proteins that inhibit chromatin structure, and mutations in DNMT3B, which encodes DNA methyltransferase [5,6], resulting in hypomethylation of D4Z4 sequences. Both FSHD1 and FSHD2 cause chromatin relaxation and misexpression of the DUX4 in skeletal muscle [7,8] (Figure 1), ultimately leading to degenerative necrosis of skeletal muscle fibers.

1.3. Pathological and Physiological Functions of DUX4

DUX4 plays an important role in both physiology and disease. DUX4 is a transcription factor that is highly conserved in primates such as orangutans and humans. DUX4 participates in important regulatory roles during early development of the human embryo [9,10] (Figure 2), and thereafter is repressed in all tissues except for testis and thymus [9,11] (Figure 2). The expression of DUX4 in skeletal muscle is similar to the transcriptional procedure in the embryonic stage, but it has a damaging effect on skeletal muscle. Studies have found that a large amount of DUX4 is distributed irregularly in the myoblasts of FSHD patients; with the development of myoblasts, the expression of DUX4 is further increased, resulting in the degeneration and necrosis of muscle cells. When abnormally activated, DUX4 can induce a variety of genetic processes, such as inflammation [12], oxidative stress [13,14], and termination of muscle development. In addition, DUX4 is also associated with a variety of diseases, including viral infections [15], acute lymphoblastic leukemia [16], and tumors [17].

2. Therapeutic Approaches

Currently, there are no clinically approved drugs that can cure FSHD. Most treatments focus on alleviating symptoms and enhancing patients’ quality of life, including physical therapy, physical exercise, and scapular immobilization. Until DUX4 was identified as the causative gene of FSHD in recent years [4], therapies are shifting from non-targeted therapies to approaches that target the DUX4 gene. Previous treatments for FSHD have focused on improving patients’ existing muscle function with drugs, including prednisone, beta-2 agonists, myostatin inhibitors, and anti-inflammatory and antioxidant agents.

2.1. β2-Agonists

β2-adrenergic agonists (β2-agonists) can improve muscle mass in animals and healthy individuals [18,19]. There was a randomized, double-blind placebo clinical trial of the β2 agonist salbutamol for FSHD [20], which divided patients into placebo and intervention groups: Patients were treated with salbutamol 8.0 mg twice daily or 16.0 mg salbutamol twice daily for one year, and their muscle function was assessed with the primary assessment being the Maximum Voluntary Isometric Contraction Test (MVICT), and the secondary assessments including changes in manual muscle testing (MMT) strength, grip strength, function, and muscle mass. The results showed no improvement in patients’ overall strength, but grip strength was improved in both dose treatment groups, and muscle mass increased in the high-dose group, while the muscle volume and mass in the placebo group decreased, indicating that salbutamol can improve the muscle function of FSHD to some extent though it’s not obvious. However, it is difficult to confirm whether the disease progress can be delayed for a long time, and the desensitization of β2-adrenoceptors caused by long-term administration may lead to the weakening of drug targeting and affect the curative effect.

2.2. Myostatin Inhibitors

Myostatin was discovered as a new member of TGF-β family in 1997 [21]. It is an important negative regulator of muscle growth [22]. Myostatin is mainly expressed in skeletal muscle, and the level in non-muscle tissue is low [21,23]. Preclinical trials have been conducted to treat myopathy by inhibiting myostatin. In research on treating SMA model mice with myostatin [24], it was found that the muscle strength of gastrocnemius, tibialis anterior, and triceps brachii of mice after treatment was improved, and the exercise ability was also improved. In addition, there was a phase I/II randomized, double-blind placebo trial that studied an intervention in 42 FSHD patients using MYO-029 [25], an antibody that targets muscle growth inhibitors, and the study assessed muscle strength and function by manual muscle testing/(MMT) and quantitative muscle testing (QMT), the result showed that some of the patients had an increase in muscle mass that was not accompanied by muscle function improvement. In conclusion, the therapeutic effect of myostatin in FSHD was not satisfactory. In recent years, with the deepening of the understanding of myostatin, a variety of myostatin drugs have entered the clinical trial stage, which is widely used in various diseases, including muscular dystrophy, cachexia, aging-related muscular atrophy, type II diabetes [26,27,28,29].

2.3. Anti-Inflammatory and Antioxidant Treatment

There has been various histological evidence that the markers of inflammation and oxidative damage are increased in the muscles of FSHD patients through the muscle biopsy of FSHD patients [30,31,32,33], and the level of proinflammatory factors such as TNFα is detected in the muscles of FSHD patients, which is related to the spontaneous contraction ability of skeletal muscle [14]. Oxidative stress is one of the characteristics of various muscular diseases such as DMD, myotonic dystrophy (DM1), and limb girdle muscular dystrophies (LGMD) [34,35,36]. In vitro studies [37,38] have shown that FSHD myoblasts are usually able to repair moderate oxidative damage under normal conditions, but they are unable to complete it when the level of oxidative stress is too high. In addition, two prominent extramuscular features of FSHD (retinal telangiectasia and sensorineural hearing loss) are also closely related to oxidative stress [39,40]. Therefore, targeting the pathological damage in patients’ muscles by reducing inflammation and oxidative stress is one of the effective treatment strategies.

Glucocorticoid is a kind of corticosteroid that has strong anti-inflammatory properties. It was first discovered in the blood of patients with Cushing’s disease in 1949, and high levels of cortisol have been found to alleviate inflammation in patients with arthritis [41]. Clinical trials have been conducted to evaluate the effects of prednisolone in FSHD patients [42,43,44]; muscle biopsies indicate that after the intervention, patients either experienced a worsening of their condition following initial improvement or showed no significant changes, the study was inconclusive and there was a large amount of variability between individuals. Considering the side effects of long-term use of glucocorticoids, including osteoporosis, immunosuppression, and adrenal atrophy, this strategy cannot be used as a viable long-term solution.

Conventional antioxidant therapy can alleviate pathological levels in FSHD patients both in vitro [45,46,47] and in vivo [48,49], although with modest efficiency. Studies have utilized three different antioxidant compounds—vitamin C, coenzyme Q10, and mitoTEMPO—to treat the myotubes of FSHD patients, detecting the levels of reactive oxygen species (ROS) before and after the intervention [48]. The results suggested that all three compounds reduce the ROS level of myotubes with similar efficiency, which proves that the pathological mechanism of redox directly targeting FSHD can have a direct and beneficial impact on myogenesis of patients, although this mechanism is not clear. In addition, nutritional supplements such as vitamin E, zinc gluconate, and selenomethionine can enhance the antioxidant defense of FSHD patients, which can significantly improve the maximum voluntary contraction ability and endurance of bilateral quadriceps.

Since DUX4 gene was identified as the pathogenic gene of FSHD, the therapeutic strategy of FSHD is changing to targeted therapy [50]. More studies have targeted DUX4 to treat FSHD. FSHD is caused by the abnormal expression of DUX4, so effectively inhibiting the expression of DUX4 may cure FSHD. Based on the pathological mechanism of FSHD, both upstream and downstream of the DUX4 are considered potential therapeutic targets. We sort out the latest targeted therapies as follows.

3. Genetic Approaches

3.1. Epigenetic and Molecular Pathway for DUX4

The expression of DUX4 can be inhibited by enhancing the epigenetic suppression of D4Z4 repeats or by inhibiting upstream signals. Overexpression of SMCHD1 in myotubes of FSHD1 and FSHD2 can inhibit DUX4 expression [51], indicating that specific delivery of SMCHD1 in muscle tissue may be a treatment scheme, and small molecule drugs for enhancing the activity of SMCHD1 are being developed. In recent years, through the screening of chemical libraries and genomes, many cascade reactions have been revealed, and compounds that can inhibit DUX4 expression have been identified, such as bromine domain and terminal external (BET) inhibitors, β2 adrenergic receptor agonists, phosphodiesterase (PDE) inhibitors, p38 inhibitors, and Wnt agonists [52,53,54,55,56] (Figure 3). As for molecule drugs, only one small molecule drug has entered clinical trials in Europe and America: losmapimod, an inhibitor of p38 signal, which is also the first drug targeting DUX4. At present, phase I clinical trials [57] (

Table 1. Overview of research on the suppression of DUX4 using different therapeutic strategies and their main results, future directions, and potential. (↑: up regulation. ↓: down regulation).

Genetic Approaches
	Route	Results	Potentials	Future Focus
ASOs	PMO targeting-DUX4 Marsollier et al. Chen et al. [59,60]	DUX4 transcript ↓ target gene ↓	The synthesis is relatively easy, and the cost is low. ASOs can be delivered in many ways, including intravenous injection and local injection, with high flexibility.	An accurate delivery system ensures more efficient delivery efficiency. To evaluate the long-term drug tolerance of patients and reduce the side effects on patients. Reduce off-target effect and immunogenic reaction.
	LNA and (2′MOE) gapmerASOs targeting-DUX4 Lim et al. [61,62]	Immortalized FSHDpatient-derived muscle cells: DUX4 transcript ↓ target gene ↓ Mouse model: DUX4 transcript ↓
	cEtASO targeting-DUX4 Bouwman et al. [63]	DUX4 transcript ↓ DUX4 protein ↓ The pathology of skeletal muscle in mice was improved.
RNAi	siRNA targeting-DUX4 Lim et al. [64]	DUX4 transcript ↓	Compared with other methods, it can effectively reduce the expression of DUX4. Able to exert rapid therapeutic effects.	Enhance the precision of RNAi drug delivery. Reduce-immunogenic reactions.
	siRNA targeting-DUX4 [65]	Primary myoblasts: DUX4 protein ↓ DUX4 downstream target ↓
	siRNA targeting- FRG1(Candidate genes causing FSHD in animals) Wallace et al. [66]	FRG1 transcript ↓ Muscle mass and Pathology improved.
Crispr	CRISPR/dCas9 targeting D4Z4 sequence Himeda et al. [67]	DUX4 transcript ↓	Precise gene editing. Capable of providing long-term therapeutic effects. Not only can it be used for deleting or replacing genes, but also for regulating gene expression.	Targeting precision: Next-generation CRISPR tools will provide higher targeting precision and reduce off-target effects. Multiplex Editing: Future developments may lead to CRISPR technologies capable of simultaneously targeting multiple gene loci. Reduce off-target effects and immunogenic responses.
	CRISPR/ dCas9 targeting D4Z4 activators(BAZ1A, BRD2, KDM4C, SMARCA5) Himeda et al. [68]	Myocardial cells derived from FSHD: DUX4 transcript ↓ target gene ↓
	Correcting SMCHD1 mutation with Crispr/dCas9 Goossens et al. [69]	SMCHD1 transcript ↑ DUX4 transcript ↓ target gene ↓
	CRISPR/Cas13 targeting DUX4 Rashnonejad et al. [70]	DUX4 transcript ↓ The pathology of skeletal muscle was improved.
Small compounds drugs	BET inhibitors Campbell et al. [53]	DUX4 transcript ↓ target gene ↓	Convenient administration method. Good cell membrane permeability.	Develop efficient screening platforms to discover potential small-molecule drugs Determine effective doses while minimizing side effects.
	p38 inhibitors(I) Mellion et al. [57]	FSHD myotubes:DUX4 transcript ↓ Skeletal muscle cell necrosis ↓
	p38 inhibitors(IIb) Tawil et al. [57]	losmapimod is safe and well tolerated in adults.

) have been carried out. Through oral administration of losmapimod to healthy volunteers and FSHD patients, the results show that losmapimod can reduce the expression of DUX4 gene in the myotubes of FSHD patients in a dose-dependent manner and inhibit the death of skeletal muscle cells. Losmapimod is well tolerated in patients. The latest phase IIb clinical trial (Table 1) of losmapimod (ReDUX4) has been completed [58], which is one of the largest clinical trials of FSHD intervention so far. The study is a 48-week randomized, double-blind, placebo trial, and the subjects were recruited from 17 neurological centers across the United States, France, Canada, and Spain, comprising a total of 80 FSHD1 patients. The preclinical experiment evaluated the safety and effectiveness of losmapimod in FSHD patients. The results showed that repeat dosing of losmapimod was safe and well tolerated in adults. This preclinical trial was helpful in designing the phase III study of losmapimod in the treatment of FSHD, which may make losmapimod a candidate drug for the treatment of FSHD. After the next round of clinical trials, this may make losmapimod a candidate drug for the treatment of FSHD.

3.2. Inhibition of DUX4 at mRNA Level

The transcription of DUX4mRNA can be directly reduced by targeting RNA level, including antisense oligonucleotide (ASOs) or RNA interference (RNAi) therapy (Figure 3). Oligonucleotides can inhibit DUX4 expression in many ways; the most widely tested is an antisense oligonucleotide (AOs). ASO has been proven to successfully target DUX4 pre-mRNA in immortalized FSHD myocytes and restrain its polyadenylation, resulting in the degradation of DUX4 mRNA [71]. Marsollier et al. [59] and Chen et al. [60] (Table 1) tested the efficiency of different phosphorodiamidate morpholino oligomers (PMOs) targeting DUX4 transcripts. Both studies identified two PMOs that effectively suppressed DUX4 and target gene expression in FSHD myotubes cultures. Lim et al. (Table 1) [61,62] showed the use of locked nucleic acid (LNA) and 2′-O-methoxyethyl (2′-MOE) gapmer ASOs that support the breakdown of DUX4 mRNA by RNase H in immortalized myotubes, and in vitro experiment, LNA and 2′-MOE ASOs was injected into tibialis anterior of FLExDUX4 mice. The results showed that both ASOs could reduce the DUX4 level of injected muscle. Additionally, a study [63] tested a cEtASO targeting the open reading frame of exon 1 of DUX4 transcripts in ACTA1-MCM mice (expressing low levels of DUX4). In this study, the DUX4 ASO was administered via subcutaneous injection for systemic delivery. The results showed significant reductions in DUX4 mRNA; protein levels in the quadriceps, triceps brachii, gastrocnemius, and tibialis anterior muscles; and improvements in skeletal muscle pathology. Unfortunately, there are currently no clinical trials for ASO-based treatments for FSHD, but these findings suggest that systemic delivery of ASOs targeting DUX4 is a promising strategy.

Another method is RNA interference, which specifically realizes mRNA degradation or destruction through complementary binding with the target RNA sequence. (RNAi) therapies utilizing small interfering RNA (siRNA) were used to target the 3′ untranslated region transcribed from pLAM, the coding region, and the region upstream of the DUX4 transcription start [64,65,72] to degrade the level of DUX4mRNA.To date, FRG1 is the only candidate gene identified to induce FSHD in animal models. A study has developed FRG1-targeted microRNA vectors (miFRG1) [66] (Table 1); in vitro experiments demonstrated that the expression of FRG1 was significantly reduced in mice with FRG1 overexpression. Additionally, the in vivo delivery of miFRG1 via AAV6 further decreased FRG1 expression and improved the muscle quality and histopathology of the mice, thereby validating the feasibility of developing RNAi-based gene therapy for the treatment of FSHD.

3.3. Crispr

The advent of Crispr/cas9 technology has brought hope to the treatment of FSHD, which can accurately edit the error genes. Hineda et al. [67] first reported the improvement of pathogenic gene expression in FSHD using CRISPR (Table 1). The study overcame the technical barriers of infecting primary myoblasts using a centrifugation-based continuous infection technique, successfully delivering CRISPR/Cas9 to target the D4Z4 repeat sequence in FSHD, and altered chromatin and DUX4-fl expression, demonstrating the efficacy of CRISPR technology in correcting the epigenetic dysregulation in FSHD. They used the same method to suppress DUX4 activators [68] BAZ1A, BRD2, KDM4C, and SMARCA5, confirming that targeting DUX4 activators reduced DUX4 expression in FSHD cardiomyocytes (Table 1). They also treated FLExDUX4 mice using AAV delivery of CRISPR/Cas9 combined with local muscle injection [73]. The results showed a reduction in DUX4 mRNA and decreased expression of DUX4 target genes following treatment. It is important to note that this method does not directly inhibit the DUX4 gene, but rather improves downstream expression by suppressing the epigenetic factors of DUX4. Crispr has been used to correct the FSHD2-related SMCHD1 mutation [69] (Table 1; Figure 3), which is a missense variant of intron 34, and an extra-frame 53-bp pseudoexon has been introduced into the final transcript. Crispr/Cas9 with guide RNA (gRNA) targeting the flanking intron sequence of the pseudoexon restored the reading frame of SMCHD1 and increased the expression of wild-type SMCHD1 in myotubes of primary and immortalized patients, resulting in the decrease of DUX4 mRNA expression. Crispr can also be used for targeted regulation of gene expression. In a myotube study of myoblast differentiation in primary patients treated, 45% DUX4 knock-down was achieved [67], and an increasing trend of chromatin inhibition of DUX4 at the contraction site was observed. Crispr/Cas9 was also used in genome-wide knockout screening to identify genes that can inhibit DUX4 cytotoxicity [74]. A recent study [70] developed AAV6-CRISPR-Cas13 to silence DUX4 mRNA, showing that DUX4 mRNA was reduced by over 50% in FSHD mice after delivery and improved histopathological outcomes. The study also considered the host immune response, finding a significant immune response in wild-type mice, including immune cell infiltration and proinflammatory factors. In summary, previous studies have clearly demonstrated that CRISPR can target and reduce DUX4 expression in FSHD muscle, but it is essential to minimize the immune response.

Some new therapies have been developed vigorously in recent years, but only in the clinical trial stage, so there is still a long way to go before clinical application. We analyze the advantages and disadvantages of various treatment schemes in clinical application and discuss the problems that must be solved when they are applied in clinic.

4. Path to Clinic

4.1. Small Molecular Drugs

It is one of the promising strategies for synthesizing small molecular drugs by targeting molecular signal pathways. Compared with biological agents, small molecule drugs are easier to synthesize, lower in cost, and usually more easily absorbed by patients, with stable properties and no immunogenicity. In addition, this kind of treatment mode is more mature in clinic and safer than other treatment approaches.

Small molecule drugs may cause both off-target toxicity and on-target toxicity; the latter is particularly troublesome for targets that are widely expressed and play a key role in cells. The p38 inhibitor losmapimond demonstrated this; losmapimond, one of the most anticipated small molecule drugs at present. It inhibits DUX4 expression by targeting p38 pathway. The paradox is that p38 is also involved in regulating multiple stages of myogenesis [75,76,77]; hence, the dose of losmapimond is required to repress the expression of DUX4 without affecting muscle regeneration, but it’s not easy to control. Fortunately, even slight changes in upstream factors, such as epigenetic regulatory factors, can significantly inhibit the expression of DUX4 [68], which may indicate that lower doses can also have therapeutic effects and be safer while reducing off-target and target toxicity. Targeted drugs such as BET inhibitors and losmapimond have been clinically approved by the FDA, and although they have been shown to be harmless for some symptoms, they are not the safest and most effective treatments in the long term.

4.2. Oligonucleotide Drugs

Oligonucleotide therapies can directly inhibit DUX4 expression, targeting based on the target sequence, realizing personalized treatment, and can be stopped at any time if adverse events are encountered. However, this therapy carries the risk of off-targeting and cytotoxicity, inefficient muscle cell uptake, and less efficient drug delivery to tissues except the liver. All oligonucleotide drugs have the potential for cytotoxicity and immunogenicity. miRNAs are inherently less specific than ASOs, increasing the risk of off-targeting. Oligonucleotide drugs are costly for the treatment of FSHD, and there is currently no evidence to demonstrate their long-term effectiveness.

A variety of oligonucleotide drugs have been approved for the treatment of muscular atrophy such as DMD. This approach has reached a relatively advanced stage for treating clinical indications, and the development of oligonucleotide therapy for FSHD is actively underway. In the process of oligonucleotide therapy going to clinic, two problems that urgently need to be solved are the choice of delivery carrier and precise drug dosage. When drugs are delivered to patients through weakly targeted carriers, autoimmune reactions will cause serious inflammatory reactions such as myositis and myocarditis, and the liver toxicity caused by off-target needs particular attention. Studies have shown that high-dose injection of AAV9 drugs has caused serious toxicity in rhesus monkeys and piglets, and acute liver failure and shock have occurred [78]. Therefore, to alleviate the extramuscular reaction, it is necessary to optimize the delivery vector, including transforming capsid protein and modifying regulatory elements, which can not only improve the transduction efficiency and targeting of the vector but also reduce the immune response. At the same time, it is still necessary to strictly control the drug dosage and assess the off-target toxicity. In a word, to cure FSHD clinically with oligonucleotide drugs, the toxic effects must be minimized when providing therapeutic effects.

4.3. Crispr

It can accurately edit the pathogenic mutant genes, and it is possible to achieve a permanent cure for all forms of FSHD through one-time treatment. In the long run, Crispr is more economical than other gene therapies because it can be achieved in a single dose and will work for the rest of patients’ lives. However, delivery of Crispr/cas9 requires the participation of vectors, and currently, the main vector used is the AAV vector. In most populations, there are neutralizing antibodies against AAV, which create an immune response and render the drug ineffective [79]. As we mentioned in the last section, high-dose drug delivery using a virus vector can cause off-target effects and adverse events related to hepatotoxicity. Off-target Crispr will lead to irreversible changes in genetic material, and editing non-target fragments may lead to dysfunction of key proteins. At present, variants of Cas9 have been developed that can only disrupt single-stranded DNA, while targeting double-stranded DNA allows for precise editing of that region, significantly reducing mutations in non-targeted regions [80]. Additionally, research has developed Cas13 to target and cleave RNA instead of DNA, thereby avoiding the potential risks of permanent off-target gene editing associated with DNA-targeting systems [70]. The use of Crispr tools requires strict specificity testing, and the rapid development of controllable specificity editing improves the security of Crispr tools [81]. At present, the urgent clinical problem may be to reduce immunogenicity and increase the efficiency of delivering skeletal muscle while detargeting the liver.

5. Limitations

The individual differences of patients with FSHD have not been fully considered in many studies, which leads to a simple understanding of the treatment effect and may cause us to doubt the feasibility of the treatment plan. Furthermore, the absence of long-term follow-up data in many studies restricts our ability to evaluate the enduring effects and safety of the treatments discussed. Lastly, while multiple treatment strategies were considered, the review may not have fully explored the advantages and disadvantages of each approach, warranting further investigation in future studies.

6. Conclusions and Prospect

The disease mechanism of FSHD was once a mystery. With the in-depth study of FSHD in recent years, considerable progress has been made in the study of the disease mechanism. The treatment of the disease has shifted from relieving symptoms to targeted treatment and is moving towards clinic, making it possible to cure FSHD. The treatment based on oligonucleotide and Crispr have proved the feasibility of DUX4 knock-down in reversing the pathology of FSHD, but these therapies still face many problems before they are applied to patients. In this paper, we elaborate on the emerging therapies in recent years and discuss the pathway to clinical application of these approaches, which needs to consider the drug delivery, efficacy, and safety before they can be truly applied to patients. In addition, the early exploration of symptomatic relief strategies in FSHD has also achieved certain results that are equally important. Stem cell therapy has become an important tool in the field of regenerative medicine due to its potential for self-renewal and multi-lineage differentiation. It also plays a significant role in the advancement of treating myopathy. In recent years, numerous studies have focused on developing stem cell therapies to repair damaged muscles and restore muscle function, particularly with mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), which have gained attention in FSHD treatment research. MSCs and iPSCs hold promise for replacing damaged muscle of FSHD patients with healthy muscle cells.

In fact, FSHD patients exhibit significant individual variability; therefore, we believe that all treatment plans should fully consider these individual differences. Based on the severity of the patient’s symptoms, treatment should not only involve editing the DUX4 gene or inhibiting DUX4 expression but also focus on alleviating the patient’s muscle symptoms, such as using β2 agonists or myostatin inhibitors or employing stem cell therapies to replace and rejuvenate damaged muscle.

Although we know that the underlying cause of FSHD may be genetic defects, a combination of various treatment options can provide the greatest benefits for patients. In a word, although many treatment strategies, including gene therapy and stem cell therapy, are still in development or preclinical stages, any ultimate treatment plan for rare diseases requires substantial, reliable preclinical data. Therefore, these studies and data are of great value. In the process of overcoming challenges associated with gene therapies mentioned in this review, the combined use of multiple therapies will bring more significant improvement to FSHD patients and comprehensively enhance their muscle function and self-care ability.