A therapeutic leap: how myosin inhibitors moved from cardiac interventions to skeletal muscle myopathy solutions.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of ß myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity's role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

Potential small effector molecules restoring cellular defects due to sialic acid biosynthetic enzyme deficiency: Pathological relevance to GNE myopathy.

GNEM (GNE Myopathy) is a rare neuromuscular disease caused due to biallelic mutations in sialic acid biosynthetic GNE enzyme (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase). Recently direct or indirect role of GNE in other cellular functions have been elucidated. Hyposialylation of IGF-1R leads to apoptosis due to mitochondrial dysfunction while hyposialylation of ß1 integrin receptor leads to altered F-actin assembly, disrupted cytoskeletal organization and slow cell migration. Other cellular defects in presence of GNE mutation include altered ER redox state and chaperone expression such as HSP70 or PrdxIV. Currently, there is no cure to treat GNEM. Possible therapeutic trials focus on supplementation with sialic acid, ManNAc, sialyllactose and gene therapy that slows the disease progression. In the present study, we analyzed the effect of small molecules like BGP-15 (HSP70 modulator), IGF-1 (IGF-1R ligand) and CGA (cofilin activator) on cellular phenotypes of GNE heterozygous knock out L6 rat skeletal muscle cell line (SKM­GNEHz). Treatment with BGP-15 improved GNE epimerase activity by 40 % and reduced ER stress by 45 % for SKM­GNEHz. Treatment with IGF-1 improved epimerase activity by 37.5 %, F-actin assembly by 100 %, cell migration upto 36 % (36 h) and atrophy by 0.44-fold for SKM­GNEHz. Treatment with CGA recovered epimerase activity by 49 %, F-actin assembly by 132 % and cell migration upto 41 % (24 h) in SKM­GNEHz. Our study shows that treatment with these small effector molecules reduces the detrimental phenotype observed in SKM­GNEHz, thereby, providing insights into potential therapeutic targets for GNEM.

Understanding pathophysiology of GNE myopathy and current progress towards drug development.

GNE myopathy is a rare genetic neuromuscular disease that is caused due to mutations in the GNE gene responsible for sialic acid biosynthesis. Foot drop is the most common initial symptom observed in GNE myopathy patients. There is slow progressive muscle weakness in the lower and upper extremities while the quadriceps muscles are usually spared. The exact pathophysiology of the disease is unknown. Besides sialic acid biosynthesis, recent studies suggest either direct or indirect involvement of GNE in other cellular functions such as protein aggregation, apoptosis, ER stress, cell migration, HSP70 chaperone activity, autophagy, muscle atrophy, and myogenesis. Both animal and in vitro cell-based model systems are generated to elucidate the mechanism of GNE myopathy and evaluate the efficacy of therapies. The many therapeutic avenues explored include supplementation with sialic acid derivatives or precursors and gene therapy. Recent studies suggest other therapeutic options such as modulators of HSP70 chaperone (BGP-15), cofilin activator (CGA), and ligands like IGF-1 that may help to rescue cellular defects due to GNE dysfunction. This review provides an overview of the pathophysiology associated with GNE function in the cell and promising therapeutic leads to be explored for future drug development.

Evaluation of N-Acetylmannosamine Administration to Restore Sialylation in GNE-Deficient Human Embryonal Kidney Cells.

BACKGROUND: A key mechanism in the neuromuscular disease GNE myopathy (GNEM) is believed to be that point mutations in the GNE gene impair sialic acid synthesis - maybe due to UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) activity restrictions - and resulting in muscle tissue loss. N-acetylmannosamine (ManNAc) is the first product of the bifunctional GNE enzyme and can therefore be regarded as a precursor of sialic acids. This study investigates whether this is also a suitable substance for restoring the sialic acid content in GNE-deficient cells. METHODS: A HEK-293 GNE-knockout cell line was generated using CRISPR-Cas9 and analyzed for its ability to synthesize sialic acids. The cells were then supplemented with ManNAc to compensate for possible GNE inactivity and thereby restore sialic acid synthesis. Sialic acid levels were monitored by immunoblot and high performance liquid chromatography (HPLC). RESULTS: The HEK-293 GNE-knockout cells showed almost no polysialylation signal (immunoblot) and a reduced overall (-71%) N-acetylneuraminic acid (Neu5Ac) level (HPLC) relative to total protein and normalized to wild type level. Supplementation of GNE-deficient HEK-293 cells with 2 mM ManNAc can restore polysialylation and free intracellular sialic acid levels to wild type levels. The addition of 1 mM ManNAc is sufficient to restore the membrane-bound sialic acid level. CONCLUSIONS: Although the mechanism behind this needs further investigation and although it remains unclear why adding ManNAc to GNE-deficient cells is sufficient to elevate polysialylation back to wild type levels - since this substance is also converted by the GNE, all of this might yet prove helpful in the development of an appropriate therapy for GNEM.

Pharmacokinetics and clinical efficacy of 6'-sialyllactose in patients with GNE myopathy: Randomized pilot trial.

GNE myopathy, caused by biallelic mutations in the GNE gene, is characterized by initial ankle dorsiflexor weakness and rimmed vacuoles in the muscle histopathology, resulting in reduced sialic acid production. Sialyllactose is a source of sialic acid. We performed a pilot clinical trial to analyze the pharmacokinetic properties of 6'-sialyllactose (6SL) and evaluated the safety, and efficacy of oral 6SL in patients with GNE myopathy. Ten participants were in the pharmacokinetic study, and 20 in the subsequent clinical trial. For the pharmacokinetic study, participants were administered either 3 g (low-dose) or 6 g (high-dose) of 6SL in a single dose. Plasma concentrations of 6SL, sialic acid, and sialic acid levels on the surface of red blood cells were periodically assessed in blood samples. Patients were randomly allocated to test (low- and high-dose groups) or placebo groups for the trial. Motor function, ambulation, plasma 6SL and sialic acid concentrations, GNE myopathy-functional activity scale scores, and MRI findings were assessed. 6SL was well tolerated, except for self-limited gastrointestinal discomfort. Free sialic acid in both low- and high-dose groups significantly increased at 6 and 12 weeks, but not in the placebo group. In the high-dose group, proximal limb powers improved with daily 6SL. Considering the fat fraction on muscle MRI, results in the high-dose group were superior to those in the low-dose group. 6SL may be a good candidate for GNE myopathy therapeutics as it induces an increase or reduces the decrease in limb muscle power, attenuates muscle degeneration, and improves the biochemical properties of sialic acid.

[Efficacy of Aceneuramic Acid for Distal Myopathy with Rimmed Vacuoles].

Distal myopathy with rimmed vacuoles (DMRV), also known as GNE myopathy, is a rare disease affecting the distal muscles, such as the tibialis anterior muscle. The GNE gene, which codes for a key enzyme in the sialic acid biosynthesis pathway, is mutated in a homozygous or compound heterozygous manner, and the lack of sialic acid in skeletal muscle is the critical underlying mechanism in DMRV pathogenesis. DMRV mouse models were established, and supplementation with sialic acid improved the phenotypes of the models. A phase 1 clinical trial using aceneuramic acid was conducted at Tohoku University Hospital, Japan, followed by trials using a slow-release product. A phase II/III study, subsequent extended trial, and confirmatory trial were also conducted. Regulatory approval is currently under review.

Efficacy confirmation study of aceneuramic acid administration for GNE myopathy in Japan.

BACKGROUND: A rare muscle disease, GNE myopathy is caused by mutations in the GNE gene involved in sialic acid biosynthesis. Our recent phase II/III study has indicated that oral administration of aceneuramic acid to patients slows disease progression. METHODS: We conducted a phase III, randomized, placebo-controlled, double-blind, parallel-group, multicenter study. Participants were assigned to receive an extended-release formulation of aceneuramic acid (SA-ER) or placebo. Changes in muscle strength and function over 48 weeks were compared between treatment groups using change in the upper extremity composite (UEC) score from baseline to Week 48 as the primary endpoint and the investigator-assessed efficacy rate as the key secondary endpoint. For safety, adverse events, vital signs, body weight, electrocardiogram, and clinical laboratory results were monitored. RESULTS: A total of 14 patients were enrolled and given SA-ER (n = 10) or placebo (n = 4) tablets orally. Decrease in least square mean (LSM) change in UEC score at Week 48 with SA-ER (- 0.115 kg) was numerically smaller as compared with placebo (- 2.625 kg), with LSM difference (95% confidence interval) of 2.510 (- 1.720 to 6.740) kg. In addition, efficacy was higher with SA-ER as compared with placebo. No clinically significant adverse events or other safety concerns were observed. CONCLUSIONS: The present study reproducibly showed a trend towards slowing of loss of muscle strength and function with orally administered SA-ER, indicating supplementation with sialic acid might be a promising replacement therapy for GNE myopathy. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT04671472).

Phase II/III Study of Aceneuramic Acid Administration for GNE Myopathy in Japan.

BACKGROUND: GNE myopathy is an ultra-rare muscle disease characterized by a reduction in the synthesis of sialic acid derived from pathogenic variants in the GNE gene. No treatment has been established so far. OBJECTIVE: We evaluated the safety and efficacy of oral supplementation of aceneuramic acid in patients with GNE myopathy. METHODS: This multicenter, placebo-controlled, double-blind study comprised genetically confirmed GNE myopathy patients in Japan who were randomly assigned into treatment groups of sialic acid-extended release (SA-ER) tablets (6 g/day for 48 weeks) or placebo groups (4:1). The primary endpoint of effectiveness was set as the change in total upper limb muscle strength (upper extremity composite [UEC] score) from the start of administration to the final evaluation time point. RESULTS: Among the 20 enrolled patients (SA-ER group, 16; placebo group, 4), 19 completed this 48-week study. The mean value of change in UEC score (95% confidence interval [CI]) at 48 weeks was -0.1 kg (-2.1 to 2.0) in the SA-ER group and -5.1 kg (-10.4 to 0.3) in the placebo group. The least squares mean difference (95% CI) between the groups in the covariance analysis was 4.8 kg (-0.3 to 9.9; P = 0.0635). The change in UEC score at 48 weeks was significantly higher in the SA-ER group compared with the placebo group (P = 0.0013) in the generalized estimating equation test repeated measurement analysis. In one patient in the SA-ER group, who was found to be pregnant 2 weeks after drug administration fetal death with tangled umbilical cord occurred at 13 weeks after the discontinuation of treatment. No other serious adverse effects were observed. CONCLUSIONS: The present study indicates that oral administration of SA-ER tablets is effective and safe in patients with GNE myopathy in Japan.

Recent advances in establishing a cure for GNE myopathy.

PURPOSE OF REVIEW: GNE myopathy is a rare autosomal recessive disease caused by biallelic variants in the GNE gene, which encodes an enzyme involved in sialic acid biosynthesis. No drugs are approved for the treatment of GNE myopathy. Following proof-of-concept of sialic acid supplementation efficacy in mouse models, multiple clinical trials have been conducted. Here, we review clinical trials of sialic acid supplementation therapies and provide new insights into the additional clinical features of GNE myopathy. RECENT FINDINGS: Clinical trials of sialic acid supplementation have been conducted in Europe, the USA, Japan, and South Korea. Some clinical trials of NeuAc-extended release tablets demonstrated amelioration of decline in upper extremity muscle strength; however, no significant improvement was observed in phase 3 trials in Europe and USA. A phase 2 trial of ManNAc showed slowed decline of both upper and lower extremity strength. GNE myopathy patient registries have been established in Europe and Japan, and have provided information on extramuscular manifestations such as thrombocytopenia, respiratory dysfunction, and sleep apnea syndrome. Sensitive and reliable biomarkers, and a disease-specific functional activity scale, have also been investigated. SUMMARY: We discuss recent advances in establishing a GNE myopathy cure, and discuss other prospective therapeutic options, including gene therapy.

Functional characterization of GNE mutations prevalent in Asian subjects with GNE myopathy, an ultra-rare neuromuscular disorder.

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is a bifunctional enzyme (N-terminal epimerase and C-terminal Kinase domain) that catalyses the rate limiting step in sialic acid biosynthesis. More than 200 homozygous missense or compound heterozygous mutations in GNE have been reported worldwide to cause a rare neuromuscular disorder, GNE myopathy. It is characterized by a slowly progressive defect in proximal and distal skeletal muscles with patients becoming wheel-chair-bound. There are no current approved therapies available for GNE myopathy. ManNAc therapy is currently in advanced clinical trials and has shown signs of slowing the disease progression in a phase 2 trial. The present study aims to understand the effect of GNE mutation on its enzymatic activity and identification of potential small effector molecules. We characterized different GNE mutations (p.Asp207Val, p.Val603Leu, p.Val727Met, p.Ile618Thr and p.Arg193Cys) prevalent in Asian population that were cloned, expressed and purified from Escherichia coli as full-length recombinant proteins. Our study demonstrates that full length GNE can be expressed in E. coli in its active form and analysed for the functional activity. Each mutation showed variation in epimerase and kinase activity and responded to the small effector molecules (metformin, BGP-15 kaempferol, catechin, quercetin) in a differential manner. Our study opens an area for futuristic structural determination of full length GNE and identification of potential therapeutic molecules.

Targeting GNE Myopathy: A Dual Prodrug Approach for the Delivery of N-Acetylmannosamine 6-Phosphate.

ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs phosphate masking groups capable of providing more favorable druglike properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein, we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favorable ADME properties, while also being more effective than ManNAc at increasing sialic acid levels in GNE-deficient cell lines. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.

A phase 3 randomized study evaluating sialic acid extended-release for GNE myopathy.

OBJECTIVE: To investigate the efficacy and safety of aceneuramic acid extended-release (Ace-ER), a treatment intended to replace deficient sialic acid, in patients with GNE myopathy. METHODS: UX001-CL301 was a phase 3, double-blind, placebo-controlled, randomized, international study evaluating the efficacy and safety of Ace-ER in patients with GNE myopathy. Participants who could walk ≥200 meters in a 6-minute walk test at screening were randomized 1:1, and stratified by sex, to receive Ace-ER 6 g/d or placebo for 48 weeks and assessed every 8 weeks. The primary endpoint was change in muscle strength over 48 weeks measured by upper extremity composite (UEC) score. Key secondary endpoints included change in lower extremity composite (LEC) score, knee extensor strength, and GNE myopathy-Functional Activity Scale (GNEM-FAS) mobility domain score. Safety assessments included adverse events (AEs), vital signs, and clinical laboratory results. RESULTS: Eighty-nine patients were randomized (Ace-ER n = 45; placebo n = 44). Change from baseline to week 48 for UEC score between treatments did not differ (least square mean [LSM] Ace-ER -2.25 kg vs placebo -2.99 kg; LSM difference confidence interval [CI] 0.74 [-1.61 to 3.09]; p = 0.5387). At week 48, there was no significant difference between treatments for the change in key secondary endpoints: LEC LSM difference (CI) -1.49 (-5.83 to 2.86); knee extension strength -0.40 (-2.38 to 1.58); and GNEM-FAS mobility domain score -0.72 (-2.01 to 0.57). Gastrointestinal events were the most common AEs. CONCLUSIONS: Ace-ER was not superior to placebo in improving muscle strength and function in patients with GNE myopathy. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for patients with GNE myopathy, Ace-ER does not improve muscle strength compared to placebo.

GNE myopathy: from clinics and genetics to pathology and research strategies.

GNE myopathy is an ultra-rare autosomal recessive disease, which starts as a distal muscle weakness and ultimately leads to a wheelchair bound state. Molecular research and animal modelling significantly moved forward understanding of GNE myopathy mechanisms and suggested therapeutic interventions to alleviate the symptoms. Multiple therapeutic attempts are being made to supplement sialic acid depleted in GNE myopathy muscle cells. Translational research field provided valuable knowledge through natural history studies, patient registries and clinical trial, which significantly contributed to bringing forward an era of GNE myopathy treatment. In this review, we are summarising current GNE myopathy, scientific trends and open questions, which would be of significant interest for a wide neuromuscular diseases community.

Safety, pharmacokinetics and sialic acid production after oral administration of N-acetylmannosamine (ManNAc) to subjects with GNE myopathy.

GNE myopathy is a rare, autosomal recessive, inborn error of sialic acid metabolism, caused by mutations in GNE, the gene encoding UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase. The disease manifests as an adult-onset myopathy characterized by progressive skeletal muscle weakness and atrophy. There is no medical therapy available for this debilitating disease. Hyposialylation of muscle glycoproteins likely contributes to the pathophysiology of this disease. N-acetyl-D-mannosamine (ManNAc), an uncharged monosaccharide and the first committed precursor in the sialic acid biosynthetic pathway, is a therapeutic candidate that prevents muscle weakness in the mouse model of GNE myopathy. We conducted a first-in-human, randomized, placebo-controlled, double-blind, single-ascending dose study to evaluate safety and pharmacokinetics of ManNAc in GNE myopathy subjects. Single doses of 3 and 6g of oral ManNAc were safe and well tolerated; 10g was associated with diarrhea likely due to unabsorbed ManNAc. Oral ManNAc was absorbed rapidly and exhibited a short half-life (~2.4h). Following administration of a single dose of ManNAc, there was a significant and sustained increase in plasma unconjugated free sialic acid (Neu5Ac) (Tmax of 8-11h). Neu5Ac levels remained above baseline 48h post-dose in subjects who received a dose of 6 or 10g. Given that Neu5Ac is known to have a short half-life, the prolonged elevation of Neu5Ac after a single dose of ManNAc suggests that intracellular biosynthesis of sialic acid was restored in subjects with GNE myopathy, including those homozygous for mutations in the kinase domain. Simulated plasma concentration-time profiles support a dosing regimen of 6g twice daily for future clinical trials.

[Therapeutic development for GNE myopathy.]

GNE myopathy is rare muscle disease which affect distal muscles. GNE gene, which encodes for a key enzyme in the sialic acid biosynthesis pathway, is mutated in the homozygote or compound heterozygote in the disease. The lack of sialic acid in skeletal muscle is the critical pathological process in GNE myopathy. GNE myopathy model mouse was established and supplementation of sialic acid improves the phenotype of model mouse. Phase Ⅰ clinical trial was conducted at Tohoku University Hospital using aceneuramic acid, followed by the trials using slow release product of sialic acid. Phase Ⅱ/Ⅲ study is ongoing.

Aceneuramic Acid Extended Release Administration Maintains Upper Limb Muscle Strength in a 48-week Study of Subjects with GNE Myopathy: Results from a Phase 2, Randomized, Controlled Study.

BACKGROUND: GNE Myopathy (GNEM) is a progressive adult-onset myopathy likely caused by deficiency of sialic acid (SA) biosynthesis. OBJECTIVE: Evaluate the safety and efficacy of SA (delivered by aceneuramic acid extended-release [Ace-ER]) as treatment for GNEM. METHODS: A Phase 2, randomized, double-blind, placebo-controlled study evaluating Ace-ER 3 g/day or 6 g/day versus placebo was conducted in GNEM subjects (n = 47). After the first 24 weeks, placebo subjects crossed over to 3 g/day or 6 g/day for 24 additional weeks (dose pre-assigned during initial randomization). Assessments included serum SA, muscle strength by dynamometry, functional assessments, clinician- and patient-reported outcomes, and safety. RESULTS: Dose-dependent increases in serum SA levels were observed. Supplementation with Ace-ER resulted in maintenance of muscle strength in an upper extremity composite (UEC) score at 6 g/day compared with placebo at Week 24 (LS mean difference +2.33 kg, p = 0.040), and larger in a pre-specified subgroup able to walk ≥200 m at Screening (+3.10 kg, p = 0.040). After cross-over, a combined 6 g/day group showed significantly better UEC strength than a combined 3 g/day group (+3.46 kg, p = 0.0031). A similar dose-dependent response was demonstrated within the lower extremity composite score, but was not significant (+1.06 kg, p = 0.61). The GNEM-Functional Activity Scale demonstrated a trend improvement in UE function and mobility in a combined 6 g/day group compared with a combined 3 g/day group. Patients receiving Ace-ER tablets had predominantly mild-to-moderate AEs and no serious adverse events. CONCLUSIONS: This is the first clinical study to provide evidence that supplementation with SA delivered by Ace-ER may stabilize muscle strength in individuals with GNEM and initiating treatment earlier in the disease course may lead to better outcomes.

[Sialic Acid Replacement Therapy for Distal Myopathy with Rimmed Vacuoles].

Distal myopathy with rimmed vacuoles or GNE myopathy, is an early adult-onset myopathy with slow progression that preferentially affects the tibialis anterior muscle. Severely affected patients show marked limb muscle atrophy together with respiratory dysfunction. The disease is caused by a mutation in the GNE gene that catalyzes two rate-limiting reactions in cytosolic sialic acid synthesis. Oral treatment with sialic acid metabolite prevents muscle atrophy and weakness in a mouse GNE myopathy model and a global Phase III study is currently underway. In addition, a global patient registry of neuromuscular cases is widely accepted as a useful tool to obtain epidemiological data and bolster patient recruitment for further development of this treatment strategy.

Sialyllactose ameliorates myopathic phenotypes in symptomatic GNE myopathy model mice.

Patients with GNE myopathy, a progressive and debilitating disease caused by a genetic defect in sialic acid biosynthesis, rely on supportive care and eventually become wheelchair-bound. To elucidate whether GNE myopathy is treatable at a progressive stage of the disease, we examined the efficacy of sialic acid supplementation on symptomatic old GNE myopathy mice that have ongoing, active muscle degeneration. We examined the therapeutic effect of a less metabolized sialic acid compound (6'-sialyllactose) or free sialic acid (N-acetylneuraminic acid) by oral, continuous administration to 50-week-old GNE myopathy mice for 30 weeks. To evaluate effects on their motor performance in living mice, spontaneous locomotion activity on a running wheel was measured chronologically at 50, 65, 72 and 80 weeks of age. The size, force production, and pathology of isolated gastrocnemius muscle were analysed at the end point. Sialic acid level in skeletal muscle was also measured. Spontaneous locomotion activity was recovered in 6'-sialyllactose-treated mice, while NeuAc-treated mice slowed the disease progression. Treatment with 6'-sialyllactose led to marked restoration of hyposialylation in muscle and consequently to robust improvement in the muscle size, contractile parameters, and pathology as compared to NeuAc. This is due to the fact that 6'-sialyllactose is longer working as it is further metabolized to free sialic acid after initial absorption. 6'-sialyllactose ameliorated muscle atrophy and degeneration in symptomatic GNE myopathy mice. Our results provide evidence that GNE myopathy can be treated even at a progressive stage and 6'-sialyllactose has more remarkable advantage than free sialic acid, providing a conceptual proof for clinical use in patients.