Dystrophin (DMD) pre-mRNA. Viltolarsen binds to a specific sequence in exon 53 of the dystrophin pre-mRNA, blocking the spliceosome complex's ability to include this exon in the final mRNA. This causes the splicing machinery to skip over exon 53, joining exon 52 to exon 54. This restores the open reading frame (ORF) of the dystrophin transcript in patients with certain deletions in the DMD gene (e.g., deletions that disrupt the reading frame). The result is the production of a shortened, internally deleted but still functional dystrophin protein, converting a severe Duchenne phenotype to a milder Becker-like phenotype.

One of the most prevalent and deadly muscle-wasting disorders affecting young boys is called Duchenne muscular dystrophy (DMD), which is brought on by mutations in the DMD gene. Exon skipping has become a viable DMD therapy option. In order to restore the reading frame and permit dystrophin production, antisense oligonucleotides (AONs) are engineered to cause exon skipping [1].

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As an antisense oligonucleotide, viltolarsen is not used in cell-free enzyme inhibition assays. Its activity is measured in cell-free systems by determining its ability to hybridize to its complementary RNA target sequence. Melting temperature (Tm) analysis is performed by mixing the PMO with a synthetic 25-30 mer RNA oligonucleotide corresponding to the target site. The mixture is heated (25-95degC), and the absorbance at 260 nm is monitored to determine the Tm, which is a measure of binding affinity. Viltolarsen forms a stable duplex with its target RNA (Tm > 60degC). RNase H cleavage assays are not applicable, as PMOs do not recruit RNase H; they sterically block splicing.

In cell-based assays, viltolarsen is tested in patient-derived myoblasts (muscle cells) or immortalized cell lines (e.g., H2K cells) that harbor a mutation amenable to exon 53 skipping. Cells are treated with viltolarsen at concentrations ranging from 0.1-100 uM (typically by "gymnosis," i.e., free uptake, without transfection) for 24-72 hours. Exon skipping efficiency is measured by RT-PCR of the dystrophin transcript, followed by gel electrophoresis or capillary electrophoresis to separate the full-length product from the shorter product. The percentage of exon skipping is quantified by densitometry. Functional dystrophin protein production is confirmed by Western blotting or immunofluorescence. The EC50 for exon skipping is typically in the low micromolar range (e.g., 1-10 uM). Viltolarsen also restores the expression of dystrophin-associated protein complex (DAPC) in cells.

Antisense activity of viltolarsen is initially assessed in a cell-free system using a splicing minigene construct. A plasmid containing the human DMD exon 53 and its flanking intronic sequences is engineered. The minigene is transcribed in vitro (e.g., using a T7 RNA polymerase) to produce a pre-mRNA substrate. Viltolarsen (0.1-10 uM) is added to a nuclear extract (e.g., HeLa nuclear extract) along with the pre-mRNA. After incubation for 2-4 hours, the RNA is extracted, and RT-PCR is performed using primers flanking the exon. The skipped product (exon 53 omitted) is visualized on a gel. This confirms that the PMO directly inhibits spliceosome assembly and causes skipping. This assay is more physiologically relevant than simple hybridization.

For cellular activity, primary human myoblasts or fibroblasts from DMD patients with a confirmed exon 53 skipping mutation are used. Cells are cultured in a differentiation medium to induce myotube formation. Viltolarsen is added to the culture medium (free uptake) at concentrations of 0.1-100 uM. After 48-96 hours, RNA is extracted, and exon skipping is assessed by nested RT-PCR. Primers are placed in exons 51 and 55. The product is resolved on a 2% agarose gel or by capillary electrophoresis. The percent skipping is calculated based on the intensity of the band representing the exon 53-skipped product compared to the full-length product. Western blotting for dystrophin is performed on cell lysates using a dystrophin antibody (e.g., Dys-2). Immunofluorescence staining of myotubes with a dystrophin antibody is used to visualize the sarcolemmal localization of the restored protein.

In vivo efficacy of viltolarsen is evaluated in the mdx52 mouse model, which has a deletion of exon 52 in the dystrophin gene, making it amenable to exon 53 skipping. Viltolarsen is administered intravenously (IV) or subcutaneously (SC) to mdx52 mice at doses ranging from 10-100 mg/kg, weekly or bi-weekly for 4-12 weeks. The primary endpoint is the restoration of dystrophin protein in skeletal muscles (e.g., quadriceps, diaphragm, heart), measured by Western blotting and immunofluorescence staining. Functional improvement is assessed by grip strength, treadmill running distance, and histological analysis (reduction in central nucleation, fibrosis, and serum creatine kinase levels). Viltolarsen has been shown to produce significant dystrophin protein expression (e.g., 5-10% of normal levels) and improve muscle pathology.

Viltolarsen is administered intravenously in humans. In preclinical PK studies in animals (mice, rats, monkeys), following IV or SC administration, viltolarsen shows a short plasma half-life (hours) but prolonged retention in skeletal muscle (days to weeks). It is distributed primarily to the kidney, liver, and muscle. The compound is cleared by endocytosis and lysosomal degradation. It is not metabolized by CYP enzymes, and drug-drug interactions are not expected. The major excretion route is urine. In human PK studies, viltolarsen (e.g., a 20 mg/kg weekly IV dose) shows linear PK. The Tmax at the end of infusion, and the elimination half-life is 2-3 hours for plasma, but the muscle half-life is much longer. The high molecular weight (approx. 6 kDa) and hydrophilic PMO backbone limit oral bioavailability.

In preclinical toxicology studies, viltolarsen was well-tolerated at clinically relevant doses. The most common findings in animals were injection site reactions, minimal to moderate glomerulonephritis (due to renal accumulation of PMO), and vacuolization in kidney tubules, which are reversible. No significant hepatotoxicity, cardiotoxicity, or CNS effects were observed. In human clinical trials (Phase 2 studies), the most common adverse events were pyrexia (fever), vomiting, cough, and nasopharyngitis. No serious drug-related adverse events or deaths were reported. It is not genotoxic. The FDA approved viltolarsen in 2020 for DMD patients amenable to exon 53 skipping, based on a surrogate endpoint of increased dystrophin production. The long-term safety and efficacy are being monitored.

[1]. Dzierlega K, et al. Optimization of antisense-mediated exon skipping for Duchenne muscular dystrophy. Gene Ther. 2020;27(9):407-416.

See also: Viltolarsen (note moved to).

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Viltolarsen (Viltepso) was approved by the FDA in August 2020 for the treatment of Duchenne muscular dystrophy (DMD) in patients with a confirmed mutation amenable to exon 53 skipping. It is developed by NS Pharma, a subsidiary of Nippon Shinyaku. The drug is a PMO (phosphorodiamidate morpholino oligomer) with a backbone that is resistant to nuclease degradation. Its chemical structure is all-P-ambo-(2',3'-azanediyl-P,2',3'-trideoxy-P-(dimethylamino)-2',3'-seco)(2'-N->5')(CCTCCGGTTC TGAAGGTGTT C). The CAS number is 2055732-84-6. For research use, viltolarsen is provided as a solution. The compound is not for human use in research settings.

6923.363

2055732-84-6

154562176

White to off-white solid powder

-33.1

29

149

122

465

21200

42

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1-[(2R,6S)-6-[[[(2S,6R)-2-[[[(2R,6S)-2-(2-amino-6-oxo-1H-purin-9-yl)-6-[[[(2S,6R)-2-[[[(2R,6S)-2-(2-amino-6-oxo-1H-purin-9-yl)-6-[[[(2R,6S)-2-(2-amino-6-oxo-1H-purin-9-yl)-6-[[[(2S,6R)-2-[[[(2S,6R)-2-[[[(2R,6S)-2-(2-amino-6-oxo-1H-purin-9-yl)-6-[[[(2S,6R)-2-[[[(2S,6R)-2-[[[(2S,6R)-2-[[[(2S,6R)-2-[[[(2R,6S)-2-(2-amino-6-oxo-1H-purin-9-yl)-6-[[[(2R,6S)-2-(2-amino-6-oxo-1H-purin-9-yl)-6-[[[(2R,6S)-2-(4-amino-2-oxopyrimidin-1-yl)-6-[[[(2R,6S)-2-(4-amino-2-oxopyrimidin-1-yl)-6-[[[(2S,6R)-2-[[[(2R,6S)-2-(4-amino-2-oxopyrimidin-1-yl)-6-[[[(2R,6S)-2-(4-amino-2-oxopyrimidin-1-yl)-6-(hydroxymethyl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(5-methyl-2,4-dioxopyrimidin-1-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(5-methyl-2,4-dioxopyrimidin-1-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(5-methyl-2,4-dioxopyrimidin-1-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(4-amino-2-oxopyrimidin-1-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(5-methyl-2,4-dioxopyrimidin-1-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(6-aminopurin-9-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(6-aminopurin-9-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(5-methyl-2,4-dioxopyrimidin-1-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-6-(5-methyl-2,4-dioxopyrimidin-1-yl)morpholin-4-yl]-(dimethylamino)phosphoryl]oxymethyl]-4-[[(2S,6R)-6-(4-amino-2-oxopyrimidin-1-yl)morpholin-2-yl]methoxy-(dimethylamino)phosphoryl]morpholin-2-yl]-5-methylpyrimidine-2,4-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~14.44 mM)

Solubility in Formulation 1: 50 mg/mL (7.22 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)