| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| Other Sizes |
| Targets |
Dystrophin (DMD) pre-mRNA. Golodirsen is an antisense oligonucleotide that binds to a specific sequence in exon 53 of the dystrophin pre-mRNA. By sterically blocking the spliceosome's access to this exon, it induces the exclusion (skipping) of exon 53 from the final mature mRNA. This restores the disrupted open reading frame (ORF) in patients with certain deletions (e.g., deletions of exon 50, 52, or 48-50). The mechanism converts a severe Duchenne phenotype to a milder, Becker-like phenotype by allowing the production of a shorter, internally deleted but partially functional dystrophin protein. The induction of dystrophin synthesis is the primary therapeutic effect.
|
|---|---|
| ln Vitro |
Golodirsen selectively binds to exon 53 of the dystrophin pre-mRNA, preventing it from being included in the finished mRNA product. By omitting exon 53, the mRNA reading frame is restored and a partially internally deleted dystrophin protein with intact C- and N-terminal regions is produced [2].
Cell-free assays for golodirsen are similar to those for other PMOs. It is not an enzyme inhibitor; rather, it binds to RNA. Hybridization efficiency to its complementary RNA target is measured by mixing the PMO with a synthetic RNA oligonucleotide (typically a 20-30 mer) corresponding to the target sequence in exon 53. The mixture is heated (25-95degC), and the absorbance at 260 nm is monitored to determine the melting temperature (Tm). A high Tm (>60degC) indicates stable binding. RNase H cleavage assays are not applicable, as PMOs do not recruit RNase H; they act by steric blockage. Specificity is confirmed by examining binding to mismatched RNA sequences. |
| ln Vivo |
In cell-based assays, golodirsen is tested in DMD patient-derived myoblasts that have a deletion mutation amenable to exon 53 skipping (e.g., deletion of exon 50 or exon 52). Cells are cultured in differentiation medium to form myotubes. Golodirsen is added to the medium (free uptake, as PMOs are cell-permeable without transfection, but uptake is slow) at concentrations of 0.1-100 uM for 48-96 hours. Exon skipping is measured by RT-PCR using primers in exons 51 and 55. The percentage of exon 53 skipping is quantified by capillary electrophoresis. Functional dystrophin protein is measured by Western blotting and by immunofluorescence staining to confirm membrane localization. Golodirsen restores dystrophin expression typically to 1-5% of normal levels, which is considered clinically meaningful. The EC50 for exon 53 skipping is in the low micromolar range.
|
| Enzyme Assay |
Golodirsen's activity can be assessed in a cell-free splicing assay using a minigene construct. A plasmid containing the human DMD gene exons 52-54 with flanking introns is engineered. The minigene is transcribed in vitro to produce pre-mRNA. The pre-mRNA is incubated with a nuclear extract (e.g., from HeLa cells) supplemented with ATP and an energy-regenerating system. Golodirsen (0.1-10 uM) is added to the reaction. After 2-4 hours, RNA is extracted, and RT-PCR is performed using primers spanning exons 52-54. The product is run on a gel to visualize the skipped (exon 53 absent) vs. full-length product. This assay directly demonstrates that the antisense oligonucleotide inhibits spliceosome assembly at exon 53.
|
| Cell Assay |
Cellular assays are performed using primary human myoblasts from DMD patients with mutations amenable to exon 53 skipping (e.g., exons 48-50 deletion, exon 52 deletion). Cells are plated in 6- or 24-well plates and differentiated into myotubes in DMEM containing 2% horse serum for 5-7 days. Golodirsen is added at concentrations of 1, 3, 10, 30, and 100 uM during the last 48-72 hours of differentiation. RNA is extracted using Trizol. Exon skipping is quantified by nested RT-PCR using a primary PCR (exon 51-forward, exon 55-reverse) and a secondary PCR with fluorescently labeled primers. Products are resolved by capillary electrophoresis. Percent skipping is calculated based on peak area. Dystrophin protein is quantified by Western blot using a monoclonal antibody (DYS1, DYS2, or DYS3) and normalization to alpha-actinin or GAPDH. The limit of detection is typically <1% of normal. Immunofluorescence on myotube cultures visualizes sarcolemmal localization.
|
| Animal Protocol |
In vivo efficacy of golodirsen is evaluated in the mdx52 mouse model, which lacks exon 52 and is amenable to exon 53 skipping. Mice are treated with golodirsen at doses of 3, 10, 30, or 100 mg/kg via intravenous (IV) or subcutaneous (SC) injection twice weekly for 4-12 weeks. The primary endpoint is the restoration of dystrophin protein in skeletal muscles (quadriceps, diaphragm, heart), measured by Western blot and immunofluorescence. Functional tests include grip strength, rotarod, and treadmill exhaustion tests. Serum creatine kinase (CK) levels are measured as a biomarker of muscle damage. Histological analysis of muscle sections includes H&E staining for central nucleation and fibrosis (e.g., Sirius red staining). Golodirsen has been shown to produce significant dystrophin expression (1-5% of normal) in mdx52 mice.
|
| ADME/Pharmacokinetics |
In preclinical PK studies, golodirsen, like other PMOs, has a short plasma half-life (2-4 hours in mice) but prolonged retention in muscle tissue (days to weeks). Following SC or IV administration, it is distributed primarily to the kidney, liver, and muscle. The PMO is cleared by endocytosis and lysosomal degradation. It is not metabolized by CYP450 enzymes, so drug-drug interactions are minimal. The major route of elimination is urinary excretion. In humans, the approved dosing regimen is 30 mg/kg once weekly by IV infusion. The terminal half-life in plasma is approximately 2-5 hours, but the pharmacodynamic effect (dystrophin production) is sustained. The plasma AUC increases dose-proportionally. The high molecular weight (~8,647 Da) and hydrophilicity prevent oral absorption.
|
| Toxicity/Toxicokinetics |
In preclinical toxicology studies, golodirsen was generally well-tolerated. The primary findings in animals were related to the class effect of PMOs: glomerulonephritis and tubular vacuolization in the kidney at high doses, which were reversible. No significant hepatotoxicity, neurotoxicity, or cardiotoxicity was observed. In human clinical trials (Phase 1/2 4053-101 and Phase 2/3 ESSENCE), the most common adverse events were headache, pyrexia, cough, vomiting, and nasopharyngitis. Injection site reactions and proteinuria (transient) were observed. No drug-related serious adverse events or deaths were reported. It is not genotoxic. In 2022, the FDA expanded its approval of golodirsen to include all DMD patients amenable to exon 53 skipping. Long-term safety is monitored.
|
| References | |
| Additional Infomation |
Vyondys 53 (golodirsen) was granted accelerated approval by the FDA in December 2019 for the treatment of Duchenne muscular dystrophy in patients with a confirmed mutation of the dystrophin gene amenable to exon 53 skipping. It is developed by Sarepta Therapeutics. The approval was based on a surrogate endpoint of increased dystrophin production. The CAS number is 1422959-91-8. The molecular weight is approximately 8647.00. It is a synthetic antisense oligonucleotide that restores the reading frame. For research use, it is typically provided as a lyophilized powder or solution. The compound is not for human use in laboratory research. The drug is administered as a 30 mg/kg weekly IV infusion.
|
| CAS # |
1422959-91-8
|
|---|---|
| Appearance |
White to off-white solid powder
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
H2O : ~100 mg/mL (~11.56 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 50 mg/mL (5.78 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
(Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
