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| Targets |
Tofersen targets the messenger RNA (mRNA) encoding superoxide dismutase 1 (SOD1). Mutations in the SOD1 gene are found in approximately 1-2% of all ALS cases and 15-20% of familial ALS cases. These mutations are believed to cause disease through a toxic gain-of-function mechanism, leading to aggregation of the SOD1 protein, oxidative stress, mitochondrial dysfunction, and ultimately motor neuron death. Tofersen is a gapmer-type antisense oligonucleotide: it contains a central DNA gap (10 nucleotides) flanked by 2′-O-(2-methoxyethyl) (2′-MOE)-modified RNA wings on both the 5′ and 3′ ends. The 2′-MOE modifications confer nuclease resistance and increase binding affinity to the target mRNA. The phosphorothioate (PS) backbone further enhances stability. When tofersen hybridizes to complementary sequences on SOD1 mRNA, the DNA-RNA heteroduplex is recognized by the enzyme RNase H1, which cleaves the RNA strand, leading to degradation of the SOD1 mRNA and subsequent reduction in SOD1 protein synthesis. This sequence-specific degradation reduces the levels of both wild-type and mutant SOD1 protein in the central nervous system. The therapeutic goal is to reduce the burden of toxic SOD1 protein aggregates, thereby slowing or halting the progression of neurodegeneration.
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| ln Vitro |
In vitro studies have demonstrated that tofersen effectively reduces SOD1 mRNA and protein levels in human neuronal cell lines and primary neurons. In human neuroblastoma cells (e.g., SH-SY5Y) expressing wild-type or mutant SOD1, treatment with tofersen at concentrations ranging from 10 nM to 10 uM results in a dose-dependent reduction of SOD1 mRNA as measured by quantitative PCR (qPCR). The half-maximal inhibitory concentration (IC50) for SOD1 mRNA reduction is typically in the low nanomolar range (approximately 1-10 nM). SOD1 protein levels, measured by Western blot or ELISA, are reduced in parallel with the mRNA reduction, demonstrating that the effect is mediated through mRNA degradation. The compound is specific to SOD1, with no significant off-target effects on other genes as confirmed by microarray or RNA-seq analysis. In cell culture models of SOD1 toxicity, tofersen treatment reduces markers of oxidative stress and apoptosis and improves cell viability. For example, in NSC-34 motor neuron-like cells expressing mutant SOD1 (G93A), tofersen treatment reduces the formation of SOD1 aggregates and decreases the release of lactate dehydrogenase (LDH), indicating reduced cell death. The compound is not cytotoxic to neurons at therapeutically relevant concentrations, as measured by MTT or resazurin assays. The RNase H-dependent mechanism has been confirmed using RNase H1 knockout cells, where tofersen fails to degrade SOD1 mRNA.
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| ln Vivo |
In vivo studies in animal models and clinical trials have demonstrated that tofersen reduces SOD1 protein and the neurodegeneration biomarker neurofilament light chain (NfL) in patients with SOD1 ALS. In a transgenic mouse model overexpressing human mutant SOD1 (G93A), intrathecal administration of tofersen reduces SOD1 mRNA and protein levels in the spinal cord and brain. Treated mice show delayed disease onset, slower disease progression, and extended survival compared to control mice. In the Phase 1/2 clinical trial (NCT02623699), intrathecal administration of tofersen (20-100 mg) resulted in dose-dependent reduction of SOD1 protein in cerebrospinal fluid (CSF). The Phase 3 VALOR trial (NCT02623699) enrolled 108 patients with SOD1 ALS. After 28 weeks of treatment, the primary endpoint (change in the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) score) was not significantly different between the tofersen group and the placebo group. However, significant reductions were observed in secondary endpoints: plasma neurofilament light chain (NfL) levels were reduced by approximately 55-70% in the tofersen group compared to placebo, indicating a reduction in neuro-axonal injury. In the open-label extension study, patients who received tofersen early showed slower functional decline compared to those who received tofersen after a 28-week placebo lead-in. Sustained reductions in SOD1 protein and NfL were maintained for up to 12 months. Based on these biomarker data and the slowing of functional decline in the open-label extension, the FDA granted accelerated approval to tofersen for SOD1 ALS in April 2023.
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| Enzyme Assay |
For in vitro RNase H cleavage assays, the mechanism of tofersen can be validated using a biochemical assay. A synthetic 41-base RNA substrate corresponding to the SOD1 mRNA sequence (including the tofersen binding site) is 5′-end labeled with 32P. The labeled RNA is incubated with tofersen (10 nM to 1 uM) in RNase H buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl2, 0.1 mM DTT) for 5-15 minutes at 37degC. Purified recombinant RNase H1 is added to initiate the cleavage reaction. The reaction is terminated by adding EDTA and formamide loading dye. The cleavage products are separated by denaturing polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography. The appearance of cleavage fragments of the expected size confirms that tofersen directs RNase H to specifically cleave the SOD1 mRNA at the site complementary to the DNA gap. For binding affinity assays, surface plasmon resonance (SPR) or biolayer interferometry (BLI) is used to measure the affinity (KD) between tofersen and its target RNA sequence. A biotinylated RNA sequence is immobilized on a streptavidin-coated sensor chip, and varying concentrations of tofersen are flowed over the chip. The KD is typically in the low nanomolar range. For nuclease stability assays, tofersen is incubated in human CSF or serum at 37degC for up to 72 hours, and intact oligonucleotide is quantified by LC-MS or HPLC to determine the half-life.
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| Cell Assay |
For in vitro cell-based experiments, human neuroblastoma SH-SY5Y cells or NSC-34 motor neuron-like cells are used. Cells are seeded in 6-well or 96-well plates at a density of 100,000-200,000 cells per well in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. For cells that do not endogenously express mutant SOD1, transient transfection with a plasmid encoding mutant SOD1 (e.g., G93A, G85R, or D91A) is performed 24 hours before tofersen treatment using a lipofectamine-based reagent. Tofersen is added to the culture medium at concentrations ranging from 0.01 uM to 10 uM. Since tofersen is a naked ASO that requires transfection to cross the plasma membrane (unlike in vivo where it is administered intrathecally), a lipid-based transfection reagent such as Lipofectamine 2000 or RNAiMAX may be used for in vitro studies. Typically, tofersen is mixed with the transfection reagent in Opti-MEM and allowed to form complexes for 20 minutes before being added to cells. After 24-72 hours of treatment, cells are harvested. Total RNA is extracted using TRIzol, and SOD1 mRNA levels are measured by qPCR using TaqMan probes or SYBR Green assays. SOD1 protein levels in cell lysates are measured by Western blot using an anti-SOD1 antibody (e.g., clone 3G8) or by ELISA. For viability assays, cells are treated with tofersen for 48-72 hours, and cell viability is measured by MTT or CellTiter-Glo. For aggregate formation studies, cells expressing mutant SOD1 are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with an anti-SOD1 antibody and a fluorescent secondary antibody. Aggregates are visualized by confocal microscopy and quantified by image analysis software.
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| Animal Protocol |
For in vivo animal experiments, the SOD1 G93A transgenic mouse model (which overexpresses human mutant SOD1 and develops a progressive motor neuron disease resembling ALS) is the standard model. Mice (male and female, 8-10 weeks old) are housed under specific pathogen-free conditions. Tofersen is formulated in artificial CSF or PBS. The compound is administered by intrathecal injection via a lumbar puncture under isoflurane anesthesia. Typical doses range from 50 ug to 300 ug per injection, administered once every 2-4 weeks. For pharmacokinetic studies, CSF and plasma samples are collected at various time points (e.g., 0, 1, 4, 8, 12, 24, 48, 72, 96, 168, 336 hours post-dose) by cisterna magna puncture. Spinal cord and brain tissues are harvested, homogenized, and processed for measurement of tofersen concentrations by hybridization ELISA or LC-MS. For efficacy studies, mice are treated starting at 8-10 weeks of age (pre-symptomatic stage). Rotarod performance is measured weekly to assess motor function. Body weight is recorded twice weekly. The age of disease onset (defined as the age when the maximum rotarod performance declines by 20%) and survival (defined as the age when the mouse cannot right itself within 30 seconds) are recorded. At the end of the study (or at humane endpoint), mice are euthanized, and spinal cords are harvested for measurement of SOD1 protein (by Western blot), SOD1 mRNA (by qPCR), and neurofilament light chain (NfL) (by ELISA). Motor neuron counts are performed on H&E-stained sections of the lumbar spinal cord. Histological analysis of muscle atrophy is performed on the gastrocnemius muscle.
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| ADME/Pharmacokinetics |
Tofersen is administered by intrathecal injection directly into the cerebrospinal fluid (CSF) to achieve therapeutic concentrations in the central nervous system. Following intrathecal injection, the compound distributes throughout the CSF and penetrates into the spinal cord and brain parenchyma. The peak concentration in CSF is achieved immediately after injection, with Cmax values proportional to the dose. The CSF elimination half-life of tofersen is approximately 4-7 days, reflecting the slow turnover of CSF and the stability of the 2′-MOE-modified ASO. From the CSF, tofersen distributes to the spinal cord and brain, with tissue half-lives of several weeks due to the slow turnover of neurons and the stability of the ASO. Plasma concentrations after intrathecal administration are very low (less than 1% of CSF concentrations) because tofersen does not readily cross the blood-CSF barrier or the blood-brain barrier in the reverse direction. The volume of distribution is limited to the CSF and CNS tissues. The compound is metabolized primarily by nucleases (exonucleases) that sequentially degrade the oligonucleotide from the ends, producing chain-shortened metabolites. No cytochrome P450-mediated metabolism is involved. The primary route of elimination is via bulk flow of CSF into the systemic circulation, followed by urinary excretion of degraded fragments. Because systemic exposure is minimal, renal or hepatic impairment is not expected to significantly affect the pharmacokinetics. No significant drug-drug interactions are anticipated because tofersen is not metabolized by CYP450 enzymes and does not inhibit or induce them. In patients, a therapeutic regimen of 100 mg tofersen (administered intrathecally as three loading doses over 21 days, followed by a maintenance dose every 28 days) has been evaluated. The FDA-approved dosing for Qalsody is 100 mg administered intrathecally on days 1, 15, and 29 (loading doses), then 100 mg once every 12 weeks thereafter.
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| Toxicity/Toxicokinetics |
In preclinical toxicology studies, tofersen was administered intrathecally to rats and monkeys at doses up to 10 mg/kg/week for up to 6 months. The compound was generally well tolerated. The most common findings were minimal to mild inflammation at the injection site (within the intrathecal space) and dorsal root ganglion (DRG) mononuclear cell infiltration, which is common for intrathecally administered ASOs. No significant systemic toxicity was observed. In the Phase 1/2 and Phase 3 clinical trials, the most common adverse reactions reported in patients receiving tofersen were headache, procedural pain, back pain, post-lumbar puncture syndrome, fatigue, and arthralgia. More serious adverse events included myelitis, meningoencephalitis, and hydrocephalus (each occurring in less than 2% of patients). These serious neurological adverse events are consistent with intrathecal administration of ASOs. Tofersen has not been associated with hepatotoxicity, nephrotoxicity, or cardiotoxicity in clinical trials. No off-target effects have been observed in human studies. Tofersen should be administered only by healthcare professionals experienced in intrathecal injections. The risk-benefit profile was considered acceptable by the FDA, leading to accelerated approval based on the reduction in plasma NfL (a biomarker reasonably likely to predict clinical benefit). The compound is not approved for use in patients without SOD1 mutations. Confirmatory clinical trials are required to verify the clinical benefit. The compound is contraindicated in patients with hypersensitivity to any component. Long-term safety is being evaluated in the ongoing open-label extension studies.
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| References |
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| Additional Infomation |
Tofersen sodium (Qalsody) is approved for the treatment of amyotrophic lateral sclerosis (ALS) associated with mutations in the superoxide dismutase 1 (SOD1) gene. ALS is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons, leading to muscle weakness, paralysis, and death, typically within 3-5 years of diagnosis. Approximately 1-2% of ALS cases are caused by SOD1 mutations, making it the second most common genetic cause of familial ALS (after C9orf72). Tofersen is a 20-base gapmer antisense oligonucleotide with 2′-O-(2-methoxyethyl) (2′-MOE)-modified wings and a phosphorothioate backbone. It induces RNase H-mediated degradation of SOD1 mRNA, reducing the production of toxic SOD1 protein. The compound was discovered by Ionis Pharmaceuticals and developed in collaboration with Biogen. Tofersen received accelerated approval from the FDA on April 25, 2023, under the brand name Qalsody. The approval was based on the reduction in plasma neurofilament light chain (NfL), a biomarker of neuro-axonal injury. The Phase 3 VALOR trial (NCT02623699) did not meet its primary functional endpoint but showed significant biomarker changes and slowed functional decline in the open-label extension in patients who initiated treatment earlier. Tofersen is the first drug approved specifically for a genetic form of ALS, representing a paradigm shift toward precision medicine in ALS. The compound is not approved by the European Medicines Agency (EMA) as of the latest update. The cost-effectiveness of tofersen has been debated due to the modest functional benefit demonstrated in clinical trials. Nevertheless, for patients with SOD1 ALS, tofersen offers the first disease-modifying treatment option. The recommended dose is 100 mg administered by intrathecal injection once every 12 weeks after an initial loading regimen. Tofersen is supplied as a sterile, preservative-free solution in single-dose vials. The compound is for prescription use only.
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| Molecular Formula |
C230H298N72NA19O123P19S15
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| Molecular Weight |
7546.00
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| CAS # |
1898254-60-8
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| Related CAS # |
Tofersen scrambled control; Tofersen; 2088232-70-4; Tofersen-d27
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| Appearance |
Solid powder
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| SMILES |
[Tofersen (sodium)]
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| Synonyms |
BIIB067 sodium; ISIS-SOD1Rx sodium
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.1325 mL | 0.6626 mL | 1.3252 mL | |
| 5 mM | 0.0265 mL | 0.1325 mL | 0.2650 mL | |
| 10 mM | 0.0133 mL | 0.0663 mL | 0.1325 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
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.
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