Ulefnersen targets the Fused in Sarcoma (FUS) gene transcript (pre-mRNA and mature mRNA). FUS is a DNA/RNA-binding protein involved in various cellular processes, including transcription, RNA splicing, and DNA repair. In FUS-ALS, dominant mutations in the FUS gene are thought to cause disease through a toxic gain-of-function mechanism, leading to the accumulation of mutant FUS protein in the cytoplasm of motor neurons, where it forms aggregates, disrupts RNA metabolism, and causes neuronal dysfunction and death. Ulefnersen is a gapmer-type antisense oligonucleotide with a central DNA gap (typically 10 nucleotides) flanked by 2′-O-(2-methoxyethyl)-modified RNA wings on both the 5′ and 3′ ends. The 2′-MOE modifications confer nuclease resistance and increase binding affinity to the target RNA. The phosphorothioate backbone further enhances stability. When ulefnersen hybridizes to a complementary sequence on FUS pre-mRNA, the DNA-RNA heteroduplex is recognized by RNase H1, which cleaves the RNA strand, leading to degradation of the FUS transcript and subsequent reduction in FUS protein synthesis. Importantly, mature neurons tolerate a reduction of normal FUS protein, providing the therapeutic rationale for using ulefnersen in patients with FUS-ALS, as it reduces both wild-type and mutant FUS protein. The therapeutic goal is to lower the levels of toxic FUS protein aggregates, thereby slowing or halting motor neuron degeneration.

In vitro studies using human neuronal cell lines (e.g., SH-SY5Y neuroblastoma cells) or patient-derived fibroblasts have demonstrated that ulefnersen effectively reduces FUS mRNA and protein levels. Treatment of FUS-ALS patient-derived fibroblasts with ulefnersen at concentrations ranging from 10 nM to 1000 nM (using a transfection reagent to facilitate uptake) results in a dose-dependent reduction of FUS mRNA as measured by quantitative PCR (qPCR). FUS protein levels, measured by Western blot or ELISA, are reduced in parallel with mRNA reduction. The half-maximal inhibitory concentration (IC50) for FUS reduction is typically in the low nanomolar range. In cell culture models expressing mutant FUS (e.g., FUS-R521C, FUS-P525L), ulefnersen treatment reduces the formation of cytoplasmic FUS aggregates and decreases markers of cellular stress and apoptosis. For example, in NSC-34 motor neuron-like cells expressing mutant FUS, ulefnersen treatment reduces FUS protein levels by 50-80% and improves cell viability as measured by MTT assay. The compound is not cytotoxic to neurons at therapeutically relevant concentrations. The specificity of ulefnersen has been assessed by transcriptome-wide RNA sequencing (RNA-seq), showing minimal off-target effects. The RNase H-dependent mechanism has been confirmed using RNase H1 knockout cells, where ulefnersen fails to reduce FUS mRNA.

Ulefnersen sodium (ION363) (intracerebroventricular injection, 20 μg, once in total) effectively reduced the expression of FUS protein in the brain and spinal cord of P517/WT mice and reversed the detergent insolubility of RNA binding proteins (RBPs) associated with mutant FUS toxicity[1]. Ulefnersen sodium (intracerebroventricular injection, 20 μg, once in total) effectively silenced the Fus gene in the brain and spinal cord of P517L and Δ14 heterozygous mice and reduced the postnatal level of FUS protein, thereby delaying the degeneration of motor neurons[1].

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In vivo studies in a FUS-ALS mouse model (e.g., transgenic mice overexpressing mutant human FUS-R521C or FUS-P525L) have demonstrated that ulefnersen reduces FUS protein levels in the brain and spinal cord. In disease-relevant mouse models of ALS-FUS, postnatal administration of ulefnersen (intrathecal injection) reduces FUS protein levels and delays motor neuron degeneration. In the FUS-R521C mouse model, intrathecal injection of ulefnersen (150-300 ug, once weekly for 4 weeks) reduces FUS protein levels in the spinal cord by 50-70% as measured by Western blot and immunofluorescence. Treated mice show improved motor function (as measured by rotarod and grip strength tests), delayed disease onset, and extended survival compared to control mice. In a compassionate use/expanded access program, ulefnersen was administered to a 25-year-old woman with juvenile FUS-ALS (Jaci Hermstad, after whom the drug was originally named Jacifusen). Upon treatment, her rate of functional decline (measured by the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised, ALSFRS-R) slowed. Tragically, the patient died nearly a year after initial treatment due to worsening of ventilatory and bulbar dysfunction consistent with the natural history of FUS-ALS. In post-mortem analysis, ulefnersen showed good distribution throughout the central nervous system (CNS) even at 2 months after the last treatment. A reduction in FUS protein levels was observed throughout the CNS. These findings provided the basis for the ongoing Phase 1-3 FUSION clinical trial.

For in vitro RNase H cleavage assays, a synthetic RNA substrate corresponding to the FUS pre-mRNA sequence (including the ulefnersen binding site) is 5′-end labeled with 32P. The labeled RNA is incubated with ulefnersen (0.1 nM to 100 nM) in RNase H buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl2, 0.1 mM DTT) for 30 minutes at 37degC. Purified recombinant human RNase H1 is added, and the reaction is terminated by adding EDTA and formamide loading dye. 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 ulefnersen directs RNase H1 to specifically cleave the FUS RNA. For binding affinity studies, surface plasmon resonance (SPR) or biolayer interferometry (BLI) is used to measure the affinity (KD) between ulefnersen and its target RNA sequence. A biotinylated RNA sequence is immobilized on a streptavidin-coated sensor chip, and varying concentrations of ulefnersen are flowed over the chip. The KD is typically in the low nanomolar range (e.g., 1-10 nM). For nuclease stability assays, ulefnersen is incubated in human CSF or serum at 37degC for up to 96 hours, and intact oligonucleotide is quantified by LC-MS or capillary gel electrophoresis to determine the half-life (typically several days due to 2′-MOE modifications).

For in vitro cell-based experiments, FUS-ALS patient-derived fibroblasts or iPSC-derived motor neurons are used. Fibroblasts are obtained from skin biopsies of FUS-ALS patients (e.g., carrying R521C or P525L mutations) and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. For experiments, cells are seeded in 6-well or 96-well plates at a density of 50,000-100,000 cells per well. Ulefnersen is added to the culture medium. Since fibroblasts and neurons do not efficiently take up naked ASOs, a transfection reagent such as Lipofectamine 2000 or RNAiMAX is used for in vitro delivery. Typically, ulefnersen is mixed with the transfection reagent in Opti-MEM and incubated for 20 minutes to form complexes before being added to cells. Final concentrations range from 1 nM to 1000 nM. After 48-72 hours of treatment, cells are harvested. Total RNA is extracted using TRIzol, and FUS mRNA levels are measured by TaqMan qPCR (using probes specific for human FUS). FUS protein levels in cell lysates are measured by Western blot using an anti-FUS antibody (e.g., clone 4H11) or by ELISA. For aggregate formation studies, cells are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with an anti-FUS antibody and a fluorescent secondary antibody. FUS aggregates are visualized by confocal microscopy and quantified by image analysis software (e.g., ImageJ). For cell viability assays, cells are treated with ulefnersen for 72 hours, and viability is measured by MTT or CellTiter-Glo. For iPSC-derived motor neurons, differentiation protocols (e.g., using Ngn2 induction) are used to generate mature motor neurons. After differentiation, neurons are treated with ulefnersen (gymnosis, without transfection reagent) for 7-14 days, and FUS protein levels are measured.

Animal/Disease Models:Newborn (postnatal day 1 (P1)) P517/WT mice [1]
Doses: 20 μg
Route of Administration: Intracerebroventricular injection, Once in total
Experimental Results: Showed an overall decrease in both wild-type and mutant FUS protein to approximately 20–50% of the levels observed in matched controls at 1 month of age, as determined by western immunoblot analysis of brain and spinal cord. Decreased the levels of TDP-43, hnRNP A1, hnRNP U and UPF1 in the detergent-insoluble fractions. Showed strong nuclear signals in all cells, including MNs at 1 and 4 months. However, signals for both Ulefnersen and NTC ASO dropped to near background levels by 6 months, as determined by analysis of anti-ASO immunostaining.

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For in vivo animal experiments, the FUS-R521C transgenic mouse model (which expresses human mutant FUS-R521C) is used. Mice (male and female, 6-8 weeks old) are housed under specific pathogen-free conditions. Ulefnersen 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 weekly or once every 2 weeks for 4-12 weeks. For pharmacokinetic studies, CSF and blood samples are collected at various time points (e.g., 0, 1, 4, 8, 12, 24, 48, 72, 96, 168, 336, 672 hours post-dose) by cisterna magna puncture for CSF and tail vein for blood. Spinal cord and brain tissues are harvested, homogenized, and processed for measurement of ulefnersen concentrations by hybridization ELISA or LC-MS. For efficacy studies, motor function is assessed weekly by rotarod (time spent on an accelerating rod, typically 4-40 rpm over 5 minutes) and grip strength (peak force measured by a grip strength meter). Body weight is recorded twice weekly. The age of disease onset (defined as the age when rotarod performance declines by 20%) and survival (defined as the age when the mouse cannot right itself within 30 seconds or reaches humane endpoint) are recorded. At the end of the study, mice are euthanized, and spinal cords are harvested for measurement of FUS protein (by Western blot), FUS 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. Immunohistochemistry for FUS aggregates and glial markers (GFAP for astrocytes, Iba1 for microglia) is performed to assess neuroinflammation.

Ulefnersen is administered by intrathecal injection to achieve therapeutic concentrations in the cerebrospinal fluid (CSF) and central nervous system. Following intrathecal injection, the compound distributes throughout the CSF and penetrates into the spinal cord and brain parenchyma. In the compassionate use/expanded access program, ulefnersen showed good distribution throughout the CNS in post-mortem analysis, with detectable drug levels in the spinal cord and brain even at 2 months after the last dose. The CSF elimination half-life of ulefnersen is estimated to be approximately 7-14 days for 2′-MOE-modified ASOs, reflecting the slow turnover of CSF and the stability of the ASO. From the CSF, ulefnersen 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 ulefnersen 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 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 ulefnersen is not metabolized by CYP450 enzymes. In the ongoing Phase 1-3 FUSION trial (NCT04768972), ulefnersen is administered by intrathecal injection every 12 weeks with an additional loading dose at 4 weeks after the first administration. The primary outcome measure is a change from baseline in functional impairment as measured by the combined assessment of ALSFRS-R total score, time to rescue or discontinuation due to function deterioration, and ventilation assistance-free survival.

In the preclinical toxicology studies of ulefnersen in rodents and monkeys, intrathecal administration was generally well tolerated. The most common findings were minimal to mild mononuclear cell infiltration in the meninges and dorsal root ganglia (DRG), which is typical for intrathecally administered ASOs. No significant systemic toxicity was observed. In the clinical compassionate use/expanded access program, ulefnersen was well tolerated. No serious adverse events or deaths during the study were directly linked to ulefnersen. One patient experienced a mild adverse event that resolved without intervention. The favorable safety profile was a key factor in advancing ulefnersen to the ongoing clinical trial. In the Phase 1-3 FUSION trial, as of the latest update, ulefnersen has been well tolerated among patients, with no serious adverse events reported that were attributable to the drug. Adverse events reported have been consistent with those expected from intrathecal injection procedures, including post-lumbar puncture headache, back pain, and procedural pain. No deaths related to ulefnersen have been reported. Because ulefnersen is an investigational drug, long-term safety data are not yet available. Standard preclinical toxicology assessments (e.g., genotoxicity, carcinogenicity, reproductive toxicity) have not been publicly disclosed. As with all intrathecally administered ASOs, there is a theoretical risk of immune-mediated reactions, but no evidence of such has been reported to date. Patients receiving ulefnersen are monitored for signs of meningoencephalitis or myelitis. The ongoing trial includes safety monitoring as the primary objective for the early phases. Ulefnersen is not approved for clinical use; it is an investigational drug available only through clinical trials.

[1]. Korobeynikov VA, Lyashchenko AK, Blanco-Redondo B, Jafar-Nejad P, Shneider NA. Antisense oligonucleotide silencing of FUS expression as a therapeutic approach in amyotrophic lateral sclerosis. Nat Med. 2022;28(1):104-116.

Ulefnersen sodium (ION363, also known as Jacifusen) is an investigational antisense oligonucleotide being developed by Ionis Pharmaceuticals for the treatment of FUS-ALS, a rare and aggressive form of amyotrophic lateral sclerosis caused by mutations in the FUS gene. FUS-ALS typically affects younger individuals (often adolescents and young adults) and is characterized by rapid progression, bulbar onset, and poor prognosis. The drug was originally named Jacifusen after Jaci Hermstad, the first person to receive this treatment under the FDA compassionate use program. The compassionate use case study demonstrated slowed functional decline and CNS distribution with FUS protein reduction in post-mortem analysis, leading to the initiation of the Phase 1-3 FUSION trial. The FUSION study is a multi-center, three-part trial that started in 2021. Part 1 is a randomized, double-blind, placebo-controlled phase (2:1 randomization, ulefnersen vs. placebo) for 60 weeks, followed by an open-label extension where all participants receive ulefnersen for 84 weeks. Participants may continue to receive ulefnersen for up to three years. The study is currently recruiting in 25 locations in 16 countries. Participants must be at least 10 years old, have signs and symptoms of ALS, and a confirmed FUS variant. The estimated primary completion is 2026, with full study completion in 2028. The primary outcome measure is a change from baseline in functional impairment measured by joint rank analysis. Secondary outcomes include quality of life, slow vital capacity, handheld dynamometry, and neurofilament light chain levels. Ulefnersen has received orphan drug designation from the FDA. As of the latest update, no regulatory approval has been granted. The compound is for research use only and is not for human therapeutic applications outside of clinical trials. Ulefnersen represents a promising precision medicine approach for a devastating neurological disease.

C230H302N68O126P19S13NA19

7477.40

2589926-27-0

Ulefnersen; 2589926-25-8

Solid powder

[Ulefnersen (sodium)]

ION-363 sodium

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, 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: ≥ 25 mg/mL

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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