Breaking barriers with tofersen: Enhancing therapeutic opportunities in amyotrophic lateral sclerosis

Abstract Background and purpose Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily affects adults, characterized by muscle weakness resulting from the specific death of motor neurons in the spinal cord and brain. The pathogenesis of ALS is associated with the accumulation of mutant superoxide dismutase 1 (SOD1) proteins and neurofilaments in motor neurons, highlighting the critical need for disease‐modifying treatments. Current therapies, such as riluzole and edaravone, provide only symptomatic relief. Recently, tofersen gained approval from the US FDA under the brand name Qalsody as the first and only gene therapy for ALS, addressing a significant pathological aspect of the disease. Methods We carried out a literature survey using PubMed, Scopus, National Institutes of Health, and Biogen for articles published in the English language concerned with “amyotrophic lateral sclerosis”, pathophysiology, current treatment, treatment under clinical trial, and the newly approved drug “tofersen” and its detailed summary. Results A comprehensive review of the literature on the pathophysiology, available treatment, and newly approved drug for this condition revealed convincing evidence that we are now able to better monitor and treat ALS. Conclusions Although treatment of ALS is difficult, the newly approved drug tofersen has emerged as a potential therapy to slow down the progression of ALS by targeting SOD1 mRNA, representing a significant advancement in the treatment of ALS.


INTRODUC TI ON
Amyotrophic lateral sclerosis (ALS), also known as Charcot's disease or motor neuron disease, is a typical neuron degenerative condition defined by the gradual decline of motor neurons in the brain and spinal cord.The neurologist Jean-Martin Charcot first identified the syndrome in 1869 [1,2].In ALS there is a specific loss of motor neurons which leads to subsequent paralysis and ultimately results in the death of the affected individual [3].In adults there is onset of progressive malfunction and subsequent death of upper motor neurons (UMN) and lower motor neurons (LMN) in the motor cortex, brainstem, and spinal cord in ALS.Progressive bulbar palsy (PBP) is another name given to the disorder as the first indications appear in skeletal muscles with bulbar innervation.While primary lateral sclerosis is mainly a UMN manifestation, progressive muscular atrophy (PMA) manifests as dysfunction of LMN specifically affecting skeletal muscles in the trunk or limbs.All four limbs and the bulbar region suffer weakness and muscular atrophy as a result of ALS [2,[4][5][6].The initial weakening in ALS typically initiates in the limb muscles, particularly in the distal muscles rather than the proximal ones [7,8].ALS is specifically selective for motor neurons, and mainly spares cognitive, sensory, and autonomic nervous system functions.The only skeletal muscles left untouched are those that control eye movement and the pelvic region [3,9].Although the rate of disease development varies, most patients die from neuromuscular respiratory distress within 2-3 years of their initial symptoms [7,10].Around 10% of ALS cases are categorized as familial, whereas 90% are believed to be sporadic [11,12].In most countries, ALS is a very uncommon condition that affects 1-2 persons per 100,000 individuals annually; the prevalence of the disease is around 5 cases per 100,000 individuals, which illustrates the rapid mortality of the condition.According to statistics from the USA and UK, ALS kills more than 1 in 500 individuals [1,11].ALS is believed to arise from a combination of different factors including genetic, environmental, and malfunctions associated with the aging process [13].
In 1993, the gene for Cu/Zn superoxide dismutase 1 (SOD1) was identified as the first gene linked to ALS, responsible for 20% of familial ALS (fALS) cases and 1% to 2% of sporadic ALS (sALS) cases.These allelic variations are probably not the cause of ALS [2,15].It is still unclear exactly how SOD1 contributes to the pathogenesis of ALS.New research has revealed that the SOD1 protein, which is associated with ALS, may have additional functions beyond its known role as an antioxidant enzyme.Recent findings indicate that SOD1 could play crucial roles in activating the transcription of nuclear genes and acting as an RNA-binding protein.These newly identified functions are highly significant in understanding the underlying mechanisms of ALS and its development [15,16].The aggregation of SOD1 protein, forming insoluble structures, disrupts various cellular processes that are detrimental to neurons and contributes to their death in ALS.These abnormal processes include mitochondrial malfunction, stress in endoplasmic reticulum, and problems with protein degradation.In individuals with ALS who have SOD1 mutations, accumulations of SOD1 inclusions specifically occur in the anterior motor neurons located in the spinal cord [16,17].On April 25, 2023, the US Food and Drug Administration (FDA) approved tofersen under the brand name Qalsody for the treatment of ALS in adults with a SOD1 gene mutation.Tofersen (BIIB067), an antisense oligonucleotide, targets the SOD1 gene in individuals with ALS caused by SOD1 mutations.The specific mechanism of action of tofersen involves modulating the production of SOD1 protein.Tofersen is a synthetic, single-stranded DNA molecule that is designed to bind to the messenger RNA (mRNA) derived from the SOD1 gene.By targeting the SOD1 mRNA, tofersen works through an antisense mechanism.It binds to the mRNA sequence and triggers a process known as RNase H-mediated degradation.This degradation prevents the translation of SOD1 mRNA into SOD1 protein, thereby lowering the amount of mutant SOD1 protein in affected cells [18].

PATHOPHYS IOLOGY OF AL S
The precise mechanisms of neuron degeneration in ALS remain unknown; however, several cellular and molecular processes have been connected to disease development.These include mitochondrial dysfunction, problems with axonal transport, toxic protein aggregates, impaired protein breakdown involving autophagy or proteasome (or both), the spread of pathological proteins in a prion-like manner, oxidative stress, increased metabolism, excitotoxicity, decreased support from non-neuronal cells in terms of neurotrophic factors, inflammation, defects in RNA metabolism, and RNA toxicity [19].
There have been more than 20 identified genes that contribute to the development of ALS or increase the risk of disease development.In European populations, four specific genes (C9orf72, SOD1, TARDBP, and FUS) are responsible for causing the disease in up to 70% of individuals diagnosed with fALS.These genes have been found to have a substantial influence on the development and progression of ALS within these populations.Over 170 identified mutations associated with ALS have been found in different regions of the 153-amino acid SOD1 polypeptide.Interestingly, many of these mutated variants still exhibit partial or even full dismutase activity.Surprisingly, there is no clear relationship between the decrease in enzymatic activity and the age at which the disease starts or how quickly it progresses.As a result of these discoveries, researchers have concluded that a decline in SOD1 dismutase activity is not the only primary contributor to the onset of ALS.Instead, it is thought that the disease is caused by one or more toxic characteristics displayed by the different mutant versions of SOD1.These toxic properties could arise from structural changes, altered interactions with other cellular components, or aggregate generation that disrupts normal cellular processes and leads to neuron degeneration [1, 20,21].The main pathways for ALS pathophysiology are described in the following sections.

Mutation in SOD1 gene
ALS is associated with a mutation in the SOD1 gene which results in the toxic gain of function.This mutation in the SOD1 gene causes the formation of mutant SOD1 proteins.These mutated SOD1 proteins accumulate within the motor neurons and glial cells, leading to protein aggregation and the formation of toxic protein clumps, and cause dysfunction in the axonal transport system.How exactly these intracellular aggregates are toxic to motor neurons remains unclear, although several possible cytotoxic mechanisms have been proposed, including; (i) Coaggregation with vital cellular constitutes, (ii) Inhibition of normal proteosomic function, and (iii) Mechanical or biochemical effects on the cell, such as the disruption of axonal transport systems [1, 22,23].

Oxidative stress
The accumulated SOD1 proteins, together with other factors such as accumulation of Ca + ions, can contribute to oxidative stress within the motor neurons.This oxidative stress can lead to mitochondrial dysfunction and neuronal cell death through neuroinflammation [1, 24,25].

Glutamate excitotoxicity
In recent decades, studies have suggested that glutamate excitotoxicity has a potential role in the pathogenesis of ALS.Glutamate excitotoxicity is mediated by excessive activation of postsynaptic receptors.On the postsynaptic side there is increased expression of NMDA receptors, which are permeable to influx of Ca 2+ ions, resulting in increased intracellular Ca 2+ concentration and activation of Ca 2+ -dependent enzymatic pathways that mediate neuronal death.Glutamate excitotoxicity may also result in production of free radicals that can further damage intracellular organelles and thereby cause cell death.The pathophysiology of ALS is illustrated in Figure 1 [1, 26,27].

CURRENTLY AVAIL AB LE TRE ATMENTS FOR AL S
Currently, there are only two drugs that have been officially approved and labeled for the treatment of ALS, namely riluzole and edaravone.However, it is important to note that edaravone is only available in a limited number of countries globally.Specifically, it is accessible in the USA, Japan, Canada, and Switzerland within Europe.Key details such as structure, mechanism of action, dosage, and side effects of the two currently available drugs are listed in Table 1 [4,28,29].Although the cost of tofersen is high compared with existing drugs, it offers many advantages.

OTHER DRUG S UNDER CLINIC AL TRIAL FOR THE TRE ATMENT OF AL S
Certain drugs have been used for the treatment of non-neurological conditions for many decades and may also possess potential therapeutic effects in neurological disorders.Key details such as structure, trial code, mechanism of action, trial participants, and sponsor for all the drugs currently under clinical trial are listed in Table 2. neuron-neuroblastoma hybrid cells.TUDCA protects these cells against the harmful effects of nitric oxide (NO) toxicity.TUDCA has also gained interest in the context of ALS due to its ability to inhibit apoptosis through its interference with the mitochondrial pathway of cell death.This inhibition leads to a reduction in the production of harmful oxygen radicals, as well as a decrease in endoplasmic reticulum stress and caspase activation.Two phase 2 studies showed promising results in ALS which justified initiating a phase 3 trial [4,30,31].

Tamoxifen
Tamoxifen, a long-standing approved treatment for breast cancer, acts by selectively influencing estrogen receptors.Apart from its anticancer effects, tamoxifen also exhibits neuroprotective characteristics by regulating damage caused by inflammation and promoting autophagy.To evaluate its potential in treating ALS, a phase 2 study was carried out involving 18 ALS patients who were followed for 12 months.The study employed a randomized, double-blind, placebo-controlled approach [4,32].

Levosimendan
Levosimendan was originally developed for the treatment of heart failure in the field of cardiology.Its mode of action involves selectively binding to troponin C, which then sensitizes both fast and slow skeletal muscles.Furthermore, intravenous administration of levosimendan has been demonstrated to enhance the efficiency and contractility of the diaphragm in healthy individuals.These promising results regarding diaphragm muscle function led to a study being conducted in individuals with ALS.The study utilized a randomized, double-blind, placebo-controlled, crossover design with three periods, followed by an additional 6-month open-label follow-up phase [4,33,34].

Masitinib
Masitinib is an orally administered tyrosine kinase inhibitor that is used for treating mastocytosis.This compound has shown promising results in animal models of ALS with SOD1-G93A mutations.The interest in using masitinib for ALS stems from its ability to protect neurons through its immunomodulatory properties.It specifically targets activated microglia and mast cell activity in both the central and peripheral nervous systems, thereby exerting a neuroprotective effect.
One notable study among several publications investigating masitinib in ALS was a double-blind, placebo-controlled, randomized phase 2/3 study.This study involved 394 patients who were either given riluzole (100 mg/day) plus placebo or masitinib at doses of 4.5 or 3.0 mg/kg/day.The positive results obtained from this study TA B L E 1 Currently available treatments for amyotrophic lateral sclerosis.

Riluzole
Riluzole is a glutamate antagonist.The exact mechanism is not completely understood.Pharmacological properties that may be related to its effects: 1. Inhibiting the release of glutamate 2. Inactivating voltage-dependent sodium channels 3. Capacity to interfere with intracellular processes that follow transmitter binding at excitatory amino acid receptors

Oral tablets. Recommended dose 50 mg/12 h
Most commonly observed: dose-related nausea, asthenia, gastrointestinal problems, and elevated liver enzyme levels [28] Edaravone Edaravone is thought to eliminate hydroxyl radicals and lipid peroxides by acting as a scavenger of free radicals and reactive oxygen species [6].While its mechanism of action in ALS remains unclear, edaravone is thought to act to decrease oxidative damage to neurons, especially motor neurons and nearby glial cells that are prone to damage in ALS.Drug company officially states that the mechanism of action is unknown Intravenous (IV) infusion.Cycle 1 of the recommended treatment is an IV infusion of 60 mg/day for 14 days, followed by a 14-day drugfree period, and then daily dosage for 10/14 days, then another 14day drug-free period (cycles 2-6) Urine glucose.The level of glucose in the blood exceeds the ability of the kidneys to absorb it [29] TA B L E 2 Drugs currently under clinical trial for the treatment of amyotrophic lateral sclerosis.Peking University Third Hospital [37] Abbreviations: ALS, amyotrophic lateral sclerosis; NO, nitric oxide; ROCK, Rho-associated protein kinase; SOD1, superoxide dismutase 1.

Drug
prompted the suggestion that a larger study be conducted.The primary endpoint of the study was to measure the decline in the ALS Functional Rating Scale-Revised (ALSFRS-R) score over the course of 48 weeks [4,35].

Deferiprone
There is sufficient literature supporting the involvement of iron in neuron degenerative diseases, particularly in ALS.Iron is described as a cofactor for several enzymes involved in the functioning of motor neurons, including mitochondrial aerobic metabolism.
Furthermore, studies have shown increased levels of serum iron, ferritin, and transferrin in ALS patients compared to healthy individuals.
Postmortem studies have also indicated the accumulation of iron in the central motor tract of ALS patients, which has strengthened the hypothesis of iron's role in ALS.
The FAIRALS study is an ongoing clinical trial conducted by the French ALS Centre of Lille.It is a randomized, double-blind, placebo-controlled study that aims to evaluate the effectiveness of the iron chelator deferiprone in the treatment of ALS.The trial is expected to include 372 participants, and the treatment period will span 12 months.Half the participants will be assigned to receive a daily dose of 600 mg deferiprone, while the other half will receive a placebo.The study is registered under the identifier NCT03293069 [4,36].

Fasudil
Fasudil is a type of small molecule inhibitor known as a Rhoassociated protein kinase inhibitor (ROCK-inhibitor).Its initial development focused on its potential application in treating vasospasm that occurs after subarachnoid hemorrhage.It has been found that ROCK plays a crucial role in triggering the signaling pathway for axonal degeneration, which is activated when specific receptors bind to axonal growth inhibitory molecules.with the aim of determining the appropriate dosage of the ROCK inhibitor.Fasudil, the ROCK inhibitor of interest, will be administered intravenously twice daily for 20 days of treatment.The trial will consist of three arms: one receiving 30 mg/day fasudil, another receiving 60 mg/day fasudil, and a placebo group serving as a control [4,37].

MECHANIS M OF AC TION OF TOFER S EN
Tofersen is an antisense oligonucleotide that has been developed to specifically target SOD1 mRNA.This antisense oligonucleotide utilizes the process of RNase H-dependent degradation of SOD1 mRNA, resulting in a decrease in the production of SOD1 protein.

PHARM ACO KIN E TIC S OF TOFER S EN
The pharmacokinetic data for tofersen is comparable in healthy individuals and people with ALS.Tofersen is injected intrathecally into the CSF, allowing it to distribute from the CSF to the tissues of the central nervous system (CNS).The maximum concentration of tofersen in the CSF is achieved with the third dose, which is the final dose of the loading period.Following the loading phase, there is minimal to no accumulation of tofersen in the CSF with monthly dosing after the loading phase.Tofersen is delivered into the systemic circulation from the CSF, and it takes, on average, 2-6 h for it to reach its peak plasma concentration (T max ).With a monthly maintenance dose, tofersen did not accumulate in the plasma.Autopsy examination of CNS tissue from patients (n = 3) treated with intrathecal tofersen revealed its distribution within the CNS tissues.Tofersen is neither a substrate for cytochrome P450 (CYP450) enzymes nor is it an inducer or inhibitor of CYP450 enzymes.It is predicted that tofersen is mainly metabolized through exonuclease (3′ and 5′)-mediated hydrolysis.The primary elimination pathway has not been fully elucidated [38].

PHARM ACO DYN AMIC S Effect of tofersen on total CSF SOD1 protein
In clinical studies, the level of total SOD1 protein was assessed.
Clinical Study Part C (NCT02623699) showed that the total CSF SOD1 protein decreased by 35% in the tofersen-treated group compared to a 2% decline in the placebo group at day 196.

Effect of tofersen on neurofilament protein
A blood-based biomarker of axonal damage and neurodegeneration, plasma neurofilament, has been studied in clinical trials.Mean plasma neurofilament was reduced to 55% in the tofersen-treated group compared to a 12% increase in the placebo group at day 196 of Clinical Study Part C (NCT02623699).

Cardiac electrophysiology
There is no clinically significant lengthening of the QTc interval at the maximum dose regimen of tofersen [38].

CLINIC AL TRIAL S OF TOFER S EN
Various clinical trials for the study of tofersen have been conducted by Biogen.Phase 1-2 studies were conducted involving 50 patients, the primary objective being to evaluate the safety SOD1 has a redox potential and can convert the superoxide anion into hydrogen peroxide (H 2 O 2 ) [2, 7, 14].Moreover, since 1993, more than 200 different mutations of SOD1 have been reported.Of these mutations, 108 are missense mutations which affect 66 codons in which one amino acid is exchanged with another while the full length of the polypeptide remains the same.These missense mutations are regarded as the root cause of ALS.Of 11 nonsense mutations which shorten the length of the polypeptide, only two mutations have been associated with the disorder.Six mutations are silent DNA variants in which the sequence and length of the polypeptide do not change.
Tauro-ursodeoxycholic acid (TUDCA) is a well-known medication used by gastroenterologists to treat chronic cholestatic liver and gallstone diseases.It can be taken orally and can cross the blood-brain barrier.TUDCA has been found to have neuroprotective properties in cells that express mutant SOD1 (specifically, mutations A4V and G93A) which are involved in motor F I G U R E 1 Pathophysiology of amyotrophic lateral sclerosis.
that has shown significant improvement in survival and motor function in mutant SOD1 model when administered presymptomatically 10 context of neurodegenerative diseases and have shown both neuroprotective and pro-regenerative effects in models of Parkinson's disease (PD).In the case of ALS, ROCK inhibitors have shown significant improvements in survival and motor function in the SOD1(G93A) mouse model when administered at a presymptomatic stage.A phase 2a clinical trial is expected to commence soon in ALS, involving multiple centers and employing a randomized, double-blind, placebo-controlled, prospective design with a mixed backbone structure consisting of 5-10-5 MOE gapmer.It is composed of 19 inter-nucleotide linkages, with 15 of them being 3′-O to 5′-O phosphorothioate diesters, and the remaining four being 3′-O to 5′-O phosphate diesters.Among the 20 sugar residues, 10 are 2-deoxy-D-ribose, while the other 10 are 2′-O-(2methoxyethyl)-D-ribose (MOE).The arrangement of the residues is such that there are five MOE nucleosides positioned at both the 5′ and 3′ ends of the molecule, creating a gap of 10 2′-deoxynucleosides in between.Additionally, the cytosine and uridine bases within nal fluid (CSF) surrounding the spinal cord.It enters motor neurons and binds specifically to SOD1 mRNA and forms an RNA-DNA hybrid.After forming an RNA-DNA hybrid, tofersen activates RNase H-dependent enzyme.This RNA-DNA hybrid is a substrate for the RNase H-dependent hybrid and it cleaves RNA strands resulting in the degradation of mutant SOD1 mRNA.This leads to the reduction of SOD1 mRNA in the motor neurons which ultimately leads to reduction in the mutant SOD1 protein and of neurofilament light chains in plasma, as there are fewer templates of SOD1 mRNA for the translation process to produce mutant SOD1 protein, which

F I G U R E 2 3
and pharmacokinetic data and to assess the SOD1 concentration change from baseline at day 85.These trials were conducted at different 18 sites in USA, Canada, and four countries in Western Europe (Germany, France, Belgium, and the UK).Five doses of tofersen and a placebo were provided intrathecally over the Structure of tofersen.Clinical trials of tofersen.