Cutting-edge treatments in amyotrophic lateral sclerosis: the role of molecular pathogenesis in targeted therapies

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the selective loss of motor neurons (MNs), leading to progressive muscle weakness, atrophy, and ultimately paralysis. This review provides a comprehensive overview of the molecular mechanisms underlying ALS pathogenesis, the genetic mutations associated with both familial and sporadic forms of the disease, and the latest therapeutic strategies aimed at mitigating disease progression. mutations in genes such as C9orf72, SOD1, TARDBP, and FUS have been implicated in ALS, with an intricate interplay of protein misfolding, oxidative stress, mitochondrial dysfunction, excitotoxicity, and neuroinflammation contributing to motor neuron degeneration. While current FDA-approved treatments such as Riluzole and Edaravone offer only modest benefits and do not significantly halt disease progression. Emerging therapies, including gene therapies (e.g., antisense oligonucleotides (ASOs) and CRISPR/Cas9, stem cell-based approaches, and neurotrophic factor supplementation, are demonstrating promising results in preclinical and early-phase clinical trials. novel approaches aim to target, modulate, and promote regeneration, renewed hope for future ALS treatments. However, several challenges remain, including effective delivery methods, safety concerns, and the inherent complexity of ALS pathology, ongoing research continues to explore these innovative interventions with the goal of improving clinical outcomes for patients. This review highlights the importance of personalized therapeutic approaches and underscores the necessity of continued innovation in ALS research, with the ultimate goal of developing disease-modifying therapies and, potentially, a cure for this fatal condition.

Highlights

Amyotrophic Lateral Sclerosis, Neurodegenerative Diseases, C9orf72 Mutation, SOD1 Mutation, Riluzole This comprehensive review article presents an in-depth analysis of the molecular pathogenesis of Amyotrophic Lateral Sclerosis (ALS), emphasizing the complex interplay of genetic mutations, protein misfolding, oxidative stress, mitochondrial dysfunction, excitotoxicity, and neuroinflammation that collectively drive motor neuron degeneration. By systematically categorizing ALS subtypes based on genetic, clinical, and molecular characteristics, the article elucidates the diverse mechanisms underlying both familial and sporadic forms of the disease. Notably, it highlights key genetic mutations such as those in C9orf72, SOD1, TARDBP, and FUS and details how these contribute to disease pathology through distinct molecular pathways. The review integrates recent advances in understanding ALS heterogeneity and the impact of gene-environment interactions and epigenetic factors, underscoring the necessity for personalized therapeutic approaches.

A novel aspect of this review is its comprehensive coverage of cutting-edge therapeutic strategies targeting ALS at the molecular level, including gene therapies like antisense oligonucleotides (ASOs), RNA interference, and CRISPR/Cas9 gene editing, alongside stem cell-based treatments and antibody-mediated interventions. The article critically evaluates the current state of FDA-approved drugs, such as Riluzole and Edaravone, noting their limited efficacy, while bringing to light promising preclinical and clinical trial data on novel treatments aimed at halting or reversing disease progression. Furthermore, it discusses the challenges inherent in delivering therapies across the blood-brain barrier, safety concerns, and the need for robust clinical trial designs. By integrating molecular insights with therapeutic innovations and clinical perspectives, this review advances the field by providing a valuable roadmap for future research focused on developing effective, disease-modifying treatments for ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a degenerative neurological disorder that damages the motor neurons (MNs) of the spinal cord and brain. These neurons control voluntary muscle movements such as Walking, talking, swallowing, and breathing. Muscle weakness, atrophy, and ultimately paralysis follow from motor neuron the progressive degeneration and eventual death in ALS [1, 2]. Althoughrare exceptions exist, the disorder typically does not affect cognitive abilities, sensory functions senses, or involuntary muscles. ALS is also known as Lou Gehrig’s disease, named after the great baseball player who was diagnosed with the condition [3]. In the coming years, ALS will continue to be a significant focus for public health organizations and researchers. Moreover, forecasts generated by the National ALS Registry in the United States project a modest increase in the prevalence of ALS in the years ahead. The Registry specifically projected that, at around 32,893 ALS patients in the United States in 2022, the prevalence of the disease would be 9.9 per 100,000 persons. Specifically, the Registry estimated that there were approximately 32,893 ALS patients in the United States in 2022, corresponding to a prevalence of 9.9 per 100,000 persons. By 2030, this number is expected to rise by more than 10% to over 36,000 cases [4]. A thorough 42-year population-based study examining ALS incidence and mortality in Denmark from 1980 to 2021. The study identified 5,943 ALS cases and reported an overall incidence rate of 3.4 per 100,000 people annually. Notably, the incidence rate increased 2.8 -fold when comparing 2018–2021 to 1980–1983. Over time, the mortality rate showed an upward trend, with the proportion of deaths in the final period that exceeding that of the initial period [5]. The study found that the incidence and fatality rates of ALS, both associated with increased risk in males and advancing age, have tripled over the past four decades. Given the aging population, the number of elderly ALS patients is likely to rise sharply [6]. Based on its etiology, ALS is classified into two Subtypes. Sporadic ALS (SALS) is the most common form of ALS, accounting for approximately 90–95% of all cases, and occurs without a clear genetic or familial link [7]. Several factors have been proposed to contribute to its development. Environmental elements include pesticide, agricultural chemicals, heavy metals (e.g., lead, mercury), and toxins such β-methylamino-L-alanine (BMAA) in some areas, like Guam [8, 9]. Lifestyle choices such as smoking and excessive physical exertion have also been Suggested as possible risk factors. Motor neuron degeneration may be caused, in part, by immunological and inflammatory responses marked by abnormal immune system activation [10, 11]. Furthermore a major risk factor is aging; incidence peaks between the ages of 50 and 70. Damage caused by free radicals leads to oxidative stress, which may further contribute to neuron degeneration [12]. Familial ALS (fALS), on the other hand, is a hereditary form of the disease that accounts for approximately 5–10% of cases. Usually follows an autosomal dominant inheritance pattern and is caused by inherited mutations in specific genes; however, some cases follow autosomal recessive or X-linked patterns [2, 13]. Among the first identified mutations, C9orf72 is the most common mutation linked to both ALS and frontotemporal dementia (ALS-FTD); SOD1 (superoxide dismutase 1) mutations are found in roughly 20% of familial cases; FUS (fused in sarcoma) mutations are associated with juvenile-onset and aggressive forms of ALS, while TARDBP mutations affect the TAR DNA-binding protein involved in RNA processing. Other mutations include those in ALS2, SETX, VCP, UBQLN2, and MATR3 all of which contribute to disease risk. The Age of onset, disease course, and related symptoms- including cognitive impairment- are influenced by this genetic diversity [14–19]. Emerging concepts in ALS etiology highlight cases that do not fit neatly into either sporadic or familial categories. Often, these examples include gene-environment interactions- that is, interactions between genetic predisposition and environmental factors like toxins or infections. Epigenetic elements, which modify gene expression without altering the DNA sequence, are also thought to play a role. This suggests a complex interplay between environmental and genetic factors in ALS development [20–23]. As a result, the molecular targets for disease-modifying therapies may vary not only across different types of ALS, but also among individual cases of SALS. Our understanding of the molecular and cellular mechanisms underlying ALS pathogenesis remains limited, which complicates the selection of appropriate therapeutic targets. It is therefore not surprising that a disease-modifying therapy for ALS is still an aspiration, and even treatments that could significantly extend patient survival or slow disease progression are currently unavailable [24, 25]. This review aims to comprehensively explore the molecular mechanisms underlying ALS, evaluate current and emerging therapeutic strategies- including gene therapy, stem cell therapy, and pharmacological interventions- and highlight the challenges and future directions in developing effective treatments for this devastating neurodegenerative disease.

Classification of ALS based on different aspects

Classification of ALS by site of onset

The site of onset defines three primary forms of ALS: limb-onset, bulbar-onset, and respiratory-onset ALS. About 70–80% of cases with limb-onset ALS, which is the most prevalent form, begins in the upper or lower extremities, with initial symptoms manifesting in these regions. Early signs may include difficulty walking, tripping, or challenges with fine motor tasks such as buttoning a shirt. Typically, these symptoms start in one leg, with muscle weakness and atrophy gradually affecting other regions over time [26–28]. About 20–25% of cases are Bulbar-onset ALS, which mostly affects the muscles involved in swallowing, chewing, and speaking. This variety is associated with faster disease progression than limb-onset ALS. Early symptoms include dysarthria, dysphagia, and drooling. Respiratory-onset ALS is Rare, accounting for 1–3% of cases and begins with symptoms like dyspnea and fatigue caused by weakened breathing muscles. Patients with this form may have respiratory failure early in the course of their disease, especially after exertion or when lying down [29–31].

Classification of ALS by clinical presentation

ALS presents in various forms, each with distinct clinical characteristics. Classic ALS is characterized by symptoms such as muscle weakness, stiffness, and atrophy, resulting from the combined degeneration of both upper motor neurons (UMNs) and lower motor neurons (LMNs). In contrast, Primary Lateral Sclerosis (PLS) develops more slowly than typical ALS and affects only the UMNs, resulting in gradual spasticity, stiffness, and slowed movements [32–34]. Affected only LMNs, Progressive Muscular Atrophy (PMA) causes symptoms including weakness, muscle atrophy, and fasciculations; In some cases, PMA may progress to classic ALS [35, 36]. Whereas Flail Leg Syndrome involves muscle weakness and wasting mostly in the legs, typically progressing more slowly than classic ALS, Flail Arm Syndrome (proximal ALS) mostly affects the shoulder and arm muscles and is characterized by slow progression and limited involvement of other regions. Oculomotor ALS is an extremely rare form in which the eye muscles are affected early, while Split-Hand Syndrome presents with a specific pattern of hand muscle wasting, primarily affecting the thumb and index finger [37–40].

Classification of ALS by genetic mutations

Genetic mutations play a significant role in the development of ALS, with several key mutations identified as major contributors. C9orf72-related ALS is the most common genetic cause and is often associated with ALS-FTD. SOD1-related ALS, the second most common genetic cause, is characterized by variable progression rates [16, 41]. FUS-related ALS tends to present in younger patients, with rapid progression in most cases. TARDBP-related ALS is associated with LMN-predominant phenotypes [42, 43]. Additionally, a characteristic feature of ALS2-related and juvenile ALS caused by mutations in the ALS2 gene is a gradual progression and onset occurring prior to the age of 25 [44].

Molecular mechanisms in pathogenesis of ALS

The molecular mechanisms underlying ALS involve a complex interplay of genetic mutations, protein misfolding, mitochondrial dysfunction, oxidative stress, excitotoxicity, and neuroinflammation, all of which contribute to motor neuron degeneration and cell death. Several genetic mutations have been implicated in ALS, each contributing to disease pathogenesis through different molecular pathways [45–47]. The most common mutation, a hexanucleotide repeat expansion in C9orf72, leads to the formation of toxic RNA foci, sequestration of RNA-binding proteins, and repeat-associated non-ATG (RAN) translation, resulting in the accumulation of dipeptide repeat proteins (DPRs) that disrupt cellular homeostasis [48, 49]. Mutations in SOD1, responsible for approximately 20% of fALS cases, result in protein misfolding, aggregation, oxidative stress, and mitochondrial dysfunction, ultimately promoting motor neuron death [50, 51]. Additionally, mutations in TARDBP (TDP-43) and FUS lead to abnormal protein aggregation and impairment of RNA metabolism, disrupting RNA processing, transport, and stress granule formation, which contributes to neurotoxicity [18, 52]. Other implicated genes, such as Charged Multivesicular Body Protein 2B (CHMP2B), TANK-binding kinase 1 (TBK1), Optineurin (OPTN), Vesicle-associated membrane protein-associated protein B (VAPB), FIG4 Phosphoinositide 5-Phosphatase (FIG4), NIMA Related Kinase 1 (NEK1), Sequestosome 1 (SQSTM1), and Spastic Paraplegia 11 (SPG11), influence ALS pathogenesis by affecting vesicle trafficking, proteostasis, and cytoskeletal dynamics [53–56]. Protein misfolding is a hallmark of ALS, leading to the accumulation of misfolded and aggregated proteins that overwhelm cellular quality control systems. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), a stress response aimed at restoring protein homeostasis. however, chronic ER stress can induce apoptosis in MNs [57, 58]. Dysfunction in protein degradation pathways, including the ubiquitin-proteasome system (UPS) and autophagy, significantly contributes to disease progression by preventing the clearance of misfolded proteins [59, 60]. Genetic mutations in C9orf72, OPTN, SQSTM1, and UBQLN2 play critical roles in these pathways and contribute to neurodegeneration [17]. Mitochondrial abnormalities also play a central role in ALS, resulting in metabolic dysfunction and neuronal energy deficits [61]. Mutations in SOD1, CHCHD10, and ALS2 disrupt mitochondrial dynamics, impair axonal transport and lead to energy depletion in MNs. In addition, oxidative stress caused by excessive reactive oxygen species (ROS) production from mutant SOD1 and dysfunctional mitochondria leads to oxidative damage to proteins, lipids, and DNA, further accelerating neuronal degeneration [62–64]. Excitotoxicity, caused by excessive glutamate stimulation of MNs, is another major contributor to ALS pathophysiology. Dysfunction of the excitatory amino acid transporter 2 (EAAT2), which is primarily expressed in astrocytes, results in impaired glutamate clearance, leading to its accumulation and overstimulation of NMDA and AMPA receptors [65–67]. This overactivation increases cytoplasmic calcium (Ca²⁺) influx into MNs, which triggers enzymatic pathways that promote apoptosis and excitotoxicity [68, 69]. Proteins such as SOD1 and TDP-43 further contribute to dysregulated calcium homeostasis, exacerbating neuronal death. Neuroinflammation also plays a crucial role in ALS progression, driven by interactions among MNs, microglia, and astrocytes [70, 71]. Activated microglia release pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), as well as ROS, which contribute to neurotoxicity. Astrocytes, which normally provide neurotrophic support and maintain synaptic homeostasis, undergo reactive changes in ALS, leading to the release of inflammatory mediators and impaired glutamate clearance. Furthermore, the Fas ligand (FASL) pathway promotes motor neuron apoptosis by triggering caspase activation and mitochondrial dysfunction [72, 73]. MNs depend on effective axonal transport for the distribution of organelles, the trafficking of vesicles, and the transport of proteins; impairments in these mechanisms are associated with the progression of ALS [74, 75]. Mutations in ALS2, VAPB, FIG4, UNC13A, and CHMP2B disrupt vesicle transport, impairing synaptic function and neuronal survival [76]. Additionally, cytoskeletal abnormalities caused by mutations in SPG11, PFN1, DCTN, and NEFH compromise axonal stability and integrity, further contributing to motor neuron dysfunction [77, 78]. Several key signaling pathways are also involved in ALS pathogenesis, influencing neurodegeneration, inflammation, and cellular stress responses. Dysregulation of the NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway contributes to chronic neuroinflammation, exacerbating motor neuron damage [79, 80]. The MAPK signaling pathway, which regulates stress responses, apoptosis, and inflammation, also plays a role in neuronal degeneration [81, 82]. Calcium-dependent pathways contribute to excitotoxicity by activating apoptosis-related cascades involving BAX, Bcl-2,Bcl-xl, BAD, and caspases [83–85]. In the Fig. 1, we illustrate a schematic representation of the molecular pathophysiology associated with ALS. Together, these interconnected molecular mechanisms drive ALS pathogenesis, highlighting the complexity of the disease and the need for targeted therapeutic interventions.

Fig. 1.

This figure summarizes the major molecular mechanisms involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). A key feature is glutamate excitotoxicity, driven by mutations in genes such as SOD1, C9orf72, and TARDBP, which reduce EAAT2 levels in astrocytes, leading to synaptic glutamate accumulation and activation of apoptotic pathways. Microglia contribute further by releasing pro-inflammatory cytokines, promoting chronic neuroinflammation. Intracellularly, oxidative stress, mitochondrial dysfunction, and impaired glial metabolism contribute to motor neuron damage. These alterations lead to protein misfolding, ER stress, and proteasomal impairment, disrupting protein homeostasis. Dysregulation of axonal transport and vesicle trafficking, due to mutations in genes like OPTN and CHMP2B, also play a role. Although autophagy is activated as a compensatory mechanism, its efficiency is often impaired in ALS. Together, these events reflect a complex, multi-system process driving motor neuron degeneration. ROS reactive oxygen species, RNS reactive nitrogen species, SOD1 superoxide dismutase 1, FUS fused in sarcoma, CHMP2B Charged Multivesicular Body Protein 2B, TBK1 TANK-binding kinase 1, OPTN Optineurin, VAPB Vesicle-associated membrane protein-associated protein B, FIG4 FIG4 Phosphoinositide 5-Phosphatase, NEK1 NIMA Related Kinase 1, SQSTM1 Sequestosome 1, SPG11 Spastic Paraplegia 11, EAAT2 excitatory amino acid transporter 2, TNF-α tumor necrosis factor-alpha, FASL Fas ligand, NF-κB Nuclear Factor kappa-light-chain-enhancer of activated B cells, MAPK mitogen activated protein kinase

Drug treatments

Although no cure exists for ALS, various treatments aim to slow disease progression, manage symptoms, and improve quality of life. The current ALS treatments are compiled here in a comprehensive review. Disease-modifying treatments in ALS primarily focus on slowing the course of the disease, delaying the onset of impairment, and increasing survival. Two FDA-approved drugs, Riluzole (Rilutek) and Edaravone (Radicava), play crucial roles in the treatment of ALS by targeting various pathophysiological mechanisms associated with the disease. The first FDA-approved medication for ALS, riluzole, has been demonstrated to modestly increase survival by 2–3 months [86]. It acts by lowering glutamate release and blocking excitotoxicity, a mechanism in which excessively high glutamate levels overstimulates and damages MNs. Usually it administered orally and has the potential to slow the progression of ALS, particularly in its early stages, by mitigating associated damage [87]. Edaravone is another crucial medication which is approved in 2017, that has shown effectiveness in reducing functional deterioration in ALS, particularly in early stages. As an antioxidant, Edaravone scavenges free radicals and lowers the oxidative stress that greatly accelerates ALS-related neural damage. While not classified as a therapeutic agent, clinical research has demonstrated that Edaravone can slow the decline in the ability to perform daily activities, such as walking and other daily tasks. Over a 10–14-day period, this medication is given intravenously with regular breaks. Although these therapies help some, they are not curative and ALS patients continue to experience a gradual decline in their motor functions [88–90]. Ongoing research on gene therapies, neuroprotective medications, and stem cell therapies may offer future hope for more effective treatments. In addition to disease-modifying therapies, symptomatic treatments are very important for enhancing the quality of life for ALS patients by managing various symptoms-such as emotional lability, spasticity, discomfort, and exhaustion- that develop as the condition progresses [91]. Baclofen (Lioresal) is frequently prescribed for the management of spasticity, a condition characterized by muscle stiffness and involuntary contractions. As a GABA-B receptor agonist, it reduces the overactivity of MNs, thereby alleviating muscle tightness and enhancing mobility [92, 93]. Likewise, an alpha-2 adrenergic agonist, tizanidine (Zanaflex), lowers muscular tone by blocking the release of norepinephrine at the spinal level, thus helping to control spasticity and improve the patient’s ability to perform everyday tasks [94]. Gabapentin (Neurontin) is frequently used off-label to manage neuropathic pain, which is a prevalent symptom associated with ALS. Effective in relieving pain related to nerve damage, gabapentin acts by binding with high affinity to the α2δ subunit of presynaptic voltage-gated calcium channels, thereby inhibiting the release of excitatory neurotransmitters. Also Used off-label, Amantadine (Symmetrel) helps control cognitive problems and fatigue commonly experienced by ALS patients. It is thought to work by altering the activity of NMDA receptors and dopamine, thereby enhancing cognitive function and mood [95–97]. Another distressing sign in ALS is pseudobulbar affect (PBA), which is characterized by uncontrollably laughing or crying. Nuedexta, which is FDA-approved for the treatment of PBA, is a combination of quinidine and dextromethorphan. Dextromethorphan is an NMDA receptor antagonist; quinidine increases the effects of dextromethorphan by preventing its breakdown. This combination has shown to be highly effective in controlling emotional reactions and significantly improving ALS patients’ emotional quality of life [98–100].

Disease-modifying therapies and symptomatic treatments together provide ALS patients with essential management options that address both disease progression and related symptoms. New therapeutic approaches, which could one day lead to more effective treatments and, ideally, a cure for ALS, are continually under investigation. Table 1 summarizes the drugs currently used to treat ALS.

Table 1.

Drugs in the treatment of ALS

Drug	Brand Name	Weight	Pharmacodynamics	Half-Life	Indication	FDA	Clinical Trial Results	Refs
Riluzole	Rilutek	234.1 g/mol	Reduces glutamate release, inhibits glutamate excitotoxicity, neuroprotective effects	12 h	First-line treatment for ALS, slowing disease progression	Yes	Proven to slightly extend survival by ~ 2–3 months	[86, 101, 102]
Edaravone	Radicava	174.2 g/mol	Antioxidant, free radical scavenger, reduces oxidative stress in ALS patients	4–6 h	Slows progression in early-stage ALS	Yes	Shown to slow progression of ALS in early stages	[90, 103, 104]
Nuedexta	Nuedexta	452.1 g/mol	Combination of dextromethorphan (NMDA antagonist) and quinidine (CYP450 inhibitor) to modulate neurotransmitter activity	12–20 h	For pseudobulbar affect (PBA) (uncontrolled laughing/crying)	Yes	Significant benefit in reducing PBA symptoms in ALS	[98, 99, 105]
Baclofen	Lioresal	213.7 g/mol	GABA-B receptor agonist, reduces spasticity and muscle tightness	2–6 h	For spasticity (muscle stiffness) in ALS	Yes	Effective for spasticity management in ALS	[106–108]
Tizanidine	Zanaflex	198.7 g/mol	Alpha-2 adrenergic receptor agonist, reduces muscle tone, spasticity	2.5–3 h	For spasticity in ALS	Yes	Clinically proven to reduce spasticity
Dantrolene	Dantrium	348.5 g/mol	Inhibits calcium release from the sarcoplasmic reticulum, reduces muscle contraction	9 h	For spasticity (off-label for ALS)	Yes	Efficacy shown in reducing spasticity in neurological conditions	[109, 110]
Amantadine	Symmetrel	149.2 g/mol	Dopamine antagonist, modulates glutamate activity, antiviral properties	10–14 h	For fatigue and cognitive issues in ALS	Yes	Evidence for fatigue and cognitive improvement in ALS patients	[111, 112]
Gabapentin	Neurontin	171.2 g/mol	Inhibits excitatory neurotransmitter release, blocks calcium channels, used for neuropathic pain	5–7 h	For neuropathic pain, off-label for ALS	Yes	Proven effective in neuropathic pain, off-label use for ALS	[95, 113–116]
Corticosteroids	Prednisone	434.5 g/mol	Anti-inflammatory, reduces immune response and swelling	3–4 h	For inflammation and symptom management	Yes	Used in inflammatory stages of ALS, may reduce symptoms	[117–120]
Lopinavir/Ritonavir	Kaletra	628.5 g/mol	Antiviral treatment for HIV, protease inhibitor, may have neuroprotective effects in ALS	5–6 h	Investigational for ALS-related neuroprotection	No	Investigational in ALS, clinical trials ongoing	[121–124]

Table 2 provides a summary of important pharmacokinetic parameters for selected drugs. These parameters encompass drug absorption, metabolism, volume of distribution, clearance, route of elimination, and recommended dosages. The information highlights key aspects of each drug’s pharmacokinetics, aiding in understanding their behavior within the body. These insights can assist healthcare professionals in optimizing therapeutic regimens for patients.

Table 2.

Comparison of Pharmacokinetic parameters of the selected drugs

Drug	Absorption	Volume of Distribution (Vd)	Metabolism	Clearance	Dosage	Refs
Riluzole	Rapid, oral administration (95%)	0.6–1.2 L/kg	Hepatic (CYP1A2, CYP3A4)	Slow, half-life 12 h	50 mg twice daily	[125–127]
Edaravone	IV infusion, bioavailability ~ 30%-oral administration	0.93 L/kg	Hepatic (glucuronidation)	Moderate, half-life 4–6 h	60 mg IV daily for 14 days, then 14 days off	[90, 104]
Nuedexta	Oral administration, peak after 2 h	2.4–3.3 L/kg	Hepatic (CYP2D6, CYP3A4)	Slow, half-life of dextromethorphan ~ 12 h	20 mg dextromethorphan/10 mg quinidine twice daily	[98, 99, 105]
Baclofen	Rapid, oral absorption (~ 70%)	2.0–4.0 L/kg	Hepatic (CYP2C9)	Moderate, half-life 3–4 h	5–10 mg 3 times daily (adjusted based on response)	[92, 93, 128, 129]
Tizanidine	Rapid, oral absorption (~ 90%)	2.4 L/kg	Hepatic (CYP1A2)	Moderate, half-life 2.5–3 h	2–4 mg 2–3 times daily, max 36 mg/day	[94, 130, 131]
Dantrolene	Oral, bioavailability ~ 20–30%	0.6–1.5 L/kg	Hepatic (CYP3A4)	Slow, half-life ~ 9 h	25–100 mg 2–4 times daily, depending on severity	[109, 132, 133]
Amantadine	Oral, bioavailability ~ 90%	2.5–3.5 L/kg	Hepatic (CYP2D6)	Moderate, half-life 10–14 h	100–200 mg daily	[134–137]
Gabapentin	Oral, bioavailability 60%	1.0–1.3 L/kg	No significant metabolism	Slow, half-life 5–7 h	300–900 mg 3 times daily	[138–140]
Corticosteroids	Oral, rapid absorption (~ 90%)	0.5–1.0 L/kg	Hepatic (CYP3A4, reduction)	Moderate, half-life varies with formulation	Prednisone: 5–60 mg daily (adjusted based on response)	[141–143]
Lopinavir/Ritonavir	Oral, bioavailability ~ 33%	4.5 L/kg	Hepatic (CYP3A4)	Moderate, half-life ~ 5–6 h	Lopinavir 400 mg/ritonavir 100 mg twice daily	[121, 144–146]

Gene therapy approaches

As mentioned above genetic mutations play a significant role in both FALS and SALS. Approximately 30–40% of FALS cases, mostly in Europe and the USA, are caused by mutations in the C9orf72 gene, making it the most common genetic factor. This mutation leads to an expansion of GGGGCC hexanucleotide repeats, resulting in both harmful gain-of-function effects and a reduction in normal protein activity. Mutations in the SOD1 gene, which typically follow an autosomal dominant inheritance pattern, account for approximately 15–20% of familial cases. These genetic alterations are key contributors to ALS, influencing the disease’s onset and progression [147]. Furthermore roughly 5% of family cases are caused by mutations in TARDBP and FUS genes; FUS mutations are generally associated with faster disease progression [148]. About 70% of cases with a familial history and a smaller fraction of sporadic cases, can be explained by more than 40 genes generally linked to ALS [147]. Zebrafish models have also been widely used in ALS research to evaluate these gene therapy strategies, offering a rapid and genetically tractable system to study motor neuron pathology and therapeutic efficacy before translation into mammalian models. In Fig. 2, we provide brief but comprehensive overview of the diverse gene therapy strategies discussed in this article. Gene therapy is a cutting-edge approach aimed at correcting, replacing, or regulating genes responsible for diseases. Two key strategies used to mitigate or eliminate the effects of deleterious genes are gene knockdown and gene knockout. Gene knockdown transiently suppresses gene expression at the RNA level, typically employing methods such as RNA interference (RNAi) to degrade or block messenger RNA (mRNA). Since this approach does not alter the DNA sequence, its effects are generally reversible, making it valuable for studying gene function as well as therapeutic applications where temporary gene suppression is desired [149–152]. In contrast, gene knockout permanently inactivates a gene by directly altering the DNA sequence, often utilizing genome-editing tools such as CRISPR-Cas9. This results in mutations that cause an irreversible loss of gene function, leading to a complete and sustained absence of gene activity. Gene knockouts are crucial for elucidating gene roles in development and disease pathogenesis and are widely used in the generation of stable disease models. Together, these techniques offer versatile options in gene therapy, depending on whether transient or permanent gene silencing is required for therapeutic or research objectives [149, 153, 154]. To enhance the efficacy of gene therapy for ALS, it is essential to have a thorough understanding of the physiological role of the causative gene and the intricate molecular pathways contribute to its pathogenicity. Therefore, it is crucial to develop approaches that specifically and effectively target the underlying pathological mechanism of ALS [155]. It is widely acknowledged that distinct gene therapy strategies exist in the context of ALS, as outlined in Table 3.

Fig. 2.

This figure provides an overview of various gene therapy strategies, including ASO, RNAi, CRISPR/Cas9 gene editing, NTFs, and Antibody-based therapies, highlighting their mechanisms of action and biological applications. ASO therapy regulates gene expression by modulating alternative splicing or inducing mRNA degradation. RNAi silences gene expression through siRNA-mediated mRNA degradation, often delivered via AAV vectors. CRISPR/Cas9 enables precise gene modifications using either HDR or NHEJ following targeted DNA cleavage. NTFs, delivered through AAV, promote neuronal survival and prevent motor neuron degeneration. Lastly, antibody-based therapies target misfolded protein aggregates, facilitating their clearance to prevent neurotoxicity. These gene therapy approaches hold significant potential for treating genetic and neurodegenerative disorders. ASO Antisense Oligonucleotides, RNAi RNA Interference, NTFs Neurotrophic Factors, AAV Adeno-Associated Virus, HDR Homology-Directed Repair, NHEJ Non-Homologous End Joining, BDNF Brain-Derived Neurotrophic Factor, GDNF Glial Cell Line-Derived Neurotrophic Factor, CNTF Ciliary Neurotrophic Factor, VEGF Vascular Endothelial Growth Factor, NDNF Neuron-Derived Neurotrophic Factor

Table 3.

Gene therapy strategies and clinical trials

Gene therapy strategy	Drug	Clinical Trial Identification	Status
Antisense oligonucleotides or RNA interference	ISIS 333,611	ClinicalTrials.gov: NCT01041222	Phase 1 Completed
	BIIB067 or Tofersen	ClinicalTrials.gov: NCT02623699	Phase 3 Completed
		ClinicalTrials.gov: NCT03070119	Extension of Phase3 Ongoing
	BIIB078	ClinicalTrials.gov: NCT03626012	Phase 1 Completed
	WVE-004	ClinicalTrials.gov: NCT04931862	Phase 1b/2a Ongoing
	ION363 (Jacifusen)	ClinicalTrials.gov: NCT04768972	Phase 1–3 Ongoing
	BIIB105	ClinicalTrials.gov: NCT04494256	Phase 1 Ongoing
CRISPR/Cas9 Gene Editing
Antibody based strategies	Tegoprubart	Clintrials.gov: NCT04322149	Phase 2 A Ongoing
AAV mediated gene therapy	AMT-162	Clintrials.gov: NCT06100276	Phase 1/2 Ongoing

Antisense oligonucleotides or RNA interference

This methodology involves the utilization of small interfering RNAs (siRNAs) or antisense oligonucleotides (ASOs) to inhibit the expression of target genes at the mRNA level, thereby decreasing the synthesis of harmful proteins [156]. ASOs offer a promising therapeutic approach for ALS by modulating alternative splicing of disease-related genes at the RNA level without altering the DNA sequence [157]. In this strategy, ASOs are designed to specifically bind to pre-mRNA and influence the splicing process, which determines how RNA segments are assembled into mature transcripts. By redirecting splicing, ASOs can correct faulty RNA processing, skip mutant exons, or restore the production of functional protein isoforms, thereby reducing toxic protein accumulation that contributes to ALS pathology [158–160]. This approach has been successfully applied in targeting genes such as SOD1 and C9orf72, where ASOs modulate splicing or reduce harmful gene products, leading to improved molecular and clinical outcomes in preclinical and early clinical studies. Thus, splice-modulating ASOs represent a precision medicine tool that addresses genetic mutations underlying ALS by fine-tuning RNA processing to alleviate neurodegeneration [161–163]. These substances can be administered as naked molecules, via viral vectors, or by employing physical or chemical mechanisms like nanoparticles to specifically target the central nervous system (CNS) [164]. A limitation of both is their inability to cross the blood-brain barrier, despite showing effective CNS distribution via intrathecal (IT) injection [165]. Direct injection into the cerebrospinal fluid (CSF) (e.g., via IT administration) has limitations such as lower drug concentration in deep brain regions, increased drug concentration in the lumbar spinal area, and the possibility of serious side effects. Additionally, repeated IT injections can lead to clinical risks, including infection or spinal cord injury [166–168].As stated earlier, SOD1 mutations are among the leading causes of ALS, particularly in Asia [169]. In ALS, it is proposed that neurodegeneration linked to SOD1 mutations arises from a combination of factors, including oxidative stress, inflammation from microglia, toxic protein clumping, and dysfunction in mitochondria and oligodendrocytes [155]. More than 170 distinct mutations have been identified in SOD1 that cause ALS [170]. Numerous mutations in SOD1 lead to disease by promoting the aggregation of misfolded proteins [171]. Consequently, following the evaluation of ASOs in animal models, the initial human clinical trial (ClinicalTrials.gov: NCT01041222) was carried out using IT injection of ASO ISIS 333,611 in SOD1-associated ALS patients [172]. The trial evaluated the safety and efficacy of IT administration of the ASO ISIS 333,611, demonstrating that it lowered SOD1 mRNA and protein levels in the spinal cord and was well tolerated by participants [172]. The subsequent phase I/II clinical trial identified as NCT02623699 utilized serial concentrations of BIIB067/Tofersen ASO, demonstrating that the maximum dose 100 mg exhibited the most significant effects in lowering CSF SOD1 concentration, particularly in rapid disease progressors. This reduction in SOD1 protein levels was maintained throughout the 12-week treatment period [173]. Due to inadequate participant enrollment in the first trial, the investigation for BIIB067 was extended to a phase 3 clinical trial, involving 72 participants. The findings demonstrated that Tofersen led to a decrease in SOD1 levels in CSF and reductions in neurofilament light chain concentrations in plasma, indicating a potential beneficial impact on slowing the progression. Further research (ClinicalTrials.gov: NCT03070119) is underway in the extended phase III clinical trial [174]. Research suggests that the pathogenicity of C9orf72 arises from a toxic gain-of-function mechanism, making it a promising target for ASO-based gene therapy [175]. A completed phase I clinical trial (ClinicalTrials.gov: NCT03626012) used IT injection of ASO BIIB078 in patients with pathogenic repeat of C9orf72. The Results showed high safety without clinical efficacy [176]. Another ASO named WVE-004 is currently in a phase 1/2 clinical trial (ClinicalTrials.gov: NCT04931862) to evaluate the safety and pharmacodynamics [147]. Multiple cohorts show that 27–33 CAG repeats in ATXN2 increase ALS odds ratios 3- to 6-fold [177]. TDP-43 cytoplasmic aggregation is a near-universal ALS hallmark. ATXN2 physically associates with TDP-43 in an RNA-dependent complex [178]; polyQ-expanded ATXN2 aberrantly sequesters TDP-43 into stress granules, accelerating neurotoxicity [179]. Based on this, clinical trials are underway to evaluate ASO therapies targeting FUS (ClinicalTrials.gov: NCT04768972) and ATXN2 (ClinicalTrials.gov: NCT04494256) [147, 180]. RNAi strategies are being explored for other genes associated with ALS. For instance, preclinical research has demonstrated that decreasing Ataxin-2 expression, produced by the ATXN2 gene, can ameliorate motor deficits and enhance survival in the presence of TDP-43 proteinopathy. These results highlight the potential of utilizing RNAi techniques to address a range of ALS manifestations [181].

CRISPR/Cas9 gene editing

CRISPR technology enables accurate modification of DNA sequences, potentially rectifying mutations directly in the genome. Although This method is still in its infancy but offers hope for lasting remedies to genetic defects that cause ALS. The primary objective of this approach is to rectify the mutated DNA to prevent any aberrant downstream pathways. Although it has not yet been applied to directly alter the disease in ALS patients, it has been utilized in studies to create cellular and animal models of the disease [148]. In 2017, Gaj and colleagues performed a study on the G93A-SOD1 mouse model of ALS. AAV9 was employed as a vector to delivery sgRNA targeting the SOD1 gene. The CRISPR-Cas9 system effectively reduced the mutant SOD1 protein levels by over 2.5-fold in the lumbar and thoracic spinal cord of the treated mice. Additionally, the decrease in mutant SOD1 protein resulted in improved motor function and reduced muscle atrophy [182]. This research was followed by additional studies, all of which demonstrated a decrease in SOD1 levels and an enhancement in motor neuron survival [183, 184]. For instance, research was conducted by using human stem cell-derived MNs with SOD1 gene mutations created by CRISPR/Cas9. They observed that mutant MNs accumulated misfolded proteins, developed axonal damage, displayed synaptic abnormalities, and exhibited dysfunctional neurotransmission. These findings highlight the cell-autonomous effects of SOD1 mutations on MNs in ALS development [185]. Collectively, these studies demonstrate the potential of the CRISPR system for personalized treatment of ALS patients. Although CRISPR technology is a powerful tool for gene editing, it faces several significant challenges that must be carefully addressed. One major concern is the occurrence of off-target effects, where unintended cuts or modifications happen at genomic sites other than the intended target, potentially causing harmful mutations or disruptions in essential genes. Additionally, CRISPR editing can induce genomic instability, cellular toxicity, and stress responses, including the involvement of tumor suppressor pathways like p53. Larger genetic rearrangements such as translocations or insertions may also occur, raising safety concerns especially regarding cancer risk. Ethical and social issues are paramount, particularly when it comes to germline editing, since changes made to embryonic or reproductive cells can be inherited by future generations, leading to unpredictable genetic and societal consequences [186–189].

AAV mediated gene therapy

Adeno-associated virus (AAV) based gene therapy represents a promising strategy for addressing ALS, especially for hereditary types caused by mutations in the SOD1 gene. This technique utilizes AAV vectors to transport genetic material that can reduce the production of mutant proteins associated with ALS [190]. AAV vectors are utilized to transport RNAi agents or additional therapeutic genes to the CNS. These vectors have the ability to traverse the blood-brain barrier [191]. UniQure’s EPISOD1 trial (Clintrials.gov: NCT06100276) is evaluating AMT-162, an AAVrh10 vector-based gene therapy for SOD1-ALS. This trial involves IT administration of the AMT-162, which is designed to silence the expression of the misfolded SOD1 protein with a specific microRNA. currently in Phase I/II the trial focuses on assessing the safety, tolerability, and exploratory efficacy of AMT-162 [192]. Neurotrophic factors (NTFs) have been extensively investigated as potential therapeutic interventions for ALS [193]. Viral vectors are utilized for gene delivery into cells, particularly for delivering the NTFs into the CNS [156, 194]. AAV-driven overexpression of Neuron-Derived Neurotrophic Factor (NDNF) has demonstrated notable impacts in ALS models. In SOD1 G93A ALS model mice, IT administration of AAV-NDNF enhanced motor function, alleviated weight loss, and prolonged lifespan. This approach also supported the survival of spinal MNs and reduced abnormal protein accumulation [195]. In summary, the main limitations of AAV vectors for ALS gene therapy include difficulty crossing the blood-brain barrier, which requires high doses; limited genetic payload capacity; immune responses that reduce effectiveness; and lack of precise targeting, leading to off-target effects. These challenges constrain the efficient and safe delivery of therapeutic genes to the nervous system [196–199].

Antibody based strategies

This approach resembles those used for other neurodegenerative disorders where antibodies target abnormal proteins like amyloid-β. Clinical trials in ALS are investigating novel antibody-based interventions that target key proteins implicated in the disease’s pathogenesis. These experimental approaches are designed to retard disease advancement and enhance patient prognosis [200]. DPRs represent a crucial pathological feature linked to the C9orf72 gene expansion, which is recognized as the most prevalent genetic factor for ALS. These proteins are produced via a mechanism known as repeat-associated non-AUG (RAN) translation [201]. A study by Nguyen et al. (2020), showed that high-affinity human antibodies can specifically target RAN proteins, like GA and GP, which arise from the C9orf72 mutation. These antibodies effectively traverse the blood-brain barrier and attach to RAN protein aggregates in the brains of C9-BAC transgenic mice, a model for ALS [202]. Moreover, a study investigated the antibody response to HERV-K (HML-2) in ALS patients. Brain, spinal cord, and CSF samples from ALS patients frequently exhibit elevated levels of HML-2 transcripts and proteins, particularly the viral envelope (Env) protein. The HML-2 Env protein is neurotoxic; therefore, blocking Env either with specific antibodies or by inhibiting its interaction with the neuronal CD98HC/β1 integrin receptor rescues neurons in experimental models [203, 204].Researchers found that individuals with ALS exhibited a stronger antibody response against HML-2 envelope proteins compared to healthy controls and patients with multiple sclerosis. Higher levels of HML-2 DNA and a broader antibody response correlated with disease duration. Interestingly, lower levels of specific HML-2 antibodies in ALS patients were associated with a more definite diagnosis and decreased survival, suggesting that the antibody response to HML-2 may have prognostic value in ALS [205]. A Phase 2 A Clinical Trial (Clintrials.gov: NCT04322149), presents findings from a study evaluating the safety and tolerability of tegoprubart, an anti-CD40L antibody, in individuals with ALS. Tegoprubart demonstrated dose-dependent target engagement, markedly lowering levels of CD40L and CXCL13, which are linked to inflammation in ALS. This reduction in pro-inflammatory biomarkers, along with the absence of any drug-related severe adverse events, suggests potential therapeutic benefits [206].

Principal stem cell therapy

The emergence of cell therapy is founded on the critical understanding that numerous diseases result from the irreversible loss or dysfunction of specific cell populations, for which traditional pharmacological or surgical treatments often provide insufficient benefit. Originally conceptualized in the early 20th century, cell therapy sought to restore or replace damaged tissues by introducing viable cells capable of regeneration or functional compensation [207]. Advances in cell culture techniques, tissue engineering, and molecular biology have since facilitated the isolation, expansion, and, more recently, the reprogramming of somatic cells. These innovations have substantially broadened the therapeutic landscape of cell-based interventions. This paradigm shift is particularly significant for addressing diseases characterized by limited endogenous regenerative capacity, including neurodegenerative disorders, cardiovascular diseases, and various forms of organ failure. Contemporary research continues to refine strategies for improving cell engraftment, survival, and functional integration to enhance clinical outcomes [208–211]. Cell therapy entails the transplantation of live cells into a patient’s body to treat or cure diseases, repair damaged tissues, restore function, or modulate immune responses. Various types of cells can be utilized, including stem cells, immune cells, or somatic cells, depending on the specific condition. Stem cell therapy is a prevalent approach in which undifferentiated cells facilitate tissue regeneration or organ repair [212–216]. While stem cell treatments provide considerable therapeutic promise, they pose significant tumorigenic and immunological risks, particularly when undifferentiated cells exist mainly because of their self-renewal and multipotency. Thorough preclinical evaluation, refined production processes, and continuous regulatory monitoring are crucial to guarantee patient safety [217]. Stem cell types encompass embryonic stem cells (ESCs), characterized by their pluripotency and ability to differentiate into nearly any cell type; adult stem cells, including mesenchymal stem cells (MSC) found in tissues such as bone marrow and adipose tissue, which can specialize into particular cell types; and induced pluripotent stem cells (iPSCs), which are adult cells reprogrammed to exhibit ESC-like properties, thereby minimizing the risk of immune rejection [217–219].

Cell therapy strategies coordinated with gene therapy

Gene-modified cell therapy entails the modification of cellular genes to improve functionality or rectify genetic abnormalities, proving beneficial in the treatment of genetic disorders and cancers [220, 221]. Immune cell therapy involves the modification of immune cells, such as T-cells or NK cells, to enhance their efficacy in combating diseases, exemplified by Chimeric Antigen Receptor T-cell (CAR-T) cell therapy for cancer [222–224]. Cell therapy may be classified as autologous, utilizing the patient’s own cells to reduce the risk of rejection, or allogeneic, employing donor cells, which often necessitates the use of immunosuppressive medications to avert rejection [225–228]. Cell therapy in ALS seeks to decelerate disease progression through the replacement of damaged neurons, enhancement of regeneration, and provision of neuroprotection. Recent developments in stem cell research and genetic engineering indicate potential therapeutic applications for ALS. As illustrated in Fig. 3, these approaches leverage multiple mechanisms, including the secretion of NTFs, immunomodulation, and direct contributions to neurogenesis and synaptogenesis, to promote recovery following neurological injury or disease [229–232]. Following the selection of suitable stem cells for ALS treatment, they are cultivated and expanded in the lab to provide a sufficient number of cells for transplantation. To guarantee their integration into the nervous system, the cells may be differentiated during this phase into MNs or other supporting cell types. If gene therapy is included, cells are modified in vitro utilizing methods like CRISPR/Cas9 to either correct ALS-causing mutations or improve their therapeutic potential [233–236]. Once ready, the cells are administered to the patient using different techniques based on their characteristics and the course of treatment. Direct spinal cord injections of neural stem cells (NSCs) or other cell types are predicted to fuse with injured MNs and promote regeneration [237]. Alternatively, MSCs might be injected intravenously, allowing them to reach the brain, spinal cord and other sites of inflammation or damage via the bloodstream [238]. Sometimes cells are targeted at particular sites of motor neuron degeneration by being injected directly into affected muscles or nerves. Patients are closely monitored following the operation for possible side effects including immunological rejection, tumor development, or infections. To evaluate the efficacy of the therapy, track disease progression, monitor muscular strength and general functioning, as well as detect any side effects to subsequent treatment plans [238–240].

Fig. 3.

The figure illustrates various therapeutic strategies for promoting neural repair and regeneration, focusing on stem cell-based approaches. These strategies include the secretion of neurotrophic factors and exosomes by stem cells (such as ESC, MSC, NSC, and iPSC), which support neuronal survival and regeneration. Additionally, stem cells contribute to immunomodulation and anti-inflammatory effects, reducing inflammation and fostering neural tissue repair. The activation of astrocytes and modulation of microglia enhance tissue recovery and create a neuroprotective environment. Targeted or systematic injections of stem cells can deliver therapeutic effects directly to damaged areas of the nervous system, such as the brain or spinal cord. Finally, these stem cells play a role in neurogenesis and synaptogenesis, promoting the formation of new neurons and synapses, which are essential for restoring function in neurodegenerative conditions. These approaches are central to regenerative medicine, aiming to restore lost functions and repair neural circuits in individuals with neurological injuries or diseases

Research limitations and ethical considerations

Using fetal tissue transplantation, early studies on cell treatment for ALS started in the 1990s with an eye toward replacing damaged MNs with fetal cells. Along with mounting ethical questions about using fetal tissue, this method encountered major difficulties, including immunological rejection and uneven functional results [241, 242]. As adult stem cells from bone marrow, fat, and umbilical cord blood were seen as safer and more ethically acceptable in the 2000s, the focus shifted to them. Although human applications were still mostly unknown, early preclinical investigations in animal models showed promise [243–245]. One of the major concerns in stem cell therapies, especially those involving pluripotent stem cells such as iPSCs and ESCs, is the risk of tumor formation, including benign tumors like teratomas and malignant tumors such as teratocarcinomas [246, 247]. This risk stems from the high proliferative and differentiation potential of these cells, as well as the expression of oncogenes during the cellular reprogramming process. Additionally, stem cell transplantation can trigger immune responses that lead to inflammation and rejection of the transplanted cells. To mitigate these risks, it is essential that stem cells are fully differentiated prior to transplantation and that appropriate immunosuppressive measures are implemented. These precautions help improve the safety and efficacy of stem cell-based therapies, thereby addressing critical challenges in their clinical application [246, 248, 249]. The discovery of iPSCs transformed stem cell research in 2006. iPSCs, derived from adult cells such as skin or blood, can be reprogrammed into pluripotent cells capable to differentiating into any cell type, including MNs, thereby negating the need for ESCs and their associated ethical problems [250, 251]. This discovery let scientists create iPSCs tailored for ALS patients, enabling a better understanding of the condition and the evaluation of potential treatments. Particularly in SOD1-mutant mice, preclinical studies using iPSCs, ESCs, NSCs, and MSCs showed promising results in ALS animal models between 2005 and 2010 [250, 252]. Under Neuralstem Inc., early investigations utilizing NSC transplantation into the spinal cord facilitated the initiation of clinical trials exploring the use of NSCs in ALS patients during the 2010s [253, 254]. These studies highlighted safety issues, especially regarding tumor development, but also showed that stem cells might survive in the spinal cord and maybe have therapeutic consequences. MSCs and adipose-derived stem cells (ASCs) attracted interest during this period because of their reduced risk of immunological rejection [255]. Gene therapy paired with cell treatment also gained popularity as researchers aimed to correct genetic abnormalities linked with ALS [16, 256, 257]. Advances in gene editing technologies such as CRISPR-Cas9 in the 2020s made precise modification of ALS-causing genes possible, thereby guiding therapy methods. Combining gene editing with stem cell transplantation is currently under investigation to potentially halt ALS progression by correcting mutations at the DNA level [258, 259].

Clinical trials, challenges, and future directions

Promising clinical studies are Currently in progress, testing gene-modified stem cells, neural precursor cells, and gene therapies targeted at particular ALS mutations such as aiming at TDP-43 aggregation in neurons [260, 261]. To lower neuroinflammation in ALS, some studies also focus on immunological modulation via stem cells—including regulatory T-cells (Tregs). Moreover, a randomized placebo-controlled phase 3 study assessed the safety and efficacy of MSCs secreting MSC-NTF in slowing ALS progression. ALS patients received IT MSC-NTF or placebo in a randomized, double-blind trial. While the primary endpoint was not met, MSC-NTF treatment was safe and well-tolerated. A subgroup analysis of patients with less severe disease (ALSFRS-R ≥ 35) suggested a potential benefit with MSC-NTF [262]. The MSC-NTF group also exhibited improvements in CSF biomarkers related to neuroinflammation and neurodegeneration, indicating a potential biological effect. These findings support further investigation of MSC-NTF as a potential ALS therapy [262, 263]. In 2021 meta-analysis, aggregated data from 11 studies (220 patients) to assess the effectiveness of various stem cell types. It found a transient positive effect on clinical progression with IT MSCs, but overall worsening of respiratory function. The authors recommend further preclinical research to optimize cell products and delivery methods, and to tailor treatments to specific patient subsets [264]. Another systematic review analyzed six controlled clinical trials, found mixed results with stem cell transplantation. While some studies showed positive effects, particularly with bone marrow MSCs, others reported no significant difference compared to controls [265]. These systematic and meta-analysis reviews emphasize the need for more well-designed clinical trials to determine the true benefit of stem cell therapy for ALS. Although cell treatments for ALS show promise, various challenges remain, including ethical questions, safety, and efficacy. Concerns regarding immunological rejection, tumor development, and long-term safety of these treatments persist [266]. One of the main challenges is ensuring that stem cells are compatible with the neural system and do not cause any negative consequences. Furthermore, although preliminary research indicates that stem cells can survive and integrate into the spinal cord, their capacity to restore neurons and thus halt or stop ALS development is still unknown [240]. The use of gene-editing technology and fetal tissue also raises major ethical questions and requires careful consideration. Moreover, before they can be generally utilized, ALS cell treatments have to go through regulatory approval procedures to guarantee their safety and efficacy. Despite these challenges, developments in genetic engineering, gene therapy, and stem cell biology offer promising prospects for the future [231, 267]. New discoveries could image as more clinical studies are conducted, potentially providing therapies that halt the progression of ALS and improve quality of life. Table 4 presents a comprehensive summary of clinical trials investigating stem cell therapies for ALS from 1996 to 2024. These trials explore various stem cell types, delivery methods, and their respective clinical outcomes. The studies included in the table assess the safety, feasibility, and preliminary efficacy of different stem cell interventions across multiple trial phases. Cell types used range from bone marrow-derived mesenchymal stem cells (BM-MSCs) and NSCs to umbilical cord-derived mesenchymal stem cells (UC-MSCs) and mesenchymal stem cells secreting MSC-NTFs. The delivery methods employed vary, with IT, intraspinal, and intramuscular administration being the most common approaches. Across these studies, participant numbers vary significantly, from small-scale Phase 1 trials with fewer than 10 participants to larger Phase 2 and 3 trials involving over 100 patients. The number of injections and dosage vary between trials, with some studies exploring single-dose administration while others investigate repeated injections at regular intervals. The results highlight varying degrees of therapeutic benefit, with some trials reporting stabilization or slowed progression of ALS symptoms, while others find no significant improvement in disease progression. Importantly, the trials emphasize the safety profile of stem cell treatments, with most reporting mild to moderate side effects, such as headaches, pain, and transient immune responses. However, no severe adverse effects directly attributable to stem cell therapy have been widely reported [240, 268].

Table 4.

Clinical trial of stem cell therapy in amyotrophic lateral sclerosis

First Author / Year	Phase /Allocation	CTI	Cell Type	Dose & Route	Patients (Control)	Injections	Clinical Outcomes	Ref
Janson et al., 2001	N/A / Unknown	N/A	Not specified	2.0 × 10⁷ or 1.0 × 10⁸ cells/IT	Not specified	Not specified	Safe; speech improvement and muscle strength gain	[269]
Mazzini et al., 2003	I / Open-label	N/A	Autologous BM-MSCs	Not specified/ IT	7 (0)	Multiple (1 mm apart in 3 rows)	4 patients showed reduction in ALSFRS-R and FVC decline	[270]
Mazzini et al., 2006	I / Open-label	N/A	Autologous BM-MSCs	Not specified/ IT	10 (0)	Not specified	No change in rate of clinical decline	[271]
Cashman et al., 2008	N/A / Unknown	N/A	Not specified	1.5–7.6 × 10⁶ cells/kg/ IV	Not specified	Not specified	Safe with no clinical benefits	[272]
Deda et al., 2009	II / Open-label	N/A	Autologous BM-MSCs	Not specified/ IT	13 (0)	Multiple injections at C1-C2 levels	7 of 13 patients improved post-procedure	[273]
Karussis et al., 2010	I/II /Randomized	NCT00781872	Autologous MSCs	Mean 54.7 × 10⁶ IT + 23.4 × 10⁶ IV/ IV-IT	17 (18)	Multiple	Safe; ALSFRS score stabilization; immunomodulatory effects	[274]
Mazzini et al., 2010	I / Open-label	N/A	Autologous MSCs	Median 75 × 10⁶ cells/IT	10 (0)	3 unilateral or 6 bilateral injections	Safe and well tolerated	[275]
Glass et al., 2012	I / Open-label	NCT01348451	Fetal NSCs	5 unilateral or 10 bilateral injections of 1.0 × 10⁵ cells/injection/ IT	12 (0)	Multiple	Safe and well tolerated	[254]
Sharma et al., 2015	N/A / Open-label	N/A	Autologous MSCs	53.6 × 10⁶ IT + 26.8 × 10⁶ IM / IT-IM	Not specified	Multiple	Increased survival duration	[276]
Mazzini et al., 2015	I / Open-label	01640067	hNSC	3 unilateral or 6 bilateral injections of 75,000 cells/injection/ IT	Not specified	Multiple	Safe with reduction in ALSFRS decline	[277]
Rushkevich et al., 2015	N/A / Non-randomized	N/A	BM-MSCs (intact and neural-induced)	IV: 0.5–1.5 × 10⁶ cells/kg; IT: 5.0–9.7 × 10⁶ cells	10 (15)	Multiple	Safe with reduction in ALSFRS decline	[278]
Oh et al., 2015	I / Not specified	NCT01363401	BM-MSCs	2 doses of 1.0 × 10^6 cells/kg/ IT	8 patients	2	Safe; reduction in ALSFRS decline; immunomodulation	[279]
Petrou et al., 2016	IIa / Randomized	NCT01777646	MSC-NTF (NurOwn)	3 cohorts: 1 × 10⁶ cells/kg IT + 24 × 10⁶ cells IM; 1.5 × 10⁶ cells/kg IT + 36 × 10⁶ cells IM; 2 × 10⁶ cells/kg IT + 48 × 10⁶ cells IM / IT-IM	Not specified	Multiple	Safe with reduction in ALSFRS and FVC decline	[280]
Kuzma-Kozakiewicz et al., 2018	I / Open-label	NTC03296501	AT-MSCs	16,000 to 56,000,000 cells / IV-IT	Not specified	Multiple	Safe and well tolerated	[281]
Berry et al., 2019	II / Randomized	NCT02017912	MSC-NTF (NurOwn)	IT: 125 × 10⁶; IM: 48 × 10⁶/ IT-IM	48 (12)	Single cycle with boosters (3–10 injections)	Safe with reduction in ALSFRS decline	[282]
Geijo-Barrientos et al., 2020	I/II / Unknown	NCT02286011	BM-MCs	Median of 499 × 10⁶ cells / IM	Not specified	Not specified	Safe; larger Compound Muscle Action Potential scan curve	[283]
Petrou et al., 2021	II / Open-label	NCT04821479	Autologous MSCs	3 injections over 3 months / IT	26 (0)	3	ALSFRS-R decline slowed in some patients	[284]
Jalil Tavakol-Afshari et al.,2021	I / Open-label study without a placebo control group	IRCT20160809029275N2	BM-MSCs	1 × 106 cells/kg BW / IT-IV	15	1	Temporary delay in the progression of ALS	[285]
Robert H Baloh et al.,2022	I/IIa / Open- label	NCT02943850	Human neural progenitor cells transduced with GDNF (CNS10-NPC-GDNF)	N/A / IT	18	1	Provide new support cells and GDNF delivery to the ALS patient spinal cord for up to 42 months post-transplantation	[286]
Merit E Cudkowicz et al., 2022	III / Randomized, double-blind, placebo-controlled	NCT03280056	(MSC-NTF) NurOwn®	100–125 × 106 / IT	196	3	Safe, promising in less severe cases, but overall clinical benefit was not statistically confirmed.	[262]
Marc Gotkine et al.,2023	I/IIa / Open-label	NCT03482050	AstroRx®	5 Petient 100 × 106 and 5 patient 250 × 106 cells / IT	10	1	A signal of beneficial clinical effect was observed over the first 3 months post single treatment	[287]
Toru Yamashita et al.,2023	II / Open-label	jRCT2063200047	MUSE	Each CL2020(15 × 106) / IV	5	6	Some functional improvements, Changes in levels of certain biomarkers	[288]
Nathan Staffet al.,2023	II / Open-label	NCT03268603	Autologous adipose-derived MSCs	10–100 × 106 / IT	75 (No control group)	4 injections total	~ 37% patients showed ≥ 25% reduction in progression rate; treatment generally safe	[289]
Zahraa Alkhazaali-Ali et al.,2024	Not specified / Open-label	IRCT20160809029275N2	BM-MSC	1 × 106 cells / kg BW / IV-IT	15	N/A	Safety evaluated; efficacy assessed by immunological and biochemical biomarkers	[290]
Zahraa Alkhazaali-Ali et al.,2025	I / Open-label	IRCT20160809029275N5	MSC	1 × 106 cell/kg BW / IV-IT	21	3	Ongoing	[291]

IM Intramuscular, IT Intrathecal, IV intravenous, BM-MSCs Bone Marrow-derived Mesenchymal Stem Cells, N/A Not Assigned, MUSE Multilineage-differentiating Stress-Enduring cells, BM-MCs Bone Marrow Mononuclear Cells, hNSC Human Neural Stem Cells, CTI Clinical Trial Number

Limitations, challenges, and perspectives

Despite significant advancements in understanding ALS pathophysiology and therapeutic innovation, current treatments remain limited in efficacy, with Riluzole and Edaravone offering only modest survival or functional benefits. Clinical trials for gene therapies, such as ASOs and CRISPR/Cas9, and stem cell interventions face challenges, including inconsistent efficacy, variable patient responses, and safety concerns like immune rejection or tumorigenic risks. The blood-brain barrier further complicates drug delivery, while the heterogeneity of ALS subtypes and genetic mutations necessitates personalized approaches, complicating broad therapeutic applicability. Ethical dilemmas surrounding gene editing and stem cell use, coupled with the need for long-term safety data, underscore translational hurdles. However, emerging technologies, including AAV-mediated gene delivery, RNA-targeted therapies, and optimized stem cell protocols, hold promise for precision medicine. Advances in biomarker identification, neuroinflammation modulation, and collaborative multidisciplinary research may accelerate the development of disease-modifying therapies. Future efforts must prioritize robust clinical trial designs, deeper mechanistic insights, and ethical frameworks to translate preclinical successes into meaningful patient outcomes, ultimately bridging the gap between experimental innovation and clinical reality.

Conclusion

ALS remains a challenging neurodegenerative disease with no definitive cure, despite significant advances in understanding its pathophysiology. The molecular mechanisms underlying ALS are multifaceted, involving genetic mutations, protein misfolding, oxidative stress, excitotoxicity, and neuroinflammation. While current therapies, such as Riluzole and Edaravone, offer modest benefits, they fail to halt disease progression significantly. The review highlights promising therapeutic approaches including gene therapies, such as ASO and CRISPR/Cas9 gene editing, which aim to address genetic mutations at their source. Additionally, stem cell therapies, particularly those involving MSCs and NSCs, have shown potential in regenerating MNs and providing neuroprotective benefits. Despite encouraging preclinical and early-phase clinical trial results, further studies are necessary to confirm the long-term safety and efficacy of these treatments. Ultimately, continued research into the molecular mechanisms of ALS, alongside the development of novel therapeutic strategies, is crucial for providing ALS patients with better treatment options and, potentially, a cure in the future. The ongoing exploration of gene therapy, stem cell-based interventions, and immunomodulation remains vital to the pursuit of more effective disease-modifying therapies.

Abbreviations

ALS

Amyotrophic lateral sclerosis

MSCs

Mesenchymal stem cells

NSCs

Neural stem cells

BMAA

β-methylamino-L-alanine

fALS

Familial ALS

SALS

Sporadic ALS

ALS-FTD

ALS and frontotemporal dementia

SOD1

superoxide dismutase 1

FUS

Fused in sarcoma

LMNs

Lower motor neurons

UMNs

Upper motor neurons

RAN

repeat-associated non-ATG

PLS

Primary lateral sclerosis

PMA

Progressive muscular atrophy

CHMP2B

Charged multivesicular body protein 2B

TBK1

TANK-binding kinase 1

OPTN

Optineurin

VAPB

Vesicle-associated membrane protein-associated Protein B

FIG4

FIG4 phosphoinositide 5-phosphatase

NEK1

NIMA related kinase 1

SQSTM1

Sequestosome 1

SPG11

Spastic paraplegia 11

ER

Endoplasmic reticulum

UPR

unfolded protein response

UPS

Ubiquitin-proteasome system

ROS

Reactive oxygen species

EAAT2

Excitatory amino acid transporter 2

TNF-α

Tumor necrosis factor-alpha

IL-1β

Interleukin-1 beta

IL-6

Interleukin-6

FASL

Fas Ligand

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B Cells

PBA

Pseudobulbar affect

NIV

Non-invasive ventilation

siRNAs

Small interfering RNAs

ASOs

Antisense oligonucleotides

mRNA

messenger RNA

CNS

Central nervous system

CSF

Cerebrospinal fluid

MNs

Motor neurons

AAV

Adeno-associated virus

CAR-T

Chimeric antigen receptor T-cell

NTFs

Neurotrophic factors

NDNF

Neuron-Derived neurotrophic factor

DPRs

Dipeptide repeat proteins

ESCs

Embryonic stem cells

IPSCs

Induced pluripotent stem cells

ASCs

Adipose-derived Stem Cells

Tregs

Regulatory T-cells

BM-MSCs

Bone marrow-derived mesenchymal stem cells

UC-MSCs

Umbilical cord-derived mesenchymal stem cells

Author contributions

“R.R. and G.A. writing—review and editing, project administration""A.T. writing—original draft preparation""M.A. and A.M. supervision, validation, and investigation""S.S. data curation and formal analysis""E.S. and J.T. validation, project administration and supervision""M.H and F.J. data curation and resources""S.S. resources""A.B. validation and project administration “"H.R. performed writing—review and editing, project administration, validation, supervision, and investigation”.

Funding

None.

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors consent to publish the article.

Competing interests

The authors declare no competing interests.