Amyotrophic lateral sclerosis: a neurodegenerative disorder poised for successful therapeutic translation

Amyotrophic lateral sclerosis (ALS) is a devastating disease caused by degeneration of motor neurons. As with all major neurodegenerative disorders, development of disease-modifying therapies has proven challenging for multiple reasons. Nevertheless, ALS is one of the few neurodegenerative diseases for which disease-modifying therapies are approved. Significant discoveries and advances have been made in ALS preclinical models, genetics, pathology, biomarkers, imaging and clinical readouts over the last 10–15 years. At the same time, novel therapeutic paradigms are being applied in areas of high unmet medical need, including neurodegenerative disorders. These developments have evolved our knowledge base, allowing identification of targeted candidate therapies for ALS with diverse mechanisms of action. In this Review, we discuss how this advanced knowledge, aligned with new approaches, can enable effective translation of therapeutic agents from preclinical studies through to clinical benefit for patients with ALS. We anticipate that this approach in ALS will also positively impact the field of drug discovery for neurodegenerative disorders more broadly.

Motor neuron injury in ALS is considered to result from multiple interacting pathophysiological mechanisms that culminate in widespread network disruption. The heterogeneity of ALS suggests that different mechanisms may play more or less prominent roles in individual patients. Advances in preclinical disease modelling (Box 1 ) are needed to capture this complexity. To develop neuroprotective therapies, an advantageous approach could include upstream targeting of the initiating factor (for example, by gene silencing or gene replacement), and one or more drugs that ameliorate multiple facets of the disease pathophysiology. This section reviews recently emerging insights into contributory pathophysiological mechanisms (Fig. 1 ) and highlights novel therapeutic strategies. Fig. 1 ALS pathophysiology, genetic causes and risk factors. Advances in large-scale genomic analysis have uncovered a variety of causative genes and risk factors for amyotrophic lateral sclerosis (ALS). These gene variants map onto key pathogenic mechanisms relevant to all motor neuron cellular compartments as well as neighbouring cells such as glia and interneurons. In this way, these mechanisms are genetically validated, enabling a greater confidence in their targeting for therapeutic benefit. Some of these mechanisms have emerged only in recent years due to new genetic information, including gene changes highlighting dysregulation of RNA processing and metabolism. There is significant overlap of some genes with those found in closely related disorders such as frontotemporal dementia (for example, C9orf72 , CHCHD10 , SQSTM1 , TBK1 , CCNF , FUS , TARDBP , OPTN , UBQLN2 , TUBA4A , ATAXN2 , VCP and CHMP2B ). This suggests a closer relationship to broader neurodegenerative disorders, and indeed many of the pathways depicted are relevant in, for example, Alzheimer disease. ER, endoplasmic reticulum. For a complete list of the ALS loci, genes and associated proteins, see Supplementary Table 2 . Box 1 Advances in preclinical disease modelling in ALS Rational preclinical screening cascades for drug discovery rely on disease models which enable some level of confidence in translation of preclinical efficacy to the clinic. For neurodegeneration in particular, the identification of models which predict translational success is still a significant issue, with >90% of trials in neurodegenerative diseases failing for lack of efficacy 240 . Amyotrophic lateral sclerosis (ALS) is an interesting case as, until recently, it was the only neurodegenerative disease for which disease-modifying therapies were available to validate disease models. Recent advances, such as the ability to create patient-derived central nervous system (CNS) cell types in the laboratory and the advent of a battery of new genetic models of ALS, hold great promise for improving efficacy predictions. Historical approach The identification of mutations in SOD1 in 1993 (refs. 31 , 241 ) enabled the generation of the first SOD1 -mutant transgenic mouse models of ALS 242 . These models show a predominant motor phenotype with spinal motor neuron degeneration and loss, despite ubiquitous high-level over-expression of mutant SOD1 via its endogenous promoter 243 . Guidelines on the use of ALS models, particularly the SOD1 -mutant model, were published in 2010 (ref. 244 ). Testing of therapeutic agents previously shown to extend survival using these new approaches failed to show any benefit 243 suggesting that much of the published literature was not reproducible. Further limitations relate to the fact that this model is based on a genetic mutation which does not broadly represent ALS cases, best evidenced by a lack of TDP-43 proteinopathy in SOD1 -mutant mice 245 , in contrast to the vast majority (>97%) of patients with ALS 12 . However, the SOD1 -mutant rodent models still have value as part of a rational pharmacology screening cascade. They display motor neuron degeneration driving a disease phenotype of muscle weakness and atrophy, as in clinical ALS. They recapitulate, and indeed have been used to validate, many of the disease mechanisms implicated in human disease (Fig. 1 ) and in some cases have been shown to respond to approved therapies such as riluzole and edaravone 246 , 247 . In this way, the model is best viewed as a test bed for therapeutic approaches that target specific disease mechanisms hypothesized to be driving the disease phenotype. This allows the generation of pharmacokinetic–pharmacodynamic data in relation to mechanism as well as the development of both translational (for example, compound muscle action potential, plasma neurofilament light) and target engagement biomarkers which can be used to enhance translational approaches. New genetic mouse models with broader relevance The explosion in genetic characterization of ALS (Table 1 ; Supplementary Table 2 ) has enabled new models to be developed. With respect to mouse models, the most significant work relates to m

Oxidative stress may result from excess levels of reactive oxygen species (ROS), reactive nitrogen species or impaired functioning of antioxidant defence systems. ROS contribute significantly to neuronal injury and central nervous system (CNS) ageing by changing the structure and function of biomolecules including proteins, lipids, DNA and RNA. There is abundant evidence indicating a key role for oxidative damage in the pathophysiology of ALS 45 – 47 . Altered oxidative stress biomarker profiles have been reported in models of ALS and in human ALS biosamples 48 . There is also evidence for impaired oxidative stress defence systems in ALS, including dysregulation of glutathione homeostasis 49 and the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (ARE) cytoprotective system 50 , 51 . Oxidative stress can contribute to and exacerbate multiple other pathophysiological processes that are involved in motor neuron injury 45 . Biochemical and cell-based assays have identified oxidative stress as a signalling cue which promotes acetylated TDP-43 aggregates, while TDP-43 acetylation impairs RNA binding and promotes accumulation of insoluble, hyperphosphorylated TDP-43 species that resemble the pathological inclusions found in the CNS of individuals with ALS 52 . Mislocalization of both TDP-43 and FUS occur in cellular models of ALS exposed to oxidative stress, underpinning alterations in RNA processing 53 . Chronic oxidative stress promotes GADD34-mediated phosphorylation of TDP-43, a hallmark of TDP-43 proteinopathy 54 . Cytoplasmic aggregation of TDP-43 sequesters specific microRNAs and proteins, including nuclear genome-encoded mitochondrial proteins, leading to dysregulation of mitochondrial function that further augments oxidative stress 55 . Thus, an accelerating cycle of oxidative stress, protein aggregation and mitochondrial dysfunction is established. Oxidative stress is also involved in the crosstalk between motor neurons and neighbouring astrocytes and microglia 56 , 57 . For example, there is evidence that astrocytes release glutamate via upregulation of the cysteine–glutamate antiporter in response to increased oxidative stress and this in turn may augment excitotoxic injury to motor neurons 56 . Effective alleviation of oxidative stress could potentially ameliorate multiple facets of the pathobiology of motor neuron degeneration. A retrospective analysis of drugs evaluated in mSOD1 mice indicated that drugs that specifically target oxidative stress could be the most promising therapeutic candidates for the prevention of motor neuron degeneration 58 . Alleviation of oxidative stress is likely to be the primary mechanism of action of the free radical scavenging drug edaravone.

Mitochondrial dysfunction has been proposed as a central determinant of the pathophysiology of ALS. Altered energy production, excess generation of ROS, disruption of mitochondrial axonal transport, altered mitochondrial structure and dynamics, perturbation of mitophagy and calcium buffering, and triggering of apoptosis have been extensively described in ALS model systems and patient biosamples 73 – 75 . Defects in the specialized domains on the ER mitochondrial-associated membranes have been reported to impair neuronal calcium homeostasis, mitochondrial dynamics, ER function and autophagy, culminating in axonal degeneration 76 . Mutations in specific ALS genes have been linked to compromise of mitochondrial function through various mechanisms 77 and multiple abnormal ALS disease-associated proteins have been shown to interact directly with mitochondria, impairing their function. For example, mutant SOD1 aggregates in the mitochondrial intermembrane space and reduces the activity of the electron transport chain (ETC) complexes. The DPR poly-GR produced in C9orf72 -mutant ALS binds to the mitochondrial complex V component ATP5A1 and enhances its ubiquitination and degradation 78 . The C9ORF72 protein localizes to the inner mitochondrial membrane and regulates cellular energy homeostasis by stabilizing the translocase of inner mitochondrial domain containing 1 (TIMMDC1) protein, a crucial factor for the correct assembly of the oxidative phosphorylation complex. The function of mitochondrial complex 1 is impaired in patient-derived neurons from patients with C9orf72 -mutant ALS 79 . There is evidence that TDP-43 has a role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts, a function that is perturbed in the presence of mutations 80 . TDP-43 aggregates also sequester specific microRNAs and proteins that are nuclear genome-encoded mitochondrial proteins, and abnormal levels of expression cause dysregulation of mitochondrial function which in turn augments oxidative stress 55 . Potential therapeutic strategies targeting mitochondrial biology have been attempted. Several drugs targeting mitochondrial function and/or ROS such as coenzyme Q10, dexpramipexole, olesoxime and creatine all showed promise in preclinical models, but were unsuccessful in clinical trials 81 – 84 . Tauroursodeoxycholic acid (TUDCA) modulates the mitochondrial pathway of neuronal injury by reducing ROS formation, inhibiting BAX translocation, cytochrome c release and caspase 3 activation 85 . A recently published phase II clinical trial involving 137 patients with ALS evaluated treatment with TUDCA combined with sodium phenylbutyrate over a 6-month period 86 , 87 and showed evidence of slowing of the rate of decline on the ALS functional rating scale–revised (ALSFRS-R) score and increased survival. A European trial of TUDCA alone is ongoing (EudraCT 2018-002722-22). A recent preclinical study, using models harbouring SOD1 and TARDBP mutations, demonstrated that inhibition of protein phosphatase 1 prevented mitochondrial fragmentation, ETC impairment, disruption of axonal transport and cell death 88 and this pathway is worth exploring further. 31 P-MRS has been used to provide evidence for bioenergetic dysfunction in the brain and muscle of patients with ALS and provides a potential biomarker tool for use in clinical trials targeting bioenergetic dysfunction 89 .

Neuroinflammation is a pathological hallmark evident in preclinical models and the CNS of patients with ALS both histologically and in imaging studies 120 . Astrocytes maintain the integrity of the blood–brain barrier and modulate inflammatory signalling through the release of pro-inflammatory cytokines (interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)) or anti-inflammatory molecules (prostaglandin E2 and transforming growth factor (TGF)-β) 121 – 123 . Astrocytes derived from fibroblasts from patients with ALS are toxic to cocultured motor neurons 124 . The exact mechanisms of this toxicity have not yet been established although impaired bioenergetic support through lactate release, and pro-nerve growth factor–p75 receptor signalling, may contribute 125 . Microglia can adopt a toxic M1 phenotype or a neuroprotective M2 phenotype 126 – 128 . In mutant SOD1 -transgenic mice, the microglia switched from the M2 to the M1 phenotype after disease onset 129 . Peripheral blood monocytes from patients with ALS are more readily activated and differentiated to a pro-inflammatory M1 phenotype, and represent a potential target for immunomodulatory therapy 130 . Microglial NLRP3 inflammasome activation has been reported as a key contributor to neuroinflammation in ALS. NLRP3 inhibition could potentially become a therapeutic target to ameliorate microglia-driven neuroinflammation and disease progression in ALS 57 . The nuclear factor-kappa β (NF-κB) protein has been reported as a master regulator of inflammation in ALS 131 . NF-κB signalling was activated within glia during disease progression in mutant SOD1 mice. Notably, NF-κB signalling has been proposed to regulate microglial activation in ALS, as the localization of NF-κB activity and deletion of NF-κB signalling in microglia rescued motor neurons from microglia-mediated death and extended survival in ALS mice by impairing pro-inflammatory microglial activation 132 . The responses of glia to TDP-43 pathology have been studied 133 – 135 . TDP-43 proteinopathy has been reported to cause inflammation by triggering cytoplasmic release of mitochondrial DNA which activates the cytoplasmic DNA-sensing cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. It is possible that inhibition of cGAS–STING could mitigate inflammatory pathology in ALS 136 , 137 . Transcriptomic analysis of the motor cortex identified 1,573 differentially expressed inflammatory genes in sALS and indicated specific inflammatory molecular signatures for different patient subgroups, with the potential for personalized therapeutic targeting 138 . The C9ORF72 protein may modulate inflammation 139 and there is evidence that reduced C9ORF72 levels cannot suppress inflammation mediated by the STING pathway 140 . Biochemical and imaging biomarkers of glial activation have the potential to be used as pharmacodynamic indices in clinical trials. Patients with ALS exhibit higher cerebrospinal fluid (CSF) levels of chitotriosidase (Chit-1) and chitinase-3-like protein 1 (CHI3L1) than neurological disease controls and healthy controls, which correlate with the rate of disease progression 121 . Glial activation in vivo has been measured using translocator protein (TSPO) PET imaging and dynamic 18 F-DPA714 or 11 C-PBR28 PET–MRI 141 .

Disrupted RNA metabolism appears to play a key role in the pathophysiology of ALS. Both TDP-43 and FUS are RNA-binding proteins (RBPs) with major roles described in multiple aspects of RNA biology including splicing, transcription, stability, export and microRNA biogenesis 153 . Both proteins have binding sites for over 5,000 genes and regulate expression levels of hundreds of mRNAs. The most profound mutation-associated changes in expression occur in genes with very long introns and roles in synaptic function 154 , 155 . C9orf72 mutations lead to the accumulation of RNA foci which sequester a range of RBPs including TDP-43 and RNA export factors 155 , 156 . This sequestration has inevitable downstream effects on RNA metabolism 149 including dysregulated splicing. Targeting the repeat expansion in C9orf72 with antisense oligonucleotides (ASOs) was shown to prevent the formation of RNA foci and downstream effects 157 , 158 and is under clinical investigation (Table 2 ). The sequestration of RBPs to C9orf72 RNA foci also licenses them for nuclear export, allowing the pathological transcripts to escape to the cytoplasmic ribosomal machinery, resulting in the production of toxic DPRs 159 . This has become a potential target for therapeutic intervention by inhibition of the nuclear export adaptor SRSF1 (ref. 159 ). Table 2 Selected drugs in clinical trials for ALS Drug (company) Mechanism of action Development stage Trial identifiers Antioxidants Verdiperstat (Biohaven Pharmaceuticals) Myeloperoxidase inhibitor Phase II/III a,b NCT04297683 , NCT04436510 RT001 (Retrotope) Provides resistance to membrane lipid peroxidation Phase II NCT04762589 AP-101 (AL-S Pharma) Anti-SOD1 monoclonal antibody Phase II NCT05039099 Cell therapy Lenzumestrocel (Corestem) Mesenchymal stem cells that express anti-inflammatory and immune-modulating factors Approved in South Korea NCT04745299 NurOwn (BrainStorm Cell Therapeutics) Mesenchymal stem cells differentiated into astrocyte-like cells that express neurotrophic factors Phase III (completed) c NCT03280056 , NCT04681118 AstroRx (Kadimastem) Pluripotent stem cell therapy Phase I/II (completed) NCT03482050 RAPA-501 (Rapa Therapeutics) Autologous hybrid T cell therapy (regulatory T cells and helper T cells) Phase I/II NCT04220190 AlloRx (Vitro Biopharma) Umbilical cord-derived allogeneic mesenchymal stem cell therapy Phase I NCT05003921 Genetic therapy Tofersen (Biogen, Ionis Pharmaceuticals) Antisense oligonucleotide against SOD1 Phase III (completed) c NDA submitted NCT02623699 ION-363 (Ionis Pharmaceuticals) Antisense oligonucleotide against FUS Phase III NCT04768972 WVE-004 (WaVe Life Sciences) Antisense oligonucleotide against C9orf72 Phase I/II NCT04931862 ION-541 (Ionis Pharmaceuticals) Antisense oligonucleotide against Ataxin2 Phase I NCT04494256 Mitochondrial dysfunction AMX0035 (Amylyx Pharmaceuticals) Combination of sodium phenylbutyrate and taurursodiol that reduces nerve cell death Approved in the USA and Canada NCT03127514 , NCT05021536 SBT-272 (Stealth BioTherapeutics) Mitochondria-targeted novel peptide and peptidomimetic Phase I NA Neuroinflammation Masitinib (AB Science) CSF1R kinase inhibitor Phase III NCT03127267 Ibudilast (MediciNova) Phosphodiesterase inhibitor Phase II/III NCT04057898 3K3A-APC (ZZ Biotech) Recombinant engineered activated protein C Phase II NCT05039268 Aldesleukin (Clinigen, Novartis) Recombinant human IL-2 Phase II/III (completed) NCT02059759 ANX-005 (Annexon) C1q-specific mAb Phase II NCT04569435 Apilimod dimesylate (AI Therapeutics) Phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve) kinase inhibitor Phase II NCT05163886 Tegoprubart (Eledon Pharmaceuticals) Anti-CD40 ligand mAb that targets CD4 + T cells and B cells Phase II (completed) NCT04322149 BLZ-945 (Novartis) CSF1R inhibitor Phase II NCT04066244 COYA-101 (Coya Therapeutics) Autologous regulatory T cell therapy Phase II NCT04055623 Fasudil (Woolsey Pharmaceuticals) Rho kinase (ROCK) inhibitor Phase II NCT05218668 Latozinemab (Alector) Microglia-activating recombinant human anti-sortilin (SORT1) mAb Phase II NCT05053035 Pegcetacoplan (Apellis Pharmaceuticals) Complement C3 regulator Phase II NCT04579666 PrimeC (NeuroSense Therapeutics) Combination of celecoxib (cyclooxygenase-2 inhibitor) and ciprofloxacin (DNA gyrase inhibitor) Phase II NCT04165850 , NCT05357950 RNS60 (Revalesio) Neuroprotective G protein-coupled receptor modulator Phase II (completed) NCT03456882 CORT-113176 (Corcept Therapeutics) Glucocorticoid receptor antagonist Phase I (completed) NCT04994743 DNL-788 (Denali) RIPK1 inhibitor Phase I (completed) NCT04982991 Proteostasis Trehalose (Seelos Therapeutics) Repurposed disaccharide that may prevent mutant protein aggregation Phase II/III a NCT04297683 , NCT05136885 IFB-088 (InFlectis BioScience) Inhibitor of PPP1RlsA (GADD34), involved in the unfolded protein response Phase II EudraCT 2021-003875-32 Trametinib (GENUV) Mitogen-activated protein kinase (MEK) inhibi

Pathophysiological mechanisms currently being targeted in preclinical development and clinical trials for ALS are highlighted in Fig. 2 . Fig. 2 The major ALS pathophysiological targets currently being pursued. We have identified over 40 active clinical programmes (Table 2 ) and over 50 late-stage preclinical programmes (Table 3 ) and show here how these efforts map onto the pathophysiological landscape of amyotrophic lateral sclerosis (ALS). Among clinical programmes, some trends emerge. Neuroinflammation is the best represented therapeutic target covered by a variety of approaches, with the complement system representing a particular target of interest. Genetic therapies using antisense oligonucleotide (ASO) approaches also dominate the clinical space targeting specific ALS genes, although Ataxin2 modifiers may be more broadly applicable in relation to TDP-43 proteinopathy. Genetic therapies are also prominent in preclinical efforts. Other notable areas include proteostasis and cell therapies. Considering the most up to date understanding of pathomechanisms and genetics, several areas are poised for further exploration in sporadic ALS more broadly, including RNA metabolism and axonopathy. AAV, adeno‐associated virus; mAb, monoclonal antibody; MSC, mesenchymal stem cell; TUDCA, tauroursodeoxycholic acid. We identified 91 development programmes for drugs approved or in clinical development for the treatment of ALS, using the AdisInsight Springer database (August 2022). A total of 53 drug development programmes had at least one significant ALS trial update within the last 12 months and were not discontinued programmes (Table 2 ). Of the 53 drug development programmes, 39 focused on novel therapeutic agents, while the other 14 were related to marketed drugs, drug repurposing or drug reformulation (including six drugs that are reformulations of edaravone or riluzole, not listed in Table 2 ). Six drugs were either marketed or in the pre-registration stage, while 11 additional drugs were under evaluation in pivotal trials. The selected 53 drugs with recent updates were manually categorized according to the mechanism of action to identify trends and gaps, and several key themes emerged. The most represented target category was neuroinflammation, with 16 therapeutic agents in trials using approaches ranging from upregulation and activation of endogenous regulatory T cells (aldesleukin) to therapeutic antibodies targeting CD4 + T cells and B cells (anti-CD40L). Several approaches targeting the complement cascade, including ANX-005 (C1q inhibitor) and pegcetacoplan (C5 convertase inhibitor) were identified, although recent trials in ALS of the anti-C5 antibody ravulizumab and the C5 inhibitor zilucoplan were terminated following interim analyses, due to lack of efficacy. Another well-represented group of compounds in trials was for genetic therapies using ASO technology to knockdown gene expression. Three of these target specific mutations in genetic subpopulations ( C9orf72 , FUS and SOD1 ) and one targets Ataxin2 , a potential modifier of TDP-43 proteinopathy, which could be relevant for almost all ALS subtypes. Depletion of ataxin 2 levels limits TDP-43-mediated toxicity in multiple model systems 178 . The ASO ION-363 is currently being evaluated in a phase III trial ( NCT04768972 ) targeting patients with ALS and mutations in the FUS gene. ION-363 was initially developed as an experimental treatment specifically for a patient with FUS -associated ALS and then provided to several patients in a compassionate use programme. The controlled phase III trial was established based on this initial programme 179 . Other well-represented approaches include glutamate excitotoxicity, although the four different approaches identified represent generic bioequivalence trials for new formulations of riluzole. Cell therapy continues to be an area of interest, although the current focus is on cell therapy that modulates the toxic microenvironment, as opposed to cell replacement. NurOwn is one of the larger recent cell therapy trials, although the pivotal phase III trial ( NCT03280056 ) did not meet statistical significance in the primary efficacy end point 180 . Additionally, troponin activators that increase muscle force for a given level of motor neuron innervation represent a symptomatic approach to improve muscle strength, rather than aiming for neuroprotection of motor neurons 181 . Approaches that are under-represented, based on current knowledge of the pathophysiology of ALS, include those targeting mitochondrial dysfunction and proteostasis. AMX0035 potentially targets both of these pathways and was the subject of a recent new drug application (NDA) and marketing authorization application filings based on data from phase II trials 86 , 87 . AMX0035 was approved in 2022 for use in the USA and Canada for the treatment of ALS. The phase III PHOENIX trial of this combination therapy is ongoing. Clinical case studies Analysis o

AMX0035 (Relyvrio) is the first combination potential disease-modifying therapy trialled in ALS, although another combination therapy of dextromethorphan hydrobromide and quinidine sulfate was approved for the symptomatic treatment of pseudobulbar affect in ALS ( NCT00573443 , NCT01799941 ; Nuedexta Prescribing Information). AMX0035 is an oral proprietary, fixed-dose combination of sodium phenylbutyrate and taurursodiol (also known as TUDCA) developed by Amylyx Pharmaceuticals as a therapeutic approach targeting multiple pathophysiological mechanisms 11 in ALS and Alzheimer disease. Sodium phenylbutyrate is a histone deacetylase inhibitor that upregulates chaperones 192 , while taurursodiol inhibits mitochondrial associated apoptosis 193 . In the phase II CENTAUR trial ( NCT03127514 ), 137 participants were randomly assigned 2:1 to receive AMX0035 or placebo for 6 months. Participants completing the 6-month randomized phase were eligible to receive phenylbutyrate and taurursodiol in an open-label extension. In a modified intention-to-treat analysis, AMX0035 was associated with a statistically significantly lower mean rate of change in the ALSFRS-R score at −1.24 points per month, compared to −1.66 points per month in those receiving placebo. Secondary outcomes did not differ significantly between the two groups 87 . Median overall survival was 25.0 months in participants originally randomized to AMX0035 and 18.5 months in those originally randomized to placebo. Initiation of active treatment at baseline resulted in a 6.5-month longer median survival compared to placebo. These trial results suggest that AMX0035 may have functional and survival benefits in ALS 86 although several aspects of the trial have been criticized 194 . Amylyx has sought market authorization from multiple regulatory authorities and has also initiated recruitment into a phase III PHOENIX trial evaluating the safety and efficacy of AMX0035 in approximately 600 patients with ALS ( NCT05021536 ). AMX0035 has received market authorization in the USA and Canada for the treatment of ALS (2022). The drug approval by Health Canada was under its notice of compliance with conditions policy, which enables therapies showing the potential to fill an unmet medical need in severe, life-threatening diseases to reach the market sooner, provided that certain conditions are met. Those conditions included the provision of data from the phase III Phoenix trial ( NCT05021536 ), as well as additional planned or ongoing studies. Several post-marketing studies were also required by the FDA for the approval of AMX0035, including carcinogenicity studies, additional drug interaction studies, and clinical pharmacokinetic studies in subjects with hepatic and renal impairment 195 . One of the active ingredients of AMX0035, taurursodiol, is currently being evaluated in a randomized phase III TUDCA-ALS trial as an add-on treatment to riluzole in patients with ALS, sponsored by Humanitas Mirasole SpA ( NCT03800524 ).

Several approaches targeting the complement cascade are currently in clinical trials for the treatment of ALS, and it may be informative to consider the recent failure of ravulizumab in the CHAMPION-ALS trial. Complement is an essential effector component of both innate and adaptive humoral immune responses activated by the alternative, lectin and classic pathways. It is well known that complement can be activated in response to CNS inflammation, enhancing tissue injury, and complement deposition has been identified in CNS tissue from patients with ALS 199 . A potential role for complement in both central motor neuron loss and peripheral neuromuscular junction injury 200 has stimulated interest in targeting the complement cascade in ALS clinical trials. Ravulizumab is a humanized anti-C5 antibody developed by Alexion and AstraZeneca for the treatment of ALS and other rare diseases. It is a terminal complement inhibitor that specifically binds to the C5 complement protein with high affinity, thereby inhibiting its cleavage to C5a and C5b, and preventing the generation of the terminal membrane attack complex C5b9 and production of the pro-inflammatory C5a fragment. Ravulizumab inhibits terminal complement-mediated intravascular haemolysis and has been approved in the USA for the treatment of paroxysmal nocturnal haemoglobinuria. The phase III CHAMPION-ALS trial ( NCT04248465 ) was conducted to evaluate the efficacy and safety of ravulizumab for the treatment of ALS. AstraZeneca discontinued the CHAMPION-ALS trial in August 2021 due to lack of efficacy, following a prespecified interim analysis. This outcome suggests that peripheral targeting of the complement terminal pathway is not beneficial in ALS. More recently, a phase II/III trial of another C5 inhibitor zilucoplan ( NCT04297683 , NCT04436497 ) was also discontinued for futility. However, further approaches target C3 convertases and utilize smaller peptidic inhibitors, which may have better access to the CNS and the potential to target an earlier phase in the complement activation pathway.

A search using the AdisInsight Springer database (August 2022) identified 91 preclinical ALS drug development programmes. Of these, 22 defined a lead therapeutic candidate, listed ALS as the lead target indication, had had at least one significant update within the previous 12 months, and were not discontinued programmes (Table 3 ). Moreover, all drug development programmes except one (a riluzole microencapsulated formulation) were focused on the development of new therapies. Of the preclinical development programmes, 11 were for genetic therapies, which represent an exciting new therapeutic approach for subgroups of patients with ALS. Table 3 Selected drugs in preclinical development for the treatment of ALS Drug (company) Approach Mechanism QRL-201 (QurAlis Corporation) Cryptic exon splicing Aims to restore expression of STATHMIN-2 (STMN2), reduced due to TDP-43 pathology; STMN2 is a protein important for neural repair and axonal stability Censavudine (Transposon Therapeutics) Endogenous retrovirus A nucleoside reverse transcriptase inhibitor aiming to inhibit transposon activation GNK-301 (GeNeuro) Endogenous retrovirus An anti-human endogenous retrovirus (HERV-K) monoclonal antibody that blocks activity of the HERV-K envelope protein ALN-SOD (Alnylam Pharmaceuticals) Genetic therapy An RNA interference therapeutic targeting SOD1 for treatment of SOD1 -mutant ALS ALPHA-0602 (Alpha Cognition) Genetic therapy Gene therapy that delivers progranulin, a neurotrophic protein regulating neuronal survival and inflammatory processes AMT-161 (uniQure) Genetic therapy AAV5 gene therapy targeting repeat-expanded C9orf72 to lower toxic RNA aggregates and prevent dipeptide protein formation AS-202 (AcuraStem) Genetic therapy PIKfyve-suppressing antisense oligonucleotide targeting TDP-43 pathology BMD-001 (BIORCHESTRA) Genetic therapy Micro-RNA antisense oligonucleotide that regulates multiple mRNAs, reduces neuroinflammation and improves neurorestoration Caveolin-1 gene therapy (Eikonoklastes Therapeutics) Genetic therapy Caveolin-1 is a membrane/lipid raft scaffolding protein that promotes neurotrophin effects and enhances neuroplasticity, mitochondrial health and neuronal adaptation to stress CTx-FUS (Coave Therapeutics) Genetic therapy AAV gene therapy targeting FUS, a nuclear DNA/RNA binding protein that regulates various steps of gene expression TW-002 (Treeway, Ferrer) Genetic therapy AAV gene therapy that upregulates glial cell line-derived neurotrophic factor VTx-001 (VectorY Therapeutics) Genetic therapy AAV5-vectorized scFv that binds and neutralizes oxidized phospholipids VTx-002 (VectorY Therapeutics) Genetic therapy AAV5-vectorized scFv that targets misfolded toxic TDP-43 species QRA-244 (QurAlis Corporation) Hyperexcitability A Kv7 ion channel opener that controls motor neuron hyperexcitability-induced excitotoxicity M102 (Aclipse Therapeutics) Various Nrf2 and HSF1 dual activator that engages multiple neuroprotective mechanisms AGS-499 (Neuromagen Pharma) Neurogenesis Small-molecule telomerase reverse transcriptase that has neurogenic and neuroprotective effects Alpha-5 (Pasithea Therapeutics) Neuroinflammation Anti-integrin mAb developed for the treatment of ALS and other neuroinflammatory disorders COYA-201 (Coya Therapeutics) Neuroinflammation T reg cell-derived exosome therapy that promotes immunosuppressive and anti-inflammatory effects in cells and tissues LP143 (LongBoard Pharmaceuticals) Neuroinflammation Cannabinoid receptor type 2 agonist, aiming to reduce microglial neuroinflammation PMN-267 (ProMIS Neurosciences) Proteostasis mAb that targets the formation of misfolded TDP-43, which aggregates in neurons ALS drugs in preclinical development were identified using the AdisInsight Springer database (August 2022). Amongst these, selected active drug programmes with the lead target indication of ALS and at least one significant update within the previous 12 months are listed in the table. AAV, adeno‐associated virus; FUS, fused in sarcoma; HSF1, heat shock factor 1; mAb, monoclonal antibody; Nrf2, nuclear factor erythroid 2-related factor 2; scFV, single-chain variable fragment; SOD1, superoxide dismutase 1; TDP-43, TAR DNA-binding protein-43; T reg cell, regulatory T cell. Preclinical drug development programmes are highlighted in Table 3 and themes emerge which indicate the potential future direction of ALS clinical trials. The best represented category is genetic therapy approaches targeting specific misfolded proteins (for example, TDP-43, FUS and SOD1). Neuroinflammation is less well represented preclinically, with three approaches documented (Alpha-5, COYA-201 and LP143). Several novel mechanisms are represented for the first time. Cortical hyperexcitability is an emerging mechanism, and QRA-244, which targets voltage-gated potassium channel Kv7 family members, represents a potential approach to address this pathophysiological mechanism. Preclinical and clinical studies highlighting the pot

COYA-201 is a T reg cell-derived exosome preparation under development by Coya Therapeutics for the treatment of ALS. Neuroinflammation contributes to neurodegeneration, which is driven by an imbalance of pro-inflammatory effector T cells and T reg cells. COYA-201 was developed using the immunosuppressive cell exosome (iscEXO) platform, derived from T reg cells and M2 macrophages, both of which are prominent anti-inflammatory and neuroprotective cells 211 . Exosomes are membrane-derived small vesicles, rich in proteins and RNAs and which can avoid potential cell-based issues such as immune rejection 212 and polarization to a pro-inflammatory cell type 213 . Compared to MSC-derived exosomes, T reg cell-derived exosomes have much higher suppressive capacity and anti-inflammatory function 213 . The first-in-human trial of COYA-201 was expected in 2022.

Target identification Major recent advances have taken place that underpin a strong pipeline of candidates for neuroprotective therapy development. Our understanding of disease pathophysiology has increased, with greater knowledge of the genetic architecture of ALS and of the multiple overlapping mechanisms that contribute to motor neuron injury in the presence of specific genetic mutations. This wealth of additional genetic and pathophysiological information means that there is no shortage of potential novel therapeutic targets. To date successful neuroprotective approaches for ALS are exclusively CNS-penetrant small molecules, which presents challenges for identifying druggable targets. Approaches that draw on the plethora of existing publicly available biological data could help to rapidly identify small molecule targets, a recent example being TargetDB 214 . Machine learning-based approaches could also be deployed to assist with target identification; for example, the use of knowledge graphs which link disparate types of data with relational inferences derived from natural language processing 215 . Such strategies have been used to identify COVID-19 therapies 216 and are being deployed in ALS to identify new targets, validated using patient-derived cell models ( BenevolentAI ). Additional therapeutic modalities are expanding the list of tractable targets, and the successful use of gene therapy (onasemnogene abeparvovec) and ASO (nusinersen) approaches for amelioration of motor neuron injury in spinal muscular atrophy provide realistic prospects for therapeutic advances in defined genetic subtypes of ALS. The limited options for large-molecule (antibody) therapeutics in neurology may be abrogated by new technologies that enable CNS transport of antibody therapeutic agents. This was recently demonstrated in mice and primates using a transferrin-targeting moiety engineered into the Fc portion of a BACE antibody 217 . This strategy is being applied in several neurodegenerative conditions including FTD (DNL53, Denali Therapeutics; NCT05262023 ). Multiple mechanisms, as described above, can contribute to the ultimate demise of motor neurons. This raises the prospect that multiple targets may need to be addressed simultaneously to achieve significant slowing of disease progression. This can be achieved with polypharmacology (for example, AMX0035) and indeed the combination of riluzole and edaravone has become the standard of care for ALS in some countries. A second approach may be to identify single upstream targets that can in turn address multiple downstream mechanisms (for example, M102 which targets the transcription factors Nrf2 and HSF1). Drug repurposing is often proposed as a potential shortcut to identifying novel therapies and has shown some success for neurological disorders, including dimethyl fumarate for multiple sclerosis 218 . However, greater consideration of the drawbacks and potential limitations of repurposing is needed 218 . The most fruitful approach may well be ‘on-target’ repurposing where the original target of the therapy is the intended target for the new indication. However, the need for CNS penetration and whether the risk–benefit and dosing level is appropriate in the new indication are often limiting issues.

It is now possible to develop preclinical screening cascades which take us far beyond unreliable readouts of survival in SOD1 -transgenic mice. A combined approached can be used, with target-specific screening in vitro validated early in orthogonal patient-derived cell assays for selected compounds. SOD1 -transgenic rodents can be used for early mechanistic pharmacokinetic–pharmacodynamic work, followed by studies focused on evaluating more global effects on motor system degeneration. Finally, validation in alternative model systems such as patient-derived in vitro models and more physiologically relevant mouse models should enhance confidence in clinical translatability. Such an approach should enable a step-change in our ability to predict therapeutic efficacy in the clinic (Fig. 3 ). Fig. 3 A rational and enhanced preclinical efficacy screening cascade for ALS. In terms of efficacy assessment and increasing confidence in translation from the preclinical to the clinical arena, an enhanced approach would take advantage of the latest developments in preclinical disease modelling in rodents and non-rodent in vivo systems, as well as patient-derived cellular models. A standard in vitro cascade is shown for a target-based approach, which is subsequently confirmed at an early stage in a relevant patient-derived phenotypic model. Such models can be used as orthogonal screens through a development programme to ensure that efficacy against a relevant disease phenotype is maintained. At later stages in vivo mammalian models can be used as purely mechanistic pharmacokinetic/pharmacodynamic systems to ensure that therapeutic approaches have the required drug metabolism and pharmacokinetics, potency and specificity in vivo. Subsequently, confidence in clinical translation can be obtained in either more extensive screening in patient-derived model systems or complex disease models. Ideally several approaches should be pursued. These complex models with behavioural readouts of motor function would incorporate ‘translational’ biomarkers that have the potential to predict likely clinical benefit, such as measurements of compound muscle action potential (CMAP) and neurofilament light. ALS, amyotrophic lateral sclerosis; CSF, cerebrospinal fluid; iPSC, induced pluripotent stem cell; MN, motor neuron; PD, pharmacodynamic; PK, pharmacokinetic.

The traditional approach has been the randomized controlled trial in which the effects of the drug candidate are compared to that of placebo, and with survival or ALSFRS-R score as the commonly used primary outcome measures. This approach is costly, time-consuming and inefficient. Many preclinical and early phase I/II trials in ALS have yielded promising results that were not replicated in phase III trials. Substantial consideration has recently been devoted to the refinement of clinical trials in ALS. The ALSFRS-R has been used extensively as an outcome measure, but violates Rasch model expectations and is limited by the multidimensionality that arises from the summation of multiple subscales, which prevents direct comparison of patients with identical total scores 233 . A new self-reported ALS disability scale has been developed, with improved responsiveness. It is a 28-item Rasch-built overall ALS Disability Scale, with each item scored as 0, 1 or 2, and which demonstrates excellent test–retest reliability 227 . Other novel outcome measures are in development including modifications to the ALSFRS-R. Multi-arm, multistage platform trials (MAMS) that incorporate biomarkers of target engagement/therapeutic efficacy are now poised to accelerate drug discovery and increase trial participation. In a MAMS trial with a sequential adaptive design, the sample size is not fixed in advance and the emerging trial data are sequentially analysed with in-built, pre-determined futility and superiority analyses. This enables ineffective treatment arms to be discontinued and studies with a positive signal to seamlessly graduate to the next trial phase. Platform trial designs (for example, Healey ALS Platform Trial and MND-SMART) have emerged to allow more rapid assessments and inclusivity for patients, and more efficient deployment of a pooled placebo arm 233 , 234 . A further European platform trial, TRICALS, will start imminently as several of the initial portfolio of trials are recruiting patients in multiple European centres. A master protocol allowing the simultaneous evaluation of multiple compounds and efficient use of a common placebo group has been successfully used in other disease areas, most notably oncology 235 . However, it is noteworthy that in these conditions robust biomarkers of disease burden are available. Such biomarkers have not yet been established in ALS, although promising candidates are now emerging; for example, neurofilaments and specific protein biomarkers in genetic therapy trials. These recent proposals to modify the traditional design of ALS clinical trials have the potential to reduce the sample size required and the placebo exposure time for trial participants, as well as the cost, burden for patients and the trial duration 232 . Event-driven trial designs may ameliorate the consequences of inaccurate baseline assumptions for trial design and the resulting unknown trial duration can be mitigated by deployment of planned interim analyses. Improved patient-reported outcome measures, including home assessments, are predicted to improve the reliability and sensitivity of trial end points. Evidence is emerging that home assessments may be a viable strategy for measuring the disease trajectory in ALS clinical trials 232 , 236 , 237 . This strategy has been accelerated by the shift towards telemedicine for the provision of clinical care to patients with ALS during the COVID-19 pandemic. Dialogue with regulatory bodies will be essential in bringing to fruition these proposed innovations for ALS clinical trials. It is noteworthy that the FDA has already published guidance for adaptive platform master protocol trial designs 238 and the EMA has established a plan to carefully explore this issue.

Significant recent advances have been made in understanding the genetic architecture and pathophysiology of ALS. A strategy has emerged where more accurate subclassification of patients can be made based on identification of risk genes beyond the traditional boundaries of sALS and fALS. This expanded genetic understanding has also enabled the validation of several pathophysiological biological pathways for therapeutic targeting and, alongside the expansion of therapeutic modalities, there is now no shortage of therapeutic hypotheses to explore preclinically. In the preclinical space, the number, variety and clinical relevance of available model systems to enable more rigorous preclinical screening have expanded. This enables validation of therapeutic hypotheses and targets across models and reduces the risk of failure due to lack of efficacy. In the clinical space, recent innovations in trial design will enhance outcome measures, patient selection and randomization, minimize the impact of clinical variability and increase statistical power. Platform trials and patient-reported outcomes have the potential to improve the pace of validation of therapeutic agents. This is particularly the case if combined with surrogate biomarkers of disease burden, which are now emerging. The development of biomarkers of target engagement and efficacy to bridge the gap from preclinical to clinical testing is a key future need, as is the application of sound pharmacological principles in therapy development. Several biomarkers (biochemical, physiological, imaging) that can be applied across the translational pathway show great promise. Of added importance is the continued impact of patient advocacy groups encouraging and financially supporting researchers and clinicians. This strong and visible advocacy has been a factor in elevating the priority of regulatory agencies to address the high unmet need of individuals facing ALS. For example, regulatory agencies appear to be more receptive to considering adequate and well-controlled phase II data as part of a streamlined approval process, as was recently shown with AMX0035. Having the attention and guidance of regulatory agencies continues to be an important factor in promoting rapid access to new therapies for patients with ALS. In summary, there is great potential for developing improved neuroprotective treatments for ALS in the near future. We advocate for academia–industry partnerships which accelerate the pace of rigorous testing of therapeutic hypotheses, incorporating the latest advances in preclinical screening, biomarker discovery and trial design. The unmet need of patients with ALS is clear and a range of tools are now poised for effective translation.

Nature Reviews Drug Discovery thanks the anonymous reviewers for their contribution to the peer review of this work.