Rise of the Genomic Medicine Era in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative condition most commonly resulting in death 2 to 5 years after onset. There is growing knowledge about the genetic underpinnings of ALS, with >40 genes identified as disease causative or as risk modifiers for ALS.¹ Advances in our understanding of ALS genetics and novel gene-editing technologies have paved the way for a new wave of gene-targeted therapeutic development.² In this review, we focus on ALS genetic abnormalities, for which there are opportunities for people with ALS to access experimental or approved gene-targeted therapies.

Approximately 10% of ALS cases are associated with a family history of ALS (familial ALS [fALS]). The remaining 90% of cases with no known family history of ALS are referred to as sporadic ALS (sALS).^3,4 Recent studies using broad sequencing strategies have demonstrated that an underlying genetic cause of ALS can be discovered in upwards of 20% of sALS cases,⁵ leading to newer nomenclature such as genetic ALS and nongenetic ALS to differentiate the 2 subgroups. With increasing ALS gene discoveries over the past decade, the ratio of genetic to nongenetic ALS is changing continually. The 5 most common monogenic causes of ALS in European populations include hexanucleotide repeat expansions in C9orf72 ([chromosome 9 open reading frame 72] 4% to 7% of ALS cases); gain-of-function sequence variations in SOD1 ([superoxide dismutase 1] 2% of ALS cases); and sequence variations in TARDBP (TAR DNA-binding protein 43), FUS (fused in sarcoma), and TBK1 (TANK-binding kinase 1), each accounting for 1% or fewer of all ALS cases.^1,4

Toxic Gain-of-Function, Missense Sequence Variations in SOD1

In a 2021 study,³ the prevalence of SOD1 ALS was estimated to be 2,876 in 22 countries across Europe, North America, Latin America, and Asia, with 946 incident cases per year, nearly half of which occurred in people with no known family history of ALS. Since the discovery of SOD1 as a genetic cause of ALS in 1993, >180 unique variants in SOD1 have been identified as causative for ALS.⁶ There is substantial heterogeneity in disease penetrance, age at onset, and expected rate of progression between variants.^6-8 The SOD1 Ala5-to-Val variant (A5V; formerly A4V) accounts for nearly half of the SOD1 ALS cases in the United States and is associated with extremely rapid disease progression (median survival of 1.2 years from onset) and high penetrance in natural history studies.^6,8 There is an additional substantial minority of rapidly progressive variants of SOD1 ALS with less than 2-year median survival.⁶ Clinical features of SOD1 ALS are heterogeneous, although there is a preponderance of limb onset and lower motor neuron–predominant disease.⁸

Neuropathology of SOD1 ALS demonstrates the presence of toxic misfolded variant SOD1 protein aggregates predominant within spinal motor neurons without TDP-43 pathology.⁹ Relative sparing of the motor cortex in humans with SOD1 ALS for both SOD1 aggregates as well as microglial activation compared with other forms of ALS has been reported, consistent with the typical clinical lower motor neuron phenotype in SOD1 ALS.⁹

Tofersen

Tofersen (Qalsody; Biogen, Cambridge, MA) is a Food and Drug Administration (FDA)–approved (accelerated approval, April 2023) intrathecally delivered gapmer antisense oligonucleotide (ASO) therapy for symptomatic SOD1 ALS. Tofersen induces RNase H–mediated degradation of SOD1 mRNA, resulting in decreased levels of SOD1 protein.^2,10 The accelerated approval of tofersen was based on favorable reduction in serum neurofilament light chain (NfL) levels in the pivotal phase 3 VALOR trial (An Efficacy, Safety, Tolerability, Pharmacokinetics and Pharmacodynamics Study of BIIB067 in Adults With Inherited Amyotrophic Lateral Sclerosis, NCT02623699), as a surrogate marker of neurodegeneration and a predictor of survival and functional decline in SOD1 ALS.^2,11 Tofersen-treated participants showed a 60% reduction in serum NfL level, compared with a 20% increase in placebo-treated participants at the end of the placebo-controlled study at week 28 (P<.001).^10,11 The prespecified primary end point of VALOR—slowing of disease progression as measured by the ALS Functional Rating Scale–Revised (ALSFRS-R) at week 28 in participants with rapid disease progression—fell short of meeting statistical significance.

The open-label extension data demonstrated a clear benefit by week 52 in multiple clinical end points in the group treated early (initially randomized to tofersen 100 mg) in VALOR compared with those initially randomized to placebo (delayed-start group). The week 52 open-label extension results showed slowed ALSFRS-R decline (−6.0 vs −9.5; P=.0272), change in slow vital capacity (−9.4 vs −18.6; P=.0159), and quantitative muscle strength measured by handheld dynamometry (−0.17 vs 0.45; P=.0186) favoring the early-start group compared with the delayed-start group.¹¹ Emerging data from the ongoing tofersen expanded access cohorts suggest halt in progression or stabilization of ALSFRS-R scores in several participants with SOD1 ALS.¹²

Overall, tofersen was safe and well-tolerated. A subset of tofersen-treated participants (7%) experienced serious neurologic adverse events, including myelitis, radiculitis, aseptic meningitis, increased intracranial pressure, and papilledema.^10,11 There were no permanent neurologic sequelae of these adverse events but there was 1 therapy termination. More than half of the tofersen-treated participants in VALOR were observed to have incidental asymptomatic rises in cerebrospinal fluid (CSF) white blood cell counts and protein levels.¹⁰

Clinical Trial for Asymptomatic SOD1 Gene Carriers

The ATLAS trial (A Study of BIIB067 [Tofersen] Initiated in Clinically Presymptomatic Adults With a Confirmed Superoxide Dismutase 1 Mutation, NCT04856982) of tofersen is the first trial to evaluate a gene-targeted therapy in fALS gene carriers before onset of ALS, with the objective of preventing phenoconversion to symptomatic disease through early intervention with a proven gene-targeted therapy. The premise of this first-of-its-kind trial in ALS stems from evidence that elevated levels of blood NfL can be detected 6 to 12 months before phenoconversion, suggesting a potential window to initiate therapy in fALS gene carriers.⁷ Participants are randomized to receive either tofersen or placebo when NfL level rises above a prespecified threshold predicted to indicate a high likelihood of impending ALS phenoconversion. The primary outcome is the percentage of individuals who develop clinically manifest ALS 12 months after randomization.⁷ Enrollment is ongoing, with study completion expected in 2027. This study will serve as confirmatory trial for the FDA to consider full approval of tofersen in the United States.

Non-ASO SOD1 Silencing Therapies

AMT-162 (uniQure, Lexington, MA) is an adeno-associated virus encoding SOD1 targeted microRNA (AAV-miR-SOD1) given as a single intrathecal infusion, which is awaiting phase 1/2 clinical trials. Two individuals with SOD1 ALS were treated with AMT-162 in an expanded access protocol. Postmortem tissue demonstrated lowering of SOD1 levels in spinal cord tissue but no effect on CSF SOD1 protein levels or clear clinical benefit was observed in either participant. One participant developed meningoradiculitis as a serious adverse event.¹³

AP-101 (AL-S Pharma AG, Zurich, Switzerland) is an intravenously delivered monoclonal antibody with high affinity binding to misfolded SOD1 protein. This medication is being tested in a global phase 2a clinical trial (A Study to Evaluate Safety, Tolerability, Pharmacodynamic (Pd) Markers and Pharmacokinetics (PK) of AP-101 in Participants With Amyotrophic Lateral Sclerosis (ALS), NCT05039099; Table).

Hexanucleotide Repeat Expansion (GGGGCC; G4C2) in C9orf72

C9orf72 is the most common inherited cause of ALS and frontotemporal dementia (FTD). C9orf72 ALS represents 30% to 50% of fALS cases and approximately 5% to 7% of sALS cases in European populations.^1,4,14 In a 2021 study,³ the prevalence of C9orf72 ALS was estimated to be 4,545 in 22 countries across Europe, North America, Latin America, and Asia, with 1,706 incident cases per year.³C9orf72 ALS commonly is associated with FTD but the motor component of disease is otherwise similar to that of sALS.¹⁴

The mechanism of ALS or FTD caused by the C9 hexanucleotide repeat expansion remains incompletely understood. Transcription of C9orf72 results in 3 primary mRNA transcripts: V1, V2, and V3. The V1 and V3 transcripts contain the hexanucleotide repeat expansion but the V2 transcript is required for physiologic function of the C9 protein.¹⁵ There are thought to be 3 primary mechanisms of damage: toxicity from expanded sense and antisense stranded mRNA resulting in sequestration of RNA binding proteins in RNA foci; toxicity attributable to 5-dipeptide repeats (DPRs) from repeat-associated non-ATG (RAN) translation of either the sense (poly-GA, poly-GR, poly-GP) or antisense (poly-PR, poly-PA, poly-GP) mRNA strands; and partial loss of function because of decreased C9orf72 protein levels (haploinsufficiency).^15,16 Elevated levels of CSF DPRs may be detected in central nervous system tissue of both asymptomatic and symptomatic C9orf72 hexanucleotide repeat expansion carriers, raising questions about the pathogenicity of all or some of the DPRs.^14,16 Nevertheless, DPRs, particularly poly-GP and poly-GA, have emerged as key biomarkers of target engagement in clinical trials for C9 ALS or FTD ASO because of the availability of validated CSF biofluid assays.¹⁶

ASO Therapies for C9orf72 ALS

Gene-targeted therapies for C9orf72 ALS have focused on depletion of C9orf72 repeat–containing mRNA transcripts using ASOs. Two recent phase 1 trials of C9orf72 ASOs have been unsuccessful and drug development aborted because of worsening clinical progression or NfL levels, despite adequate central nervous system target engagement (A Study to Assess the Safety, Tolerability, and Pharmacokinetics of BIIB078 in Adults With C9orf72-Associated Amyotrophic Lateral Sclerosis, NCT03626012; and Study of WVE-004 in Patients With C9orf72-Associated Amyotrophic Lateral Sclerosis or Frontotemporal Dementia [FOCUS-C9], NCT04931862).^17,18

BIIB078 (Biogen, Cambridge, MA), a C9orf72 ASO targeting the V1 and V3 transcripts, demonstrated target engagement as measured by 50% and 55% reductions in poly-GP and poly-GA DPRs in the highest dosage cohort.¹⁷ WVE-004 (Wave Life Sciences Ltd., Cambridge, MA), a stereopure ASO, reduced CSF poly-GP by 50% compared with baseline. Both ASOs failed to show signs of clinical efficacy after at least 6 months of treatment in small phase 1 multiple-ascending-dose clinical trials, leading to the discontinuation of further drug development.¹⁸ An n=1 expanded access protocol testing increasing dosages of afinersen (Universty of Massachusetts Chan Medical School RNA Therapeutics Institute, Worcester, MA), a C9orf72 ASO targeting V1 and V3 transcripts, showed stabilization of clinical function as measured by ALSFRS-R despite more than 4-fold increase in serum NfL level on the highest 2 mg/kg dosage administered every 3 months.¹⁵ Afinersen access is not yet available in clinical trials or expanded access protocols.

Multiple therapies targeting the downstream effects of pathogenic C9 sequence variations are progressing in clinical trials (Table).

Toxic Gain-of-Function Sequence Variations in FUS

Dominantly inherited variants in FUS are a rare cause of ALS and FTD, accounting for 3% of fALS cases and 0.3% of sALS cases in European populations. FUS ALS is more common in East Asian populations (6.4% of fALS cases and 0.9% of sALS cases).⁴FUS sequence variations may be associated with typical features of ALS or FTD and may present at very young or older age. The FUS p.525L variant is characterized by adolescent onset (juvenile ALS), and similar to SOD1 p.A5V, is associated with a very aggressive disease progression course.¹⁹ In some juvenile ALS cases, there may be preceding cognitive dysfunction, and frequently there is no known family history of ALS.^19,20

FUS ALS disease causation is thought to be primarily driven by toxic gain of function of the variant FUS protein.²¹FUS pathogenic sequence variations typically are missense variants clustered in the C-terminal region within or adjacent to the nuclear localization signal.²¹ Cytosolic mislocalization of FUS and neuronal cytoplasmic inclusions with FUS immunoreactivity without TDP-43–positive inclusions are characteristic.⁹

Ulefnersen ([ION363] Ionis Pharmaceuticals, Inc., Carlsbad, CA) is an intrathecally delivered ASO targeting an intronic segment of the FUS gene shared between mice and humans that results in antisense-mediated silencing of mRNA translation and reduction in variant and wild-type FUS protein levels.²⁰ An international phase 1-3 clinical trial of ulefnersen treatment in people with FUS ALS (12 years or older) is ongoing, with study completion expected in 2025 (A Study to Evaluate the Efficacy, Safety, Pharmacokinetics and Pharmacodynamics of ION363 in Amyotrophic Lateral Sclerosis Participants With Fused in Sarcoma Mutations, NCT04768972). A previous first-in-human n=1 study performed under compassionate use authorization in a 25-year-old individual with FUS p.525L ALS used multiple ascending doses of ulefnersen ranging from 20 mg to 120 mg for a total of 12 infusions over 10 months. This study showed marked reduction in both wild-type FUS and variant FUS protein levels to near undetectable levels in postmortem tissue. Before the participant’s death, clinical data suggested marked slowing in the rate of decline in ALSFRS-R.²⁰ Preclinical work in murine FUS models showed that ulefnersen reduced brain and spinal cord FUS protein levels by 20% to 50% and prevented degeneration of spinal motor neurons.²⁰

Risk Modifier Alleles

Multiple single-gene aberrations that are not disease causative per se but may influence the risk of developing ALS, age at onset, or rate of disease progression are being explored as potential drug targets for ASO experimental therapeutic interventions in ALS.

Intermediate-Length Repeat Expansions in ATXN2

Intermediate-length (27 to 33) trinucleotide polyglutamine (polyQ/CAG) repeat expansions in ATXN2 (ataxin-2) resulting in increased stability of ATXN2 is associated with a 3-fold increased risk of ALS compared with healthy individuals.^22,23 The clinical relevance of polyQ expansion on age at onset, survival, or disease progression is unclear. ATXN2 is an RNA-binding protein with diverse cellular functions. Important in ALS pathogenesis, ATXN2 appears to modulate cytoplasmic translocation and aggregation of TDP-43 within ATXN2-positive foci.²²

BIIB105 (Biogen, Cambridge, MA), an intrathecally delivered ASO targeting ATXN-2, is in phase 1 clinical trial for people with polyQ intermediate repeat expansion as well as a sALS without pathogenic polyQ repeats cohort (A Study to Assess the Safety, Tolerability, and Effect on Disease Progression of BIIB105 in Participants With Amyotrophic Lateral Sclerosis and Participants With the ALS Ataxin-2 [ATXN2] Genetic Mutation [ALSpire], NCT04494256).

STMN2

STMN2 (stathmin-2) is a tubulin-binding protein highly expressed in motor neurons with important roles in axonal outgrowth and regeneration. Changes in STMN2 expression attributable to cryptic splicing have been implicated in the pathogenesis of sALS. In the absence of nuclear TDP-43, STMN2 splicing is disrupted, producing nonfunctional STMN2 and affecting the downstream cellular functional cascade.²⁴ QRL-201 (QurAlis, Cambridge, MA) is an ASO acting on splicing regulatory sites to restore normal STMN2 splicing. A phase 1 clinical trial is underway in Canada (A Study Evaluating the Safety and Tolerability of QRL-201 in ALS, NCT05633459).

UNC13A

Polymorphisms in UNC13A, which encodes a key synaptic protein, are associated with an increased risk of ALS and FTD in genome-wide association studies. In the absence of nuclear TDP-43, UNC13A splicing is dysregulated, resulting in erroneous inclusion of nonconserved intronic segments in mRNA transcripts, or cryptic exons.²⁵ An ASO targeting UNC13A is in late stages of preclinical development.²⁵

Conclusion

With low- or no-cost ALS genetic panels now widely available in the US, in addition to a rapidly growing number of gene-targeted clinical trials, universal genetic testing for all people with ALS must become the standard of care, even in the absence of a positive family history of ALS. This would allow early identification of people who may benefit from early access to these therapies and trials. Broad panel-based testing frequently reveals variants of uncertain significance, some of which may be subsequently reclassified as likely pathogenic, based on continually emerging evidence of ALS association. The analysis, review, and interpretation of such inconclusive or complex test results is time-consuming and requires medical genetic expertise with access to population-based databases. To this end, genetic counselors are becoming an integral part of multidisciplinary ALS clinics for pretest and posttest counseling, family segregation studies, and variant reclassification analyses. The FDA’s approval of tofersen as the first gene-targeted therapy for ALS is the beginning of the genomic medicine era in ALS. Clinician awareness of rapidly increasing gene-targeted trials in ALS is critical for early suspicion of ALS and early referrals to multidisciplinary ALS clinics for prompt testing and intervention.