The Emerging Landscape of Targeted Therapeutics for Genetic Neuromuscular Disorders

Until the past decade, targeted therapy for neuromuscular disease seemed unattainable. While genetic testing was critical for discovering disease mechanisms and therapeutic targets, its primary application to individuals was for prognosis and assisting in family planning. More recently, however, genetic testing has played an increasingly important role in the management of neuromuscular disease as corrective strategies have emerged and evolved in sophistication and availability. Genetic testing may result in identifying a variant amenable to a corresponding therapy or clinical trial.

Since the discovery of dystrophin genetic sequence variations as the cause of Duchenne muscular dystrophy (DMD) in 1987,¹ more than 1000 genes linked to neuromuscular disorders have been identified.² For disorders without targeted therapies, neurologists can attest to the frustrating fact that management is restricted to nonspecific or symptom-based strategies. With each gene discovery, however, comes the possibility of treating the underlying cause, which could be life-prolonging or curative. In this article, we discuss the main mechanisms by which genetic variants cause neuromuscular disease, followed by a description of the different forms of genetically mediated therapies in use or in development for these disorders.

Types of Sequence Variations

Loss-of-Function

Genetic sequence variations often result in a nonfunctional protein product. This can be caused by introduction of a premature stop codon leading to a truncated protein, insertion or deletion of a splice site leading to a misspliced product, or insertion or deletion of nucleotides that leads to a frameshift, resulting in an altered and nonfunctional protein. DMD is a neuromuscular disease that can derive from any of these types of sequence variations, which result in an inability to create functional protein off the altered allele.¹ In dominantly inherited diseases, 1 mutant allele leading to haploinsufficiency is sufficient to cause disease, whereas recessively inherited diseases require biallelic sequence variations to manifest clinically. Loss-of-function disorders necessitate replacement or augmentation of the deficient protein (Figure, left panels).

Gain-of-Function

Genetic sequence variations can also lead to toxic gain-of-function, resulting in cell dysfunction or death. For instance, disorders across the neuromuscular spectrum can be caused by RNA or repeat-peptide toxicity from trinucleotide or hexanucleotide repeat expansions, exemplified by CTG repeats in the DMPK gene causing myotonic dystrophy type I and by G4C2 hexanucleotide repeats in the C9orf72 gene causing amyotrophic lateral sclerosis (ALS). Variations can also lead to protein aggregation, as is seen in pathogenic TTR variants that cause transthyretin amyloidosis and in pathogenic SOD1 or TARDBP variants that cause ALS. Consequently, treatment of these disorders requires strategies to knock down or inhibit the abnormal gene product at the gene, messenger RNA (mRNA), or protein level (Figure, right panels).

Therapeutic Approaches

Protein Replacement

Before the advent of gene therapy, direct protein replacement was the mainstay of treatment for loss-of-function disease, and remains in use for several monogenic disorders (Figure, A). An example is Pompe disease, a skeletal and cardiac myopathy caused by loss-of-function sequence variations in the gene encoding acid alpha-glucosidase. Pompe disease can be treated with twice-weekly intravenous enzyme replacement therapy with alglucosidase alfa, which was approved in 2006. Limitations of this approach include transient expression, limited therapeutic effect, and, in some cases, the development of an antibody response. Gene therapy–based approaches are under development for Pompe and other disorders.

Gene Replacement

The most straightforward gene therapy approach for loss-of-function disorders is viral-mediated gene replacement (Figure, A), achieving durable augmentation with a single treatment. The most widely used gene delivery vector is adeno-associated virus (AAV), a nonpathogenic parvovirus that has been engineered to deliver DNA to target cells without integrating into the host genome or replicating. Different AAV capsids have tropism for specific tissues and cell types, with AAV9 having the strongest targeting of the nervous system and therefore the most widespread use in neurology. AAV capsids can be engineered further for more specific and potent targeting, depending on the indication.³ Other gene delivery vehicles include lentivirus, the in vivo use of which is limited by its risk of oncogenesis by means of integration into the host genome; and lipid nanoparticles (LNPs), which can be used to deliver protein, RNA, or DNA. LNPs offer the advantages of large packaging capacity, low immunogenicity, and ease of preparation; however, barriers to use need to be addressed, including crossing of the blood–brain barrier and specific targeting within the nervous system.⁴

AAV-mediated gene replacement has been applied to a wide range of neuromuscular disorders. Onasemnogene abeparvovec-xioi (Zolgensma; Novartis, East Hanover, NJ), a systemically infused AAV9-based SMN1 gene replacement, was approved by the Food and Drug Administration (FDA) in 2019 for the treatment of spinal muscular atrophy (SMA). AAV9 is being tested with intrathecal delivery in a phase I clinical trial (Intrathecal Administration of scAAV9/JeT-GAN for the Treatment of Giant Axonal Neuropathy, NCT02362438) for replacement of GAN, the loss of function of which leads to giant axonal neuropathy.⁵ Although the dystrophin gene is too large to package in AAVs, smaller dystrophin sequences have been engineered for AAV-mediated delivery for treatment of DMD.⁶ Many more types of AAV-mediated gene delivery for treatment of neuromuscular disease are in development.

While AAV therapy is being used increasingly in the clinic, its drawbacks remain numerous. Its packaging capacity is limited to 4.8 kilobases, prohibiting delivery of larger genes. Immunogenicity—to the capsid or the transgene being delivered—has necessitated immunosuppression at the time of delivery, and sometimes for a longer period, to achieve successful transduction and endurance of targeted cells. A sizable portion of the population harbors neutralizing antibodies to the environmentally prevalent AAVs, exacerbated by cross-reactivity among serotypes. These individuals often are excluded from treatments or trials because of likelihood of transduction failure, and similarly, one treatment with an AAV-based drug likely precludes redosing.⁷ Treatment often is most effective when administered early in life. However, when an infant who receives onasemnogene abeparvovec-xioi or a child who receives a DMD treatment grows, treatment effect is diluted over time, which is an important issue when redosing is not an option.⁶ Costs of AAV production remain extremely high. In addition, intrathecal or systemic delivery of AAVs has led to toxicity to the dorsal root ganglia both preclinically⁸ and in clinical trials.⁹ Dorsal root ganglia detargeting strategies are under development.

RNA Interference

RNA interference (RNAi) is a naturally occurring strategy used by cells to reduce gene expression using small, double-stranded, noncoding RNAs that bind to target mRNA and either block its translation or induce its degradation through the RNAi-induced silencing complex (Figure, B). Types of RNAi in therapeutic use include small interfering RNAs, small hairpin RNAs, and artificial microRNAs. These typically are delivered by AAVs or, when liver targeting is appropriate, LNPs. The double-stranded RNA is processed such that the ultimate effector molecule is the mRNA-targeting guide strand; meanwhile, the passenger strand is targeted for degradation.¹⁰

Patisiran (Onpattro; Alnylam, Cambridge, MA) is an FDA-approved, LNP-delivered RNAi that targets the TTR gene for treatment of transthyretin amyloidosis.¹¹ In this case, LNP-mediated delivery is feasible because transthyretin is synthesized in the liver rather than in the nervous system. Several viral and nonviral RNAi strategies are in development for treatment of Charcot-Marie-Tooth disease type 1A by knocking down the causal duplicated PMP22 gene.¹² AAV-Rh-mediated RNAi targeting SOD1 was administered intrathecally in a small human clinical trial for SOD1-related amyotrophic lateral sclerosis (SOD1-ALS)⁹ after preclinical success in mouse models and nonhuman primates.¹³ This strategy resulted in AAV-related dorsal root ganglia toxicity in 1 of 2 individuals and further development is pending.

Aside from AAV-related issues, the primary downside of RNAi is unintended targeting. This can be caused by binding of the guide strand to regions with homologous sequences to the target mRNA, but also can be caused when the passenger strand intended for degradation instead binds to its own off-targets. In addition, excessive exogenous RNAi expression can monopolize the RNAi processing machinery and cause widespread dysregulation of native cellular RNAi.¹⁰ Careful design, use of off-target prediction software, and dosing limits help minimize these effects.

Antisense Oligonucleotide Therapy

Antisense oligonucleotides (ASOs) are short sequences of single-stranded, synthetic oligonucleotides designed to complement target mRNA with high specificity, augmenting or decreasing gene expression (Figure, C). ASOs do not cross the blood–brain barrier and therefore usually are delivered intrathecally to target the nervous system, although lipid nanoparticles also have been used for peripheral delivery. Modifications to ASOs have rendered them increasingly blood–brain barrier–penetrant and stable, increasing target engagement and administration intervals,¹⁴ and innovative strategies such as conjugation to antibodies have improved delivery for neuromuscular use further.¹⁵

In loss-of-function diseases, ASOs can increase gene expression by several mechanisms (Figure, C, left panel). One method is to induce alternative mRNA splicing by masking a splice site—a strategy used to convert SMN2 into SMN1 in nusinersen (Spinraza; Biogen, Cambridge, MA), a widely used drug for treatment of SMA. Another method is to induce skipping of erroneous genetic code or premature stop codons to preserve the majority of the deficient protein—a strategy being developed for dystrophin rescue in DMD.¹⁶ ASOs also can block microRNA binding sites to prevent microRNA-mediated gene downregulation.

In toxic gain-of-function diseases, ASOs are used to trigger RNAse H enzyme–mediated degradation of the target mRNA (Figure, C, right panel). This strategy is used in several FDA-approved treatments for neuromuscular diseases caused by aberrant protein aggregation. The intrathecally administered SOD1-targeting ASO tofersen (Qalsody; Biogen, Cambridge, MA) recently was granted early approval for the treatment of SOD1-ALS, in which mutant SOD1 protein aggregates trigger death of motor neurons. The neuropathy and cardiomyopathy that characterize transthyretin amyloidosis are stabilized (and in some cases reversed) by the subcutaneously administered ASO inotersen, which lowers levels of the TTR gene encoding aggregate-prone transthyretin.¹¹ Antibody-conjugated ASOs targeting muscle are being developed for Dux4 knockdown to treat facioscapulohumeral dystrophy (FSHD).¹⁵

The primary downsides of ASOs are their poor penetrance across the blood–brain barrier and their transient expression, generally necessitating repeated intrathecal administration, which is invasive and inconvenient. Additionally, achievement of regional or cell-type specificity is limited, although the modifications described previously are helping overcome these challenges.

Gene Editing

As opposed to the previously mentioned strategies for gene modulation at the RNA transcript level, direct editing of the genome can be accomplished through use of bacterially derived clustered regularly interspaced short palindromic repeats (CRISPR)–associated systems (Cas), the most widely used of which is CRISPR-Cas9 (Figure, D). CRISPR-Cas9 can be designed to target specific regions of the genome and induce double-stranded DNA breaks, the most common outcome of which is a frameshift-inducing base insertion or deletion (indel) that results in permanent interruption of gene expression (Figure, D, right panel). CRISPR also can be used to induce repairs at the break site (Figure, D, left panel). Since its discovery, additional CRISPR-based systems have been developed or adapted to modulate gene expression. For instance, base editing alters specific nucleotides upstream of the binding site without inducing double-stranded DNA breaks.¹⁷ Other CRISPR systems have been developed that would not be classified as gene editors but rather modulate gene expression, including CRISPR-Cas13, which targets RNA for degradation, and CRISPR-interference, which represses DNA without modifying it.¹⁷

In a 2021 clinical trial, LNP containing CRISPR-Cas9–encoding RNA were delivered systemically to target TTR in 6 individuals with transthyretin amyloidosis, resulting in durable reduction in serum transthyretin protein up to 96% (Study to Evaluate Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of NTLA-2001 in Patients With Hereditary Transthyretin Amyloidosis With Polyneuropathy [ATTRv-PN] and Patients With Transthyretin Amyloidosis-Related Cardiomyopathy [ATTR-CM], NCT04601051).¹⁸ This was successful in part because TTR is synthesized in the liver; gene editing directly targeting the nervous system remains in preclinical territory. CRISPR has been used successfully in animal models of several muscle diseases, including DMD, limb-girdle muscular dystrophy, FSHD, myotonic dystrophy, and others,¹⁹ as well as for reduction of PMP22 for treatment of Charcot-Marie-Tooth disease type 1A.¹² Base editing also has been used to knock down SOD1 in a mouse model of SOD1-ALS, resulting in modest target engagement and increased lifespan.²⁰

Limitations of CRISPR-Cas9 therapy are numerous, particularly for neurologic disease in which AAVs are used to cross the blood–brain barrier. Double-stranded breaks can allow undesirable mutagenic effects, such as AAV integration.²¹ There also can be significant off-target effects, and the bacterially derived Cas9 effector protein is highly immunogenic, both factors being exacerbated by the permanent expression that results from AAV delivery.¹⁷ Furthermore, the large editing machinery typically must be split into 2 AAV vectors, limiting efficiency and increasing the required viral load. These factors are far from trivial and resulted in the recent death of a person with DMD who received AAV-CRISPR.⁶ Meanwhile, without use of AAVs, nervous system targeting is challenging, prompting inventive but less efficient solutions, including direct intraneural delivery.¹²

Small-Molecule Therapies

Small-molecule drugs are orally bioavailable molecules in the 0.1 to 1 kDa range capable of acting on targets of interest to modulate protein function or gene expression (Figure, E). Benefits of these drugs include noninvasive administration, lower cost and easier storage than gene therapy strategies, and general reversibility based on their short half-life. Because of their small size, many can diffuse passively across the blood–brain barrier, and when they are known compounds already approved for other uses, a preexisting safety track record is likely and approval can be rapid. As of 2018, 43% of all therapies under development for neuromuscular diseases were small molecules.²²

A small-molecule drug approved for loss-of-function disease is risdiplam (Evrysdi; Genentech, South San Francisco, CA), which acts similarly to nusinersen to alter splicing and convert SMN2 into SMN1 for treatment of SMA. Risdiplam is being studied in combination with other drugs targeting SMA.²³ Small-molecule drugs also can be used in toxic gain-of-function disorders. Diflunisal (Dolobid; Merck, Lebanon, NJ) and tafamidis (Vyndaqel; Pfizer, New York, NY) are FDA-approved small-molecule drugs used to treat transthyretin amyloidosis that act by stabilizing TTR protein tetramers and preventing their monomerization and aggregation.¹¹ For FSHD, the DUX4-expression–reducing small-molecule drug losmapimod (Fulcrum Therapeutics, Cambridge, MA) is in phase III human clinical trials.²⁴ Disaggregases are under study for treatment of ALS and other neurodegenerative disorders in which protein aggregation plays a role.²⁵

Because small-molecule drugs often are discovered through high-throughput drug library screens, their mechanism of action is not always clear, and they lack the targeting specificity conferred by gene therapy. These factors increase the risk and unpredictability of side effects.

Summary

The past several years of gene discovery and therapeutic development have altered the field of genetic neuromuscular medicine profoundly. Fatal diseases, including SMA, DMD, ALS, and amyloidosis, and diseases that cause debility and pain, such as myotonic dystrophy, FSHD, and giant axonal neuropathy, now have potential for an altered prognosis. From neuron to nerve to muscle, targeted therapies use an arsenal of approaches that offer individuals, their families, and their physicians new hope for disease modification, functional improvement, and life prolongation.