Postnatal gene therapy for neuromuscular diseases – opportunities and limitations

Spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by biallelic mutations of the SMN1 gene. The resulting reduction of survival motor neuron (SMN) protein production mainly affects spinal motoneurons and leads to a progressive loss of muscle strengths. The clinical phenotype associated with SMA covers a broad spectrum of disease severity from SMA type 1 with onset during the first six months of life to cases with onset during later childhood or adolescence (SMA types 2, 3 and 4). The main reason for the differences in disease severity between patients is the number of SMN2 gene copies. SMN2 is a nearly identical gene located next to SMN1, which also produces SMN protein. However, due to a single nucleotide exchange that affects the splicing, the production of functional protein is reduced to about 10% compared to SMN1. While SMN2 has no relevance in healthy humans, it serves as a back-up gene in patients with SMA and can partially rescue the phenotype. Higher SMN2 gene copy numbers are thus associated with a milder phenotype. Two SMN2 copies are most prevalent in the population and are typically associated with the severe phenotype of SMA type 1. Without treatment patients with SMA type 1 die within the first two years of life or require permanent ventilation due to severe muscle weakness [3, 4].

Nusinersen was the first drug that has been approved for the treatment of all types of SMA. It is an antisense oligonucleotide that specifically modifies the splicing of SMN2 and leads to an increase of SMN protein production. As nusinersen does not cross the blood-brain barrier it requires repeated lumbar punctures every four months for intrathecal administration. Double-blind, sham-controlled trials have shown a clear benefit compared to the control group in infants with SMA type 1 and children with SMA type 2, respectively [5, 6]. Very recently, risdiplam has been approved as the second splicing modifier for the treatment of SMA. In contrast to nusinersen, risdiplam is a small molecule that crosses the blood-brain barrier and can be administered orally. In Europe it has been approved for the treatment of SMA patients aged two months and older [7].

The gene therapy approach for the treatment of SMA makes use of an adeno-associated viral vector type 9 (AAV9) to introduce a functional copy of the SMN1 gene. Onasemnogene abeparvovec is administered through a single intravenous infusion. Based on open-label clinical trials in infants the drug has received FDA approval for the treatment of patients with SMA up to the age of two years. For Europe, EMA has approved onasemnogene abeparvovec for the treatment of patients with up to three SMN2 copies without defining a clear age or weight limit. As the total dose used is proportional to the body weight and clinical trial experience is limited to infants, there are ongoing discussions about the potential use of the treatment in older children [8]. According to the product information it is recommended to use an immunosuppressive treatment with prednisolone starting on the day before administration for at least four weeks. Most commonly observed side effects include increase of transaminases and thrombocytopenia.

There are no randomized trials comparing the different treatments for SMA and cross-study comparison is difficult due to distinct inclusion criteria and baseline characteristics. However, all studies have shown that early initiation of treatment is most relevant to achieve best outcome. As most patients with SMA do not show any symptoms during the first weeks of life, some studies have explored the pre-symptomatic initiation of treatment and shown that this can facilitate almost normal motor development for many patients [9]. As a consequence newborn screening for SMA has been explored in several pilot projects and is introduced in an increasing number of countries [10, 11].

Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common neuromuscular disease during childhood. DMD is caused by mutations of the dystrophin gene on the X-chromosome. Due to the X-linked inheritance, DMD mainly affects boys and leads to a progressive loss of muscle strength. First symptoms, such as difficulties with climbing stairs or standing up from the floor, are typically noticed between two and four years of age. The progressive course of the disease leads to loss of ambulation between 10 and 14 years of age and respiratory failure during adolescence or early adulthood. In addition, most patients develop a dilative cardiomyopathy, which contributes to early mortality. In DMD the lack of the dystrophin protein causes disintegration of a membrane associated protein complex, which leads to cell death and replacement of muscle tissue by fat and fibrous tissue. Creatine kinase in serum, a marker of muscle necrosis, is invariably markedly increased from birth and serves a convenient screening marker for the disease. Underlying mutations include deletions or duplications of one or several exons of the dystrophin gene in about two thirds of cases and smaller mutations affecting only one or a few nucleotides in the remaining cases [12].

One treatment approach is the use of antisense oligonucleotides which bind to the splicing site of a specific exon and thus lead to so-called “exon-skipping”. If used in appropriate patients the skipping of an additional exon can help to restore the reading frame and thus allow for the production of partially functional dystrophin protein. Some of the exon skipping products have been approved by the FDA based on increased production of dystrophin observed in clinical trials. However, as clinical efficacy has not been proven convincingly, these drugs are currently not approved in Europe [13].

Ataluren is a specific treatment for premature termination codon (nonsense) mutations, which account for about of 10% of the DMD cases. Although the primary endpoint was not met in a double-blind placebo-controlled trial, subgroup analysis showed lesser decline in the treatment group compared to the placebo group [14]. Ataluren has been approved in Europe for the treatment of ambulant patients with nonsense mediated DMD, who are at least two years old.

A major limitation for the use of gene therapy for the treatment of DMD is the size of the dystrophin gene which exceeds by far the packaging capacity of AAV-based vectors. One possible solution is the construction of truncated forms of the dystrophin gene, which contain the minimal functional regions of the protein. Several of these micro- or mini-dystrophins a currently in clinical development using AAV-vector based gene therapy [15]. Due to the limitations of this traditional gene augmentation therapy, genome editing is also explored for the treatment of DMD. In most cases these strategies are designed to change a DMD out-of-frame mutation into an in-frame mutation, which is associated with the milder phenotype of Becker muscular dystrophy. Currently, genome editing technologies like CRISPR/Cas9 are studied in DMD animal models [16].

Myotubular myopathy

X-linked myotubular myopathy (XLMTM) is caused by mutations of the myotubularin 1 gene (MTM1) located on the X-chromosome. The severe form of XLMTM, which is also the most common form, is characterized by severe muscle weakness, ophthalmoplegia and respiratory insufficiency from birth. Reduced fetal movement and polyhydramnios often indicate prenatal onset. Nearly all patients require respiratory support at birth and the mortality is about 50% during the first years of life. Most patients who survive require tracheostomy [17].

An open label treatment study in 12 patients described improvements in motor and respiratory function after AAV8 based gene therapy including weaning from artificial ventilation in a significant number of patients [18]. However, later three children who had received a higher dose of this gene therapy died from fatal hepatic dysfunction and sepsis [19]. It is assumed that this might have been related to pre-existing hepatobiliary disease, which had been reported in XLMTM. The use of the therapy has therefore been temporarily stopped.