Antisense Oligonucleotides Delivered to the Mouse CNS Ameliorate Symptoms of Severe Spinal Muscular Atrophy

Central nervous system–directed antisense therapy ameliorates symptoms in a severe neuromuscular disorder in mice. Making Sense with Antisense for Spinal Muscular Atrophy Children suffering from the neuromuscular wasting disease spinal muscular atrophy (SMA) experience muscle weakness, paralysis, and altered respiratory function. The disease is caused by mutations in the gene SMN1, leading to decreased production of a protein called SMN. A deficiency in SMN protein results in loss of motor neurons in the spinal cord, defective neuromuscular junctions, and atrophy of skeletal muscles. Researchers have discovered that alternative splicing of a related gene, SMN2, to include exon 7 can result in production of sufficient SMN protein to ameliorate symptoms of the disease. Several therapeutic strategies are under development to treat SMA including gene therapy to replace the defective SMN1 gene or small-molecule drugs to boost the inclusion of exon 7 during splicing of SMN2 mRNA and so increase SMN protein production. Although these strategies have shown promise in cell lines and animal models of SMA, they have shown little success in treating human patients with the disease. In a new study, Passini and colleagues take a different therapeutic approach—they use splice switching antisense technology to boost SMN protein production. First, they designed an antisense oligonucleotide (ASO-10-27) that base pairs with an intronic splicing silencer; this frees up exon 7 so that it can be incorporated into SMN2 mRNA during splicing. By chemically modifying their antisense oligonucleotide, the authors ensured that it would be stable and less likely to cause inflammation when injected. Then, the authors tested their antisense oligonucleotide in newborn mice with a severe form of SMA. They injected ASO-10-27 into the cerebral ventricles and spinal fluid of SMA mice on the day of birth and then killed the mice 16 days later. They found four- to six-fold higher levels of SMN protein throughout the entire spinal cord of mice receiving ASO-10-27, but not in those animals that received a mismatched oligonucleotide that did not alter SMN2 mRNA splicing. This boost in SMN protein production resulted in an increase in the size and strength of muscle fibers of treated mice, which translated into improved muscle performance and motor coordination in several tests. ASO-10-27 also increased the number of spinal cord motor neurons and helped to retain the delicate structure of neuromuscular junctions. The authors calculated that the ideal therapeutic dose for ASO-10-27 to ameliorate symptoms of the disease was 8 μg/g tissue. They report that the amount of SMN protein produced peaked at day 16 after injection and then waned, disappearing completely by 30 days. This may fit with their observation that many of the treated SMA mice died at 21 days (after weaning) from breathing difficulties, perhaps due to loss of SMN protein. The authors will need to do further experiments to work out the best dosing regimen for ASO-10-27 to ensure steady production of SMN protein and amelioration of SMA symptoms over the course of a lifetime. Taking a step in this direction, the authors demonstrate that injecting ASO-10-27 into the spinal fluid of cynomolgus monkeys resulted in therapeutic levels of the oligonucleotide in primate spinal tissue. The elegant study by Passini and co-workers suggests that it makes sense to pursue development of their antisense technology for treating SMA. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene that result in a deficiency of SMN protein. One approach to treat SMA is to use antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to boost production of functional SMN. Injection of a 2′-O-2-methoxyethyl–modified ASO (ASO-10-27) into the cerebral lateral ventricles of mice with a severe form of SMA resulted in splice-mediated increases in SMN protein and in the number of motor neurons in the spinal cord, which led to improvements in muscle physiology, motor function and survival. Intrathecal infusion of ASO-10-27 into cynomolgus monkeys delivered putative therapeutic levels of the oligonucleotide to all regions of the spinal cord. These data demonstrate that central nervous system–directed ASO therapy is efficacious and that intrathecal infusion may represent a practical route for delivering this therapeutic in the clinic.