C. F. Bennett

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.

Pharmacology of a Central Nervous System Delivered 2′-O-Methoxyethyl–Modified Survival of Motor Neuron Splicing Oligonucleotide in Mice and Nonhuman Primates

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously, we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here, we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared with several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and nonhuman primates (NHPs). Intracerebroventricular bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared with i.c.v. infusion. For both methods of delivery, the duration of ISIS 396443–mediated SMN2 splicing correction is long lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development.

Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for the Treatment of Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults. DM1 is caused by an expanded CTG repeat in the 3′-untranslated region of DMPK, the gene encoding dystrophia myotonica protein kinase (DMPK). Antisense oligonucleotides (ASOs) containing 2′,4′-constrained ethyl-modified (cEt) residues exhibit a significantly increased RNA binding affinity and in vivo potency relative to those modified with other 2′-chemistries, which we speculated could translate to enhanced activity in extrahepatic tissues, such as muscle. Here, we describe the design and characterization of a cEt gapmer DMPK ASO (ISIS 486178), with potent activity in vitro and in vivo against mouse, monkey, and human DMPK. Systemic delivery of unformulated ISIS 486718 to wild-type mice decreased DMPK mRNA levels by up to 90% in liver and skeletal muscle. Similarly, treatment of either human DMPK transgenic mice or cynomolgus monkeys with ISIS 486178 led to up to 70% inhibition of DMPK in multiple skeletal muscles and ∼50% in cardiac muscle in both species. Importantly, inhibition of DMPK was well tolerated and was not associated with any skeletal muscle or cardiac toxicity. Also interesting was the demonstration that the inhibition of DMPK mRNA levels in muscle was maintained for up to 16 and 13 weeks post-treatment in mice and monkeys, respectively. These results demonstrate that cEt-modified ASOs show potent activity in skeletal muscle, and that this attractive therapeutic approach warrants further clinical investigation to inhibit the gain-of-function toxic RNA underlying the pathogenesis of DM1.

Manipulation of PK-M mutually exclusive alternative splicing by antisense oligonucleotides

Alternative splicing of the pyruvate kinase M gene involves a choice between mutually exclusive exons 9 and 10. Use of exon 10 to generate the M2 isoform is crucial for aerobic glycolysis (the Warburg effect) and tumour growth. We previously demonstrated that splicing enhancer elements that activate exon 10 are mainly found in exon 10 itself, and deleting or mutating these elements increases the inclusion of exon 9 in cancer cells. To systematically search for new enhancer elements in exon 10 and develop an effective pharmacological method to force a switch from PK-M2 to PK-M1, we carried out an antisense oligonucleotide (ASO) screen. We found potent ASOs that target a novel enhancer in exon 10 and strongly switch the splicing of endogenous PK-M transcripts to include exon 9. We further show that the ASO-mediated switch in alternative splicing leads to apoptosis in glioblastoma cell lines, and this is caused by the downregulation of PK-M2, and not by the upregulation of PK-M1. These data highlight the potential of ASO-mediated inhibition of PK-M2 splicing as therapy for cancer.

TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals

Antisense oligonucleotides (ASOs) are versatile molecules that can be designed to specifically alter splicing patterns of target pre-mRNAs. Here we exploit this feature to phenocopy a genetic disease. Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss-of-function mutations in the SMN1 gene. The related SMN2 gene expresses suboptimal levels of functional SMN protein due to alternative splicing that skips exon 7; correcting this defect-e.g., with ASOs-is a promising therapeutic approach. We describe the use of ASOs that exacerbate SMN2 missplicing and phenocopy SMA in a dose-dependent manner when administered to transgenic Smn(-/-) mice. Intracerebroventricular ASO injection in neonatal mice recapitulates SMA-like progressive motor dysfunction, growth impairment, and shortened life span, with α-motor neuron loss and abnormal neuromuscular junctions. These SMA-like phenotypes are prevented by a therapeutic ASO that restores correct SMN2 splicing. We uncovered starvation-induced splicing changes, particularly in SMN2, which likely accelerate disease progression. These results constitute proof of principle that ASOs designed to cause sustained splicing defects can be used to induce pathogenesis and rapidly and accurately model splicing-associated diseases in animals. This approach allows the dissection of pathogenesis mechanisms, including spatial and temporal features of disease onset and progression, as well as testing of candidate therapeutics.