HONG KONG – An antisense oligonucleotide (ASO) that induces skipping of exon 53 has been shown to have a favorable safety profile and promising pharmacokinetics in children with Duchenne muscular dystrophy (DMD) in a Japanese phase I trial. That warrants further study of the ASO in a phase II study, researchers led by Shin'ichi Takeda, director of the Department of Molecular Therapy National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP) in Tokyo, reported April 18, 2018, in Science Translational Medicine.

DMD is a hereditary muscle disease caused by mutations in the gene encoding for the muscular protein dystrophin. That results in loss of dystrophin function, with progressive muscle weakness, cardiomyopathy, respiratory failure and premature death.

In DMD, a gene area known as an exon is deleted, interfering with the rest of the gene being assembled. Exon "skipping" enables the cellular machinery to miss an exon using ASOs as "molecular patches" to mask the faulty exon, so it is ignored during protein production.

That results in a shortened but still functional protein, despite the mutation, and ASOs that induce exon-skipping have been shown partially to restore dystrophin expression and suppress disease progression in mouse and dog models.

In humans with DMD, dystrophin expression was observed in early stage trials of two exon 51-skipping ASOs, eteplirsen (Exondys 51, Sarepta Therapeutics Inc.) and drisapersen (Prosensa Holding NV). The latter subsequently showed no significant difference to placebo in a phase III trial, despite having shown promise in phase II and was rejected by the FDA on safety grounds. (See BioWorld Today, Sept. 23, 2013.)

Eteplirsen, however, was found to induce muscular dystrophin production in a phase III trial, and it was granted accelerated approval by the FDA as the first disease-modifying exon 51-skipping ASO for DMD. (See BioWorld Today, Sept. 20, 2016.)

More recently, the exon 53-skipping ASO, golodirsen (SRP-4053, Sarepta) was show to be effective in DMD patients with confirmed deletions of the gene and is currently in phase III trials. (See BioWorld, Sept. 7, 2017.)

Now Takeda and colleagues have performed a phase I, open-label, dose-escalation trial of the morpholino ASO NS-065/NCNP-01, which was co-developed by the NCNP and Nippon Shinyaku Co. Ltd., in order to determine its safety, pharmacokinetics and activity.

"A morpholino ASO is a type of oligomer molecule which, in this study, was used for splicing the modulation of dystrophin mRNA," explained study co-lead author Takashi Saito, a visiting scientist at the NCNP.

"Theoretically, many exons can be targeted for skipping and, while there are various exon deletion patterns in DMD, a single target exon applicable to many exon deletion patterns has a priority in ASO development. Several such agents are presently in early stage clinical or preclinical development," said Saito.

Early success

The NCNP study included 10 DMD patients, ages 6 to 16, who had deletions in the dystrophin gene that could be restored using exon 53-skipping. "We performed muscle biopsy pre- and post-treatment, then measured dystrophin using immunofluorescence and western blot analysis," he said.

Eligible patients then received NS-065/NCNP-01 at doses of 1.25 mg/kg, 5 mg/kg or 20 mg/kg weekly for 12 weeks. The primary endpoint was safety, while secondary endpoints were pharmacokinetics and successful exon skipping.

No severe adverse drug reactions were observed and no treatment discontinuations were necessary.

Significantly, the ASO induced exon 53-skipping in dystrophin-encoding mRNA in a dose-dependent manner. "The activity of NS-065/NCNP-01 was assessed by an increase of exon 53-skipping dystrophin mRNA and the recovery of shortened dystrophin protein," Saito told BioWorld.

"We could not have confirmed dose-dependency using the dystrophin protein level alone, but the mRNA level suggested that NS-065/NCNP-01 induced exon skipping in vivo," he said.

"Combined with the acceptable safety profile, this dose dependency suggested by the increased mRNA warrants a next phase clinical trial at higher dose. The phase II trial by NS Pharma, the Nippon Shinyaku US subsidiary, will investigate dosing at 40 or 80 mg/kg/week."

The ASO also increased the dystrophin/spectrin ratio in seven of 10 patients. "Spectrin is a cytoskeletal protein localized in sarcolemmal membrane," Saito explained. "Spectrin can be used as a control protein for dystrophin quantification."

Furthermore, the amount of exon skipping correlated with the maximum drug concentration in plasma and the area under the concentration-time curve in plasma, suggesting favorable pharmacokinetics.

If the ASO proves successful, "we speculate that 8 to 10 percent of DMD patients would benefit from NS-065/NCNP-01 treatment," suggested Saito. However, while "the studies required for a new drug application remain a matter for the regulatory agencies, this process might take less than a few years."

Meanwhile, the NCNP team will investigate ASOs targeting an exon other than 53, in order to expand the DMD patient population suitable for exon skipping. "Enhancing ASO efficacy by optimizing ASO sequence design or chemical modification is also important."