Researchers led by at team at NYU Grossman School of Medicine have developed an antibody-based treatment for a form of congenital myasthenia (CM), which rescued the affected mice from early death, and reversed disease relapse in adult animals. Rather than correct the flawed gene that causes the lethal muscle-weakening disease, the new strategy instead targets another protein in the same signaling pathway.

“To our knowledge, our study is the first to fully counter a lethal congenital flaw by targeted therapy, restoring synapse formation with an antibody that encourages the action of a protein downstream of the mutant gene,” said Steven J. Burden, PhD, professor in the Skirball Institute and Department of Neuroscience and Physiology at NYU Langone Health. Burden is lead author of the team’s published paper in Nature, which is titled, “Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia.”

Congenital myasthenia (CM) is a group of diseases caused by mutations in genes that play key roles in the formation, function, and maintenance of neuromuscular synapses, the authors explained. Past studies have linked many cases of CM to genetic errors in a gene called Dok7, which codes for a protein that is essential for the formation of synapses. “Mutations in Dok7, the gene encoding an adaptor protein crucial for forming and maintaining neuromuscular synapses, constitute a substantial portion (10–20%) of all CM cases,” the team continued.

Human newborns with CM often survive, but face severe, life-long muscle weakness. The few available treatments only partially abate the symptoms. “The disease is debilitating, causing weakness in limb, neck and facial muscles, and one quarter of

Dok7 CM patients require non-invasive ventilation at some point during their lifetime,” the authors wrote. “Few treatments abate the clinical symptoms, although albuterol/salbutamol, which activates adrenergic receptors, can provide benefit for a subset of Dok7 CM patients through poorly understood mechanisms.”

Dok7 acts as an adapter protein that becomes attached to a key enzyme, muscle-specific kinase (MuSK). Discovered by the Burden lab in 1993, MuSK has been studied by many labs over the past two dozen years. Once attached to MuSK, Dok7 is modified, such that new attachment sites appear in Dok7, leading to the recruitment of other proteins crucial to building the neuromuscular synapse. Past studies by the Burden lab and others had in addition shown that Dok7 is not only a target of MuSK but also a stimulator of MuSK, helping to keep MuSK activated.

The most common disease-causing mutation in the Dok7 gene produces an abnormal, shorter form of the Dok7 protein. So, in order to study how loss of part of the Dok7 protein causes defects in the structure and function of neuromuscular synapses, Burden and colleagues generated a mouse model of this common form of CM. These animals fail to develop synapses that trigger the muscle contractions required for movement, including breathing, and so die soon after birth.

Past theories held that the Dok7 mutation caused CM by removing the part of Dok7 with the attachment sites for other proteins, thereby preventing assembly of the cellular machinery for building neuromuscular synapses. However, the team’s newly reported studies determined that this common form of the disease was caused not by the loss of Dok7 attachment sites, but instead because the shortened version of Dok7 was produced in modestly lower amounts. Consequently, there was not enough Dok7 to activate MuSK.

Having identified this surprising CM mechanism, the researchers used their understanding of MuSK to design synthetic antibodies that boosted the action of MuSK. The goal was to learn whether such antibodies could rescue mice with the Dok7 mutation. Engineered versions of antibodies are widely used as therapeutics, and so could represent a more practical approach to treating Dok7 CM than replenishing muscle cells with a healthy Dok7 protein by gene therapy, the study authors suggest.

Based on an extensive knowledge of MuSK biology, study author Shohei Koide, PhD, professor in the Perlmutter Cancer Center at NYU Langone Health and Department of Biochemistry and Molecular Pharmacology, and colleagues designed a strategy to find antibodies that attached to just the right region of mouse and human MuSK in order to activate MuSK without blocking its normal functions.

The team identified several candidate antibodies, and of these, an antibody designated X17 rescued neuromuscular synapse formation, and reversed the movement deficits and early lethality of mice with Dok7 CM. The studies showed that, encouragingly, most of the antibody-treated mice grew to fertile adults. “Antibody X17 rescued motor function of Dok7 CM mice, as assessed by forelimb grip strength and rotarod assays,” they wrote “Moreover, Dok7 CM mice, injected with antibody X17, were fertile and produced offspring at the expected frequency.”

When the team then withdrew the antibody treatment, the adult CM mice relapsed and displayed movement problems, which were reversed by restarting the antibody treatment. “… following withdrawal of antibody treatment, Dok7 CM adult mice ultimately displayed motor deficits, which were readily reversed after reinitiating antibody treatment … suggesting that this therapeutic strategy may provide benefit for Dok7 CM as well as other neuromuscular diseases in humans…” they continued.

The combined study results indicate that the new antibody-based therapeutic approach rescues synapses when they first form during development, but can also reverses synaptic dysfunction in adult animals. “These experiments demonstrate full rescue from congenital lethality by targeted therapy,” they concluded. “These findings point to an unforeseen therapeutic approach to treat disease, as this strategy does not directly target the mutant protein but rather targets a wildtype protein with diminished activity, caused by mutation of an upstream gene, in this case Dok7.”

While the researchers’ ultimate goal would be to develop the capability to treat CM as soon as it is diagnosed in children or adults, their research could also point to therapeutic approaches to other diseases. Burden, who is also a professor in the Department of Cell Biology, said, “While this strategy applies directly to a rare set of neuromuscular diseases studied here, the findings suggest new approaches to more common diseases – like amyotrophic lateral sclerosis, also called ALS or Lou Gehrig’s disease, spinal muscular atrophy, and autoimmune myasthenia gravis.”