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New Evidence for Gene Replacement in a Mouse Model of Charcot-Marie-Tooth Disease

Article In Brief


“In this study, the most important thing we demonstrated is that there is a way to provide therapeutic benefit in this model, which is a very authentic and representative model of the disease. I am confident that in the next couple of years we will make significant progress towards translation.”


In a mouse model of Charcot-Marie-Tooth disease type 4C, investigators provided a proof of principle that viral gene replacement therapy targeted to Schwann cells could treat the disease type and potentially other demyelinating inherited neuropathies.

Gene replacement therapy improves histological, electrophysiological, and functional deficits in a mouse model of one form of Charcot-Marie-Tooth (CMT) disease, called CMT4C, according to study in the March 25th online issue of Brain.

“This study is a major advance,” commented Michael E. Shy, MD, professor of neurology and director of the Division of Neuromuscular Medicine at the University of Iowa Carver College of Medicine in Iowa City, who was not involved in the study. “I think it is an example of how gene replacement strategies are a rational path forward to treat autosomal recessive forms of CMT.”

CMT type 4C causes weakness, muscle atrophy, areflexia, and sensory loss, with onset usually in the first decade of life. It is caused by mutation in the SH3TC2 gene, which is expressed in Schwann cells and most likely acts as a scaffolding protein within the endocytic pathway.

“The gene appears to play a regulatory role in Schwann cell elongation, in addition to myelin formation,” explained principal investigator Kleopas Kleopa, MD, PhD, professor of neurology at the Cyprus School of Molecular Medicine in Nicosia.

“During axonal myelination, Schwann cells elongate and until they meet each other at the nodal area, but in CMT4C, they don't appear to elongate enough,” leaving more space between insulated segments, he explained. These expanded nodal gaps “probably contribute to delayed conduction, as well as causing more oxidative stress, because you need more energy to maintain the function of the sodium channels at the node,” likely increasing the risk for axonal degeneration. In addition, the myelin sheath is thinner, especially around large diameter fibers, and there is progressive demyelination.

CMT4C is inherited as an autosomal recessive disease, and diagnosis is often delayed, since parents are usually unaffected. And because it is so rare, it is likely under-recognized, Dr. Kleopa said. “We now know there are more cases of CMT4C among CMT patients who were not initially diagnosed based on testing for the most common genes.”

Study Design

Because the known mutations in SH3TC2 appeared to cause a loss of function, Dr. Kleopa, along with first author Natasa Schiza and colleagues, decided to test whether gene replacement could ameliorate the phenotype in a mouse model of CMT4C. Like their human counterparts, these mice carry two inactivating gene mutations, and display expanded nodes, hypo- and demyelination, nerve conduction slowing, and mild weakness from an early age.

Three-week-old mice received intrathecal injection of a lentiviral vector containing the human SH3TC2 gene with a myelin-specific promoter and were then examined at four and eight weeks after injection.

Dr. Kleopas had previously shown that intrathecal injection was an efficient way to transfect Schwann cells, because the intrathecal space and the endoneural space surrounding peripheral nerves are continuous. Cerebrospinal fluid exerts a pressure of about 10 mm Hg, H2O, he said, while endoneural fluid is at about 1-2 mm Hg, “so there is a pressure gradient, and probably a slow proximal-distal flow of this fluid. We think this is how the vector moves from the intrathecal space towards the nerve.”


“This study is a major advance. I think it is an example of how gene replacement strategies are a rational path forward to treat autosomal recessive forms of CMT.”


After four weeks, 38 percent of Schwann cells surrounding lumbar roots and 17 percent of Schwann cells surrounding sciatic nerves were transfected and showed elevated expression of the transgene. At eight weeks post-injection, compared with mock vector-treated mice, mice treated with SH3TC2 lasted longer on a slowly turning rotarod, comparable with wild-type mice, and showed significant improvement (though not achieving wild-type levels) in performance on a faster rotarod, in foot grip strength, and in conduction along the sciatic nerve.

Compared with mock-treated mice, SH3TC2-treated mice had a decrease in the number of demyelinated fibers and increased thickness of myelin in both the anterior lumbar roots and the middle section of the sciatic nerves. Thickness in the lumbar roots of mock-treated animals averaged 0.766 micrometers, while in those receiving the transgene it averaged 1.053 micrometers. Similar improvement was noted in sciatic nerves.

At the nodes of Ranvier, the team found that treatment with the transgene improved the architecture of the nodes, narrowing the gap between adjacent Schwann cells to almost normal.

While CMT4C is thought of as a mainly demyelinating disease, a limited number of measurements in patients has shown an elevation in neurofilament light chain (Nfl) level, a marker of axonal degeneration. The mouse model also displays elevated Nfl, and Dr. Kleopa found that this elevation was reduced in those receiving SH3TC2 by about 39 percent compared with mock-treated mice.

One important result from the study, Dr. Kleopa said, was that benefit was seen even though the transfection rates of Schwann cells were limited. “This demonstrates that gene replacement therapy has potential for treating CMT4C.” However, he added, there are important steps that will need to be taken before contemplating human trials.

“In this model we treated very early in the course of the disease. This is still indicative because of early onset pathology in this model. However, one of the important issues to address is whether we can achieve an improvement in the phenotype even after the onset of neuropathy, since that will better match the human situation.”

Dr. Kleopa plans to test that in a future trial, with mice as old as four months. And he is also planning to test adeno-associated virus (AAV) as a vector, since it, unlike lentivirus, doesn't integrate into the host genome, and should therefore be safer.

“In this study, the most important thing we demonstrated is that there is a way to provide therapeutic benefit in this model, which is a very authentic and representative model of the disease,” Dr. Kleopa said. “I am confident that in the next couple of years we will make significant progress towards translation.”

Expert Commentary

“Gene replacement strategies have been very exciting for autosomal recessive conditions in neurology,” commented Dr. Shy of the University of Iowa. This has been especially true in spinal muscular atrophy, where replacement of the SMN gene “is really changing children's lives.”

The outcomes reported here are encouraging, Dr. Shy added, especially the morphological improvements in myelination. “If you are trying to treat a disease, it is very important to see actual structural changes in nerves that can explain the clinical benefits you observe. Here, the data are very convincing.

“So I think the approach is very exciting. However, there are a couple of challenges here that are a little different than for spinal muscular atrophy. The main one is that the disease is not caused by a problem with neurons, but with Schwann cells. These coat the entire length of the axons, which can be more than a meter long, and each Schwann cell only myelinates a segment of a few hundred micrometers. The challenge is to be able to get the replacement gene into enough Schwann cells to make a difference.”

In mice, the vector only needs to travel a few centimeters along the nerve; in humans, it will need to go many times as far.

Rhys Roberts, FRCP, PhD, commented that the results of the study “are potentially quite significant,” given that there are currently no treatments for CMT4C. Dr. Roberts is consultant neurologist at Addenbrooke's Hospital in Cambridge, UK, and director of the Cambridge Motor Neuron Disease Care Centre.

“Some degree of caution is always warranted, since there are many examples of improvements in mice not translating into humans. While there are a lot of technical steps that need to be optimized before this becomes a treatment in humans, I think it is an important proof of principle, and shows that there is a therapeutic potential worth pursuing.”


Drs. Kleopa, Roberts and Shy had no conflicts.

Link Up for More Information

• Schiza N, Georgiou E, Kagiava A, et al. Gene replacement therapy in a model of Charcot-Marie-Tooth 4C neuropathy Brain 2019; Epub 2019 Mar 25.