Researchers have successfully used adeno-associated virus-mediated gene therapy to treat mice that replicated a novel mutation found in a Charcot-Marie-Tooth Type 2D patient with severe disease.
The study describing the scientists’ work, “Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models,” was published in the Journal of Clinical Investigation.
DNA is constantly subject to changes known as mutations. By changing a gene’s instructions for making a protein, a mutation can cause the protein to malfunction or to be absent entirely. As a result, mutations sometimes lead to disease. Mutations can be called recessive or dominant, depending on inheritance patterns. Dominant mutations mean disease can develop if the genetic change is present in only one of the copies of the gene. (Every person has two copies of each gene, one inherited from each parent.)
Applying gene therapy to dominant mutation-based conditions is not easy; it requires scientists to “molecularly silence” the mutated part of the gene without altering the works of the other normal (non-mutated) copy of that gene.
“However, the feasibility of gene silencing therapy to treat dominantly-inherited, mono-allelic mutations — where treatment may require reduction in the expression [levels] of a toxic mutant protein resulting from a gain-of-function (GoF) allele—is unclear,” researchers said.
Knowing this, investigators at the University of Maine developed a mouse model of Charcot-Marie-Tooth Disease Type 2D, a type of dominantly inherited neuropathy (nerve disease) , caused by a novel mutation in the glycyl tRNA-synthetase (GARS) gene. Importantly, this mutation was replicated specifically because it was observed in an infant patient with severe peripheral neuropathy, a nerve condition that causes reduced sensation, tingling, weakness, or pain in nerves of the hands, feet, legs, and arms.
Scientists set out to decrease the levels of mutant GARS protein using a technique called RNA interference (RNAi), which ultimately blocks gene expression — the process by which the genetic code of a gene is used to create a working product, like a protein.
RNA interference was engineered to target only mutant GARS messenger RNA – i.e., the molecules that carried GARS’ abnormal genetic information. After that, RNAi molecules were packed in an adeno-associated virus serotype 9 (AAV9), a virus that does not cause disease. However, due to its viral features it can easily enter (“infect”) cells and deliver its content, ultimately altering the genetic makeup of the “infected” cells.
The investigators’ gene therapy strategy almost completely prevented the neuropathy in mice treated via intracerebroventricular (ICV) injection (into the brain) at birth by reducing the speed of nerve cell degeneration, which manifested itself as increased grip strength, greater muscle percentage, and better nerve function, compared to control-treated (non-treated) animals.
In another batch of mice, AAV9-mediated treatment was administered into the lumbar spinal cord of 5- and 9-week-old mutant and non-mutant mice. At 3–4 weeks of age (corresponding to 14 human years), animals had developed neuropathy symptoms.
“Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed,” the researchers reported.
When left untreated, 5-week-old mice undergo active nerve fiber loss, while axon loss slows and muscle atrophy speeds up in 9-week-old animals. In comparison to untreated mice, animals treated at 5 weeks of age had their grip strength and nerve function improved, nerve loss rates reduced, and body weight increased starting at five weeks after treatment. Interestingly, there was no improvement in axon size compared to untreated animals. Of note, as the neurodegenerative process progresses, nerve fiber (axon) diameter tends to decrease.
The same therapeutic effects were observed in a second mouse model of Charcot-Marie-Tooth Type 2D treated with the same gene therapy approach. Importantly, the observed benefits were dose dependent, greater with ICV injection than systemic (into the blood), and persisted for at least one year.
The team concluded its findings support the feasibility of virally delivered RNA interference for target-specific silencing as a treatment strategy for dominant neuropathies caused by mutations.
“In summary, these studies demonstrate how precision animal models can be used for testing personalized therapies for rare and orphan diseases, and provide important proof-of-concept for RNAi-based gene therapy for this dominant disease. This approach could be applied to other, related disorders including other dominantly inherited peripheral neuropathies or motor neuron diseases, ” the authors wrote.