The goal of this project is to develop a gene-therapy treatment for Charcot-Marie-Tooth type 2D (CMT2D), an inherited axonal neuropathy caused by dominant mutations in glycyl tRNA synthetase (GARS). Our strategy is strongly supported by preliminary data and directly targets the mutant GARS transcripts, making it directly relevant to the disease mechanism. There are currently no treatments for CMT2D or any other inherited peripheral neuropathy, despite a cumulative incidence of 1:2500 people affected by these diseases and their relevance to the NINDS mission. Thus, there is a major unmet clinical need. There are at least 80 loci in the human genome that can cause CMT, suggesting there are many possible mechanisms, and a single drug or treatment is unlikely to work in all forms. Previous clinical and preclinical trials have focused on strategies such as promoting myelination or axonal transport to treat other forms of CMT, but there is no evidence that these processes underlie the pathophysiology of CMT2D. Our preliminary studies indicate that increasing GARS expression is not deleterious, but also does not mitigate the neuropathy. Instead, we found that an effective treatment strategy is to knockdown the mutant form of GARS, while preserving sufficient wild type expression for its essential function of charging glyince onto tRNAGly during translation. We have successfully done this using AAV9 to deliver allele-specific RNAi knockdown, selectively targeting mutant Gars transcripts. However, using this strategy requires that a new RNAi sequence be developed and tested for each mutant allele of GARS. Therefore, here we propose to further improve upon this already innovative approach by creating a single gene therapy vector that will knockdown any GARS transcript using RNAi, and simultaneously express an RNAi-resistant wild type GARS cDNA to maintain normal function. Although not without precedent, such an approach has not been previously tried for a dominant neuromuscular disease, and its success would have translational implications not just for CMT2D, but for a variety of peripheral neuropathy and motor neuron diseases. To develop and test this strategy, we propose the following aims in this phased R21/R33 proposal: In the R21 phase, Aim 1 will be to build a vector that expresses RNAi targeting GARS transcripts for degradation and expresses an RNAi-resistant wild type GARS cDNA. These sequences, which will work interchangeably in human and mouse studies, will be tested and optimized in vitro, and then packaged into AAV9 for in vivo testing.
In Aim 2, these vectors will be tested for efficacy in vivo in three existing mouse models of CMT2D. We will optimize timing, dosing, route of delivery, and outcome measures for these vectors in these models. In the R33 phase, the optimized vectors and study design will be use in appropriately powered and rigorously executed preclinical studies in the mouse models of CMT2D. The successful completion of these studies will position us for the next steps in translation, including pre-IND and IND applications and possible clinical trials and commercialization.

Public Health Relevance

Inherited peripheral neuropathies are debilitating neurological disorders for which there are currently no treatments. We are proposing a highly innovative gene therapy approach to treat Charcot-Marie-Tooth type 2D, a dominant axonal neuropathy caused by mutations in the GARS gene. This would be the first treatment for CMT2D, and would set an important precedent for using gene therapy to treat dominant neuromuscular diseases. The successful completion of these experiments will provide the preclinical data needed to approach FDA for an investigative new drug application, clinical partners for a possible trial in patients, and industry/biotech partners for possible commercialization of this approach.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
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National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
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Nuckolls, Glen H
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Jackson Laboratory
Bar Harbor
United States
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