Glycosylation represents an important post-translation modification that occurs on proteins and lipids and contributes to their structural and functional properties. The outermost end of most glycan chains on (glyco)proteins are occupied by sialic acids. Sialic acids are 9 carbon acidic sugars which strongly influence the cell-cell and cell-matrix interactions. The UDP-N-acetylglucosamine-2-epimerase/N-acetyl-mannosamine kinase (GNE) catalyzes the first two steps of the sialic acid biosynthesis. Both enzyme activities are located on one polypeptide, therefore GNE represents a bifunctional enzyme. The epimerase activity of GNE underlies a strong feedback inhibition by CMP-Neuraminic 5-acid (Neu5Ac), the end-product of the metabolic pathway. Lack of GNE is lethal.

Mutations in the GNE gene generates GNE variants, which are involved in the development of the GNE myopathy (formerly hereditary inclusion body myopathy, HIBM), which is accompanied with a progressive amyotrophia during ageing. Prominently, proximal and distal skeletal muscles are affected. After two or three progressive decades, the patients are tightly restricted in their mobility. Many GNE mutations lead to decreased enzyme activity and therefore clinical trials tried to bypass the GNE defect by supplementation of the GNE products ManNAc or N-acetylneuraminic acid. Unfortunately, these clinical trials failed so far. One bottleneck of all GNE myopathy research is the lack of feasible enzymatic assays. Therefore the aim of this application is to develop a fast, reliable and robust assay based on thin layer chromatography (TLC), which would be an important piece for the development for further therapies of GNE myopathy including gene therapy.

As a gene therapy is in development to treat GNE myopathy, the goal of this project is to develop ways to quickly and accurately measure the effects of this therapy. This requires we find a biological indicator called a biomarker; in our case, a biomarker will measure the changes in muscle sialic acid levels produced by a successful GNE myopathy gene therapy. While the ultimate goal of a clinical trial is to find improvement of muscle function, biomarkers are particularly important for the Food and Drug Administration (FDA) to approve an investigational new drug (IND) application because biomarkers can detect biological changes happening in the muscle even before improvement of function can be measured. Our previous research tested potential biomarkers in samples of human muscle to compare their ability to detect changes that would happen in a clinical trial. However, before a gene therapy can go to clinical trial, it has to be shown to be effective and safe in a mouse model to allow its use in humans. Because of this, it is critical to make sure that any biomarker that we develop can work in both mouse pre-clinical trials and human clinical trials. Our project proposes to compare the ability of our potential biomarkers to detect how GNE gene therapy changes sialic acid levels in the muscle of existing mouse models of GNE myopathy. We will also expand on our previous studies to confirm that these biomarkers also function in human muscle cells.

Adeno-associated viral vector (AAV)-based gene therapy is in development for several genetic muscular diseases, and one AAV gene therapy for a muscle disease has already been approved by the FDA. These therapies require large doses of AAV, which comes with safety concerns. This proposal aims to develop an AAV gene therapy for GNE myopathy, whereby the AAV vector is repurposed to deliver an unmutated version of the GNE gene to muscles, with a particular focus on reducing the dose of AAV required. To do that, we are developing a novel expression cassette that will lead to high levels of both GNE and glycoprotein sialylation in skeletal muscles throughout the body. By replacing the mutated enzyme (GNE) as well as the downstream products of that enzyme (glycoprotein sialylation), we aim to treat the underlying disease pathology with a safe and effective gene therapy. This project will specifically test three such gene therapies, the best of which will be tested in a dose-finding study in a future funding cycle. The data generated by this proposal and a subsequent dose-finding study can be used to support an Investigational New Drug application to the FDA in preparation for a GNE myopathy gene therapy clinical trial.

The complete function of the GNE gene in humans remains poorly understood, particularly with respect to how amino acid changes caused by missense mutations can lead to a body-wise muscle pathology. In this grant, we propose to design a biological assay to better understand the impact of GNE patient mutations on the gene’s function. This assay can be used to help classify patient mutations in GNE that are currently classified as ‘VUS’ (variants of uncertain significance); which would help towards providing a definitive genetic diagnosis to enable these patients to participate in future clinical trials (e.g. gene therapy).

GNE myopathy is a muscle disease where a mutation in the GNE gene gives rise to progressive muscle weakness and loss that has a major impact on patients’ quality of life. Our laboratory is interested in developing gene therapies for the treatment of GNE myopathy. These treatments use Adeno Associated Virus (or AAV) as a DNA delivery device to place a normal copy of the GNE gene into cells, thereby providing normal GNE gene function back to the patient. This type of gene therapy could be used to treat all patients with GNE myopathy, regardless of their particular genetic mutation. Once given, the gene therapy delivered can last for decades, perhaps a lifetime, in the patient. The current grant proposal seeks to build and test gene therapy in a new mouse model of GNE myopathy where the mouse GNE gene can be deleted in the adult animal. The mouse GNE gene is essential for development, so only deletion of the gene in the adult will allow for animals to be born. Such a new disease model is required to help investigators define the therapeutic potential of gene therapies and also drug-based therapies for GNE myopathy. It is also needed to define the dose of therapy required to correct the disease. Current models of GNE myopathy have problems that make translational research for this disease difficult. Making and testing of this new model should greatly speed needed research for this disease.

GNE Myopathy is a neuromuscular adult onset disease characterized by slowly progressive muscle weakness, caused by recessive mutations in the GNE gene. Although the function of this protein is well known, the mechanism of the disease is still not clear. To date, no reliable animal model is available for the disease, and cellular models from GNE Myopathy patients’ muscle cells (expressing the mutated protein) are less informative than expected. Therefore, we reason that models where the effect of the GNE mutation could be more dramatic may be useful to determine additional function (s) of GNE specifically in muscle. In a system where the GNE protein is totally absent there is a better chance to see changes in the processes related to the GNE gene. We propose to establish cellular models of mouse and human muscle cells that completely lack GNE expression. For this purpose we will use the Sol8 mouse muscle lineage, and two primary genuine muscle cells derived from GNE Myopathy patients muscle biopsies from our unique cell bank. By the Crispr/Cas9 methodology we will knock out the GNE gene. We will then determine the changes at various levels in order to recognize the biological and molecular pathways affected in the GNE knocked out cells and define specific biomarkers. These cells could then be treated by AAVGNE gene therapy to evaluate its potential benefits in restoring the normal affected features.

GNE Myopathy is a unique neuromuscular adult onset disease characterized by slowly progressive distal and proximal muscle weakness, caused by recessive mutations in GNE. Although the function of this protein is known to be a key enzyme in the biosynthesis of sialic acid, no clear definite explanation has been provided to account for the muscle atrophic pathology. The lack of animal models severely impair the comprehensive understanding of GNE function, and, more importantly, prevents the development of an efficient system to assess any kind of treatment, and in particular an AAV platform based GNE gene therapy. The goal of this study is to develop critical tools for this purpose. Since the lack of GNE in the entire organism results in the death of the embryo before it is born, we will develop a mouse where GNE can be knocked out only in muscle, so the mice can be born and we can see the effect of the lack of GNE in muscle. By the CRISPR/Cas9 genome editing method, we have already generated the first step towards this goal, and want to continue towards the second and final steps. Once established, these mice will be used as a system to rescue the muscle pathology by AAV-GNE gene therapy.

Developing drugs from the very beginning, especially for diseases with small number of patients, is extremely difficult. In contrast, the repositioning of approved drugs, whose safety information is already available, will allow us to bypass the slow drug developing process even for extremely rare diseases. The enzymatic activity of GNE is known to be activated by specific allosteric site mutations, raising a possibility that even the GNE with GNE- myopathy-causing mutations may possibly be activated by the modification of such allosteric sites. Our computationally predicted molecular model of GNE indicates the presence of a deep narrow cavity. Filling this cavity by a small compound is likely to inhibit the binding of CMP-NeuAc to the allosteric site, leading to the activation of the GNE.

In this study, we therefore aim to find approved small molecular agents that can fit the cavity and activate the GNE with either p.D207V in epimerase domain or p.V603L in kinase domain, which are the two most common mutations among Japanese GNE myopathy patients, by in silico drug screening based on binding affinity. We will then narrow down the candidates by measuring GNE enzyme activity and confirm the efficacy of drugs using patients’ cells including mutations frequent in other ethnic populations.

We propose the repositioning of approved drugs using in-silico approach is a fast, an effective and realistic method to treat GNE myopathy, which is the mission of NDF.