Targeting GNE Myopathy: A Dual Prodrug Approach for the Delivery of N-Acetylmannosamine 6-Phosphate.

ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs phosphate masking groups capable of providing more favorable druglike properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein, we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favorable ADME properties, while also being more effective than ManNAc at increasing sialic acid levels in GNE-deficient cell lines. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.

Novel GNE mutations in autosomal recessive hereditary inclusion body myopathy patients.

Hereditary Inclusion Body Myopathy (HIBM, IBM2, MIM:600737) is an autosomal recessive adult onset progressive muscle wasting disorder. It is associated with the degeneration of distal and proximal muscles, while often sparing the quadriceps. The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneunaminic acid (sialic acid). Affected individuals have been identified with mutations in the GNE gene. In the present study, the GNE coding region of 136 symptomatic patients were sequenced. A total of 41 patients were found to have GNE mutations. Eight novel mutations were discovered among seven patients. Of the eight novel mutations, seven were missense (p.I150V, p.Y186C, p.M265T, p.V315T, p.N317D, p.G669R, and p.S699L) and one was nonsense (p.W495X), all of which span the epimerase, kinase, and allosteric domains of GNE. In one patient, one novel mutation was found in the allosteric region and kinase domain of the GNE gene. Mutations in the allosteric region lead to a different disease, sialuria; however, this particular mutation has not been described in patients with sialuria. The pathological significance of this variation with GNE function remains unknown and further studies are needed to identify its connection with HIBM. These findings further expand the clinical and genetic spectrum of HIBM.

Serum neural cell adhesion molecule is hyposialylated in hereditary inclusion body myopathy.

Hereditary inclusion body myopathy (HIBM) is a young-adult onset autosomal recessive disorder caused by a hypomorphic rate limiting enzyme of sialic acid biosynthesis. The enzyme is UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, and is encoded by the GNE gene. HIBM causes slowly progressive muscle weakness and atrophy. Patients are typically diagnosed at 20-30 years of age, and most patients are incapacitated and wheelchair-confined by 30-50 years of age. Some sialic acid containing glycoproteins, including neural cell adhesion molecule (NCAM), are hyposialylated in HIBM muscle biopsy samples. We developed a method to allow detection of serum NCAM sialylation using Western blot, and tested serum samples from several patients and a HIBM mouse model. Preliminary results showed a clear difference in polysialylated and hyposialylated forms of NCAM extracted from serum, and showed NCAM is hyposialylated in HIBM serum samples. This initial finding may prove useful in reducing the need for serial muscle biopsies in HIBM treatment trials. Additional studies are underway to further validate this finding and to evaluate the specificity, reliability, and robustness of this potential serum biomarker for HIBM.