Preclinical Assessment of wt GNE Gene Plasmid for Management of Hereditary Inclusion Body Myopathy 2 (HIBM2)

Chris Jay,John Eager,Daniel Darvish,Phillip B Maples,Stephan Hinderlich,Neil Senzer,Gregory Nemunaitis,John Nemunaitis,Alex Tong,Julie Ogden

doi:10.4137/grsb.s728

Abstract

Hereditary Inclusion Body Myopathy (HIBM2) is a chronic progressive skeletal muscle wasting disorder which generally leads to complete disability before the age of 50 years. There is currently no effective therapeutic treatment for HIBM2. Development of this disease is related to expression in family members of an autosomal recessive mutation of the GNE gene, which encodes the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK). This is the rate limiting bifunctional enzyme that catalyzes the first 2 steps of sialic acid biosynthesis. Decreased sialic acid production, consequently leads to decreased sialyation of a variety of glycoproteins including the critical muscle protein alpha-dystroglycan (alpha-DG). This in turn severely cripples muscle function and leads to the onset of the syndrome. We hypothesize that replacing the mutated GNE gene with the wildtype gene may restore functional capacity of GNE/MNK and therefore production of sialic acid, allowing for improvement in muscle function and/or delay in rate of muscle deterioration. We have constructed three GNE gene/CMV promoter plasmids (encoding the wildtype, HIBM2, and Sialuria forms of GNE) and demonstrated enhanced GNE gene activity following delivery to GNE-deficient CHO-Lec3 cells. GNE/MNK enzyme function was significantly increased and subsequent induction of sialic acid production was demonstrated after transfection into Lec3 cells with the wild type or R266Q mutant GNE vector. These data form the foundation for future preclinical and clinical studies for GNE gene transfer to treat HIBM2 patients.

Highlights

Hereditary Inclusion Body Myopathy 2 (HIBM2) is an autosomal recessive muscle wasting syndrome with its onset between the ages of 20 and 40 years of age progressing to severe incapacitation within 5 to 15 years (Argov and Yarom, 1984)
GNE is a highly conserved gene identified as 1 of 40 genes exclusively shared by vertebrates and bacteria (Salzberg, White et al 2001). It encodes the bifunctional enzyme uridine diphosphate-N-acetylglucosamine 2—epimerase/N-acetylmannosamine kinase (GNE/MNK) which catalyzes the first two sequential sialic acid pathway reactions critical to sialic acid synthesis, and HIBM2 mutations are distributed over the whole enzyme (Argov, Eisenberg et al 2003; Krause, Schlotter-Weigel et al 2003; Broccolini, Ricci et al 2004; Huizing, Rakocevic et al 2004; Saito, Tomimitsu et al 2004)
The resulting chromatograms were compared against the GNE sequence from GenBank and the wild type had no mutations, whilst the M712T and R266Q clones contained only the expected point mutations (Sup Fig. 1)

Summary

Introduction

Hereditary Inclusion Body Myopathy 2 (HIBM2) is an autosomal recessive muscle wasting syndrome with its onset between the ages of 20 and 40 years of age progressing to severe incapacitation within 5 to 15 years (Argov and Yarom, 1984). Sialic acid dysregulation is likely to significantly contribute to disease pathogenesis, recent assessments of myoblast cellular sialylation patterns suggest the possible role of other GNE related contributing mechanisms, such as GM3 and GD3 synthase regulation and impaired apoptosis signalling It is published under the Creative Commons Attribution By licence.

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