Sustained expression and safety of human GNE in normal mice after gene transfer based on AAV8 systemic delivery

Abstract

GNE myopathy is an autosomal recessive adult onset disorder caused by mutations in the GNE gene. GNE encodes the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase, the key enzyme in the biosynthesis pathway of sialic acid. Additional functions for GNE have been described recently, but the mechanism leading from GNE mutation to this myopathy is unclear. Therefore a gene therapy approach could address all potential defects caused by GNE mutations in muscle. We show that AAV8 viral vectors carrying wild type human GNE cDNA are able to transduce murine muscle cells and human GNE myopathy-derived muscle cells in culture and to express the transgene in these cells. Furthermore, the intravenous administration of this viral vector to healthy mice allows expression of the GNE transgene mRNA and of the coexpressed luciferase protein, for at least 6 months in skeletal muscles, with no clinical or pathological signs of focal or general toxicity, neither from the virus particles nor from the wild type human GNE overexpression. Our results support the future use of an AAV8 based vector platform for a safe and efficient therapy of muscle in GNE myopathy.

Introduction

Mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase encoding gene (GNE) lead to an adult onset recessive myopathy [1]. This progressive myopathy with typical distal onset and quadriceps sparing received different names in the past: hereditary inclusion body myopathy (HIBM) [2], quadriceps sparing myopathy [3], and distal myopathy with rimmed vacuoles (DMRV, Nonaka’s disease) [4]. Upon recently it has been agreed by the vast majority of researchers and clinicians dealing with this disease to designate this condition by a single name: GNE myopathy, in order to emphasize that all three commonly used names point to the same single entity: myopathy due to GNE mutations. This disease is common particularly in the Persian Jewish community (estimated prevalence of 1:1500), but was also described in non Jewish families from the Middle East (all carrying the same founder mutation, M712T) [5]. The condition has however a worldwide distribution, with most patients being compound heterozygotes, carrying mutations either at the epimerase domain, at the kinase domain, or one in each domain of the GNE gene [6]. It is particularly frequent in Japan with two founders and many other less frequent mutations involving again both domains of the gene [7].

Although the role of GNE as a key enzyme in the biosynthetic pathway of sialic acid has been thoroughly investigated [8], the process by which the mutations in the enzyme lead to muscle disease is not understood. The only animal model relevant to GNE myopathy is a transgenic mouse model generated on a GNE^-/- background and overexpressing the D176V GNE missense mutation occurring in the epimerase domain of the enzyme (a founder mutation in Japan) [9]. This mouse shows a strong hyposialylation of most organs in vivo, and recapitulates many histopathological features of GNE myopathy. Dietary supply of sialic acid or its metabolic intermediate ManNAc could partially prevent the development of this pathology, indicating that sialic acid deficiency is part of the disease pathomechanism [10]. Recently, additional functions have been ascribed to GNE in muscle cells, such as its binding properties to alpha actinin, its localization in the Z disk [11] and its role in cytoskeleton organization [12], suggesting that other pathways may be affected in the pathophysiology of GNE myopathy. If indeed other mechanisms contribute to the development of GNE myopathy, metabolic supplementation may not correct them all.

To address more comprehensively all the potential defects occurring in muscle tissue as the result of GNE mutations, we have taken a genetic approach as a mean to treat this disorder, and have used the adeno associated virus type 8 (AAV8) based system as a GNE delivery tool. Accumulating evidence points to recombinant AAV vectors being the most promising gene delivery tool for muscle diseases [13]. Furthermore a preclinical proof of efficacy of this treatment has been shown in the GNE myopathy mouse model [14]. In the present study we have evaluated the potential of the virus carrying the human wild type GNE gene to infect human GNE myopathy derived primary muscle cells, as well as its safety and extent of expression up to 6 months after intravenous administration to mice.