Induced muscle and liver absence of Gne in postnatal mice does not result in structural or functional muscle impairment.

Background: GNE Myopathy is a unique recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, caused by mutations in the GNE gene which is a key enzyme in the biosynthesis of sialic acid. To date, the precise pathophysiology of the disease is not well understood and no reliable animal model is available. Gne KO is embryonically lethal in mice. Objective: To gain insights into GNE function in muscle, we have generated an inducible muscle Gne KO mouse. To minimize the contribution of the liver to the availability of sialic acid to muscle via the serum, we have also induced combined Gne KO in liver and muscle. Methods: A mouse carrying loxp sequences flanking Gne exon3 was generated by Crispr/Cas9 and bred with a human skeletal actin (HSA) promoter driven CreERT mouse. Gne muscle knock out was induced by tamoxifen injection of the resulting homozygote GneloxpEx3loxp/HSA Cre mouse. Liver Gne KO was induced by systemic injection of AAV8 vectors carrying the Cre gene driven by the hepatic specific promoter of the thyroxine binding globulin gene. Results: Characterization of these mice for a 12 months period showed no significant changes in their general behaviour, motor performance, muscle mass and structure in spite of a dramatic reduction in sialic acid content in both muscle and liver. Conclusions: We conclude that post weaning lack of Gne and sialic acid in muscle and liver have no pathologic effect in adult mice. These findings could reflect a strong interspecies versatility, but also raise questions about the loss of function hypothesis in Gne Myopathy. If these findings apply to humans they have a major impact on therapeutic strategies.

Generation and characterization of a novel gne Knockout Model in Zebrafish.

GNE Myopathy is a rare, recessively inherited neuromuscular worldwide disorder, caused by a spectrum of bi-allelic mutations in the human GNE gene. GNE encodes a bi-functional enzyme responsible for the rate-limiting step of sialic acid biosynthesis pathway. However, the process in which GNE mutations lead to the development of a muscle pathology is not clear yet. Cellular and mouse models for GNE Myopathy established to date have not been informative. Further, additional GNE functions in muscle have been hypothesized. In these studies, we aimed to investigate gne functions using zebrafish genetic and transgenic models, and characterized them using macroscopic, microscopic, and molecular approaches. We first established transgenic zebrafish lineages expressing the human GNE cDNA carrying the M743T mutation, driven by the zebrafish gne promoter. These fish developed entirely normally. Then, we generated a gne knocked-out (KO) fish using the CRISPR/Cas9 methodology. These fish died 8-10 days post-fertilization (dpf), but a phenotype appeared less than 24 h before death and included progressive body axis curving, deflation of the swim bladder and decreasing movement and heart rate. However, muscle histology uncovered severe defects, already at 5 dpf, with compromised fiber organization. Sialic acid supplementation did not rescue the larvae from this phenotype nor prolonged their lifespan. To have deeper insights into the potential functions of gne in zebrafish, RNA sequencing was performed at 3 time points (3, 5, and 7 dpf). Genotype clustering was progressive, with only 5 genes differentially expressed in gne KO compared to gne WT siblings at 3 dpf. Enrichment analyses of the primary processes affected by the lack of gne also at 5 and 7 dpf point to the involvement of cell cycle and DNA damage/repair processes in the gne KO zebrafish. Thus, we have established a gne KO zebrafish lineage and obtained new insights into gne functions. This is the only model where GNE can be related to clear muscle defects, thus the only animal model relevant to GNE Myopathy to date. Further elucidation of gne precise mechanism-of-action in these processes could be relevant to GNE Myopathy and allow the identification of novel therapeutic targets.

In vivo and in vitro genome editing to explore GNE functions.

GNE myopathy is an adult onset neuromuscular disorder characterized by slowly progressive distal and proximal muscle weakness, caused by missense recessive mutations in the GNE gene. Although the encoded bifunctional enzyme is well known as the limiting factor in the biosynthesis of sialic acid, no clear mechanisms have been recognized to account for the muscle atrophic pathology, and novel functions for GNE have been hypothesized. Two major issues impair studies on this protein. First, the expression of the GNE protein is minimal in human and mice muscles and there is no reliable antibody to follow up endogenous expression. Second, 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. In order to broaden our knowledge on GNE functions in muscle, we have taken advantage of the CRISPR/Cas9 method for genome editing to first, add a tag to the endogenous Gne gene in mouse, allowing the determination of the spatiotemporal expression of the protein in the organism, using well established and reliable antibodies against the specific tag. In addition we have generated a Gne knock out murine muscle cell lineage to identify the events resulting from the total lack of the protein. A thorough multi-omics analysis of both cellular systems including transcriptomics, proteomics, phosphoproteomics and ubiquitination, unraveled novel pathways for Gne, in particular its involvement in cell cycle control and in the DNA damage/repair pathways. The elucidation of fundamental mechanisms of Gne in normal muscle may contribute to the identification of the disrupted functions in GNE myopathy, thus, to the definition of novel biomarkers and possible therapeutic targets for this disease.

Pre Clinical Assessment of AAVrh74.MCK.GNE Viral Vector Therapeutic Potential: Robust Activity Despite Lack of Consistent Animal Model for GNE Myopathy.

BACKGROUND: GNE myopathy is a unique adult onset rare neuromuscular disease caused by recessive mutations in the GNE gene. The pathophysiological mechanism of this disorder is not well understood and to date, there is no available therapy for this debilitating disease. We have previously established proof of concept that AAV based gene therapy can effectively deliver the wild type human GNE into cultured muscle cells from human patients and in mice, using a CMV promoter driven human wild type GNE plasmid delivered through an adeno associated virus (AAV8) based platform. OBJECTIVE: In the present study we have generated a muscle specific GNE construct, driven by the MCK promoter and packaged with the AAVrh74 serotype for efficacy evaluation in an animal model of GNE Myopathy. METHODS: The viral vector was systemically delivered at 2 doses to two age groups of a Gne-/- hGNED207V Tg mouse described as a preclinical model of GNE Myopathy, and treatment was monitored for long term efficacy. RESULTS: In spite of the fact that the full described characteristics of the preclinical model could not be reproduced, the systemic injection of the rAAVrh74.MCK.GNE viral vector resulted in a long term presence and expression of human wt GNE in the murine muscles and in some improvements of their mild phenotype. The Gne-/- hGNED207V Tg mice are smaller from birth, but cannot be differentiated from littermates by muscle function (grip strength and Rotarod) and their muscle histology is normal, even at advanced age. CONCLUSIONS: The rAAVrh74.MCK.GNE vector is a robust tool for the development of GNE Myopathy therapies that supply the intact GNE. However, there is still no reliable animal model to fully assess its efficacy since the previously developed Gne-/- hGNED207V Tg mice do not present disease characteristics.

Upregulation of Hallmark Muscle Genes Protects GneM743T/M743T Mutated Knock-In Mice From Kidney and Muscle Phenotype.

BACKGROUND: Mutations in GNE cause a recessive, adult onset myopathy characterized by slowly progressive distal and proximal muscle weakness. Knock-in mice carrying the most frequent mutation in GNE myopathy patients, GneM743T/M743T, usually die few days after birth from severe renal failure, with no muscle phenotype. However, a spontaneous sub-colony remains healthy throughout a normal lifespan without any kidney or muscle pathology. OBJECTIVE: We attempted to decipher the molecular mechanisms behind these phenotypic differences and to determine the mechanisms preventing the kidney and muscles from disease. METHODS: We analyzed the transcriptome and proteome of kidneys and muscles of sick and healthy GneM743T/M743T mice. RESULTS: The sick GneM743T/M743T kidney was characterized by up-regulation of extra-cellular matrix degradation related processes and by down-regulation of oxidative phosphorylation and respiratory electron chain pathway, that was also observed in the asymptomatic muscles. Surprisingly, the healthy kidneys of the GneM743T/M743T mice were characterized by up-regulation of hallmark muscle genes. In addition the asymptomatic muscles of the sick GneM743T/M743T mice showed upregulation of transcription and translation processes. CONCLUSIONS: Overexpression of muscle physiology genes in healthy GneM743T/M743T mice seems to define the protecting mechanism in these mice. Furthermore, the strong involvement of muscle related genes in kidney may bridge the apparent phenotypic gap between GNE myopathy and the knock-in GneM743T/M743T mouse model and provide new directions in the study of GNE function in health and disease.

The Interaction of UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) and Alpha-Actinin 2 Is Altered in GNE Myopathy M743T Mutant.

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the gene mutated in GNE myopathy. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, the interaction of GNE with α-actinins has been investigated. Surface plasmon resonance and microscale thermophoresis analysis revealed, that in vitro, GNE interacts with α-actinin 2, and that this interaction has a 10-fold higher affinity compared to the GNE-α-actinin 1 interaction. Further, GNE carrying the M743T mutation, the most frequent mutation in GNE myopathy, has a 10-fold lower binding affinity to α-actinin 2 than intact GNE. It is possible that this decrease eventually affects the interaction, thus causing functional imbalance of this complex in skeletal muscle that could contribute to the myopathy phenotype. In vivo, using bi-molecular fluorescent complementation, we show the specific binding of the two proteins inside the intact cell, in a unique interaction pattern between the two partners. This interaction is disrupted in the absence of the C-terminal calmodulin-like domain of α-actinin 2, which is altered in α-actinin 1. Moreover, the binding of GNE to α-actinin 2 prevents additional binding of α-actinin 1 but not vice versa. These results suggest that the interaction between GNE and α-actinin 1 and α-actinin 2 occur at different sites in the α-actinin molecules and that for α-actinin 2 the interaction site is located at the C-terminus of the protein.

Survival-apoptosis associated signaling in GNE myopathy-cultured myoblasts.

GNE Myopathy (GNEM) is a neuromuscular disorder caused by mutations in the GNE gene. It is a slowly progressive distal and proximal muscle weakness sparing the quadriceps. In this study, we applied our model of mutated M743T GNE enzyme skeletal muscle-cultured myoblasts and paired healthy controls to depict the pattern of signaling proteins controlling survival and/or apoptosis of the PI3K/AKT, BCL2, ARTS/XIAP pathways, examined the effects of metabolic changes/stimuli on their expression and activation, and their potential role in GNEM. Immunoblot analysis of the GNEM myoblasts indicated a notable increased level of activated PTEN and PDK1 and a trend of relative differences in the expression and activation of the examined signaling molecules with variability among the cultures. ANOVA analysis showed a highly significant interaction between the level of PTEN and the patients groups. In parallel, the interaction between the level of BCL2, BAX and PTEN with the specific PI3K/AKT inhibitor-LY294002 was highly significant for BCL2 and nearly significant for PTEN and BAX. The pattern of the ARTS/XIAP signaling proteins of GNEM and the paired controls was variable, with no significant differences between the two cell types. The response of the GNEM cells to the metabolic changes/stimuli: serum depletion and insulin challenge, as indicated by expression of selected signaling proteins, was variable and similar to the control cells. Taken together, our observations provide a clearer insight into specific signaling molecules influencing growth and survival of GNEM muscle cells.

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

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 6months 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.