564. Correction of Hyposialylation in Hereditary Inclusion Body Myopathy, by Ex-Vivo Therapy

Abstract

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive disorder that is characterized by a late onset of muscle weakness in upper and lower limbs with sparing of quadriceps and accumulation of vacuoles and amyloid deposits in muscle tissues. Recently, HIBM was associated with mutations of the UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE) gene, located on chromosome 9. This bi-functional enzyme is responsible of the synthesis and regulation of sialic acid, the most common monosaccharide in glycoconjugated molecules of eukaryotes. It is known that sialic acids are important in a lot of cellular functions. Mutations have been identified in both domains of GNE, and in compound heterozygous form. One of the most prevalent mutations in HIBM (M712T) is located in the kinase domain of GNE. The presence of this mutation reduce the GNE enzymatic activity and thus the sialylation in muscles. Although reduced activity is found among mutated GNE gene, the process that causes muscular pathology is not yet understood. There is probability inadequate sialylation or hyposialylation of important functional proteins in HIBM muscles that causes the pathology.To investigate possible therapies of HIBM a knock-in mouse model has been produced by Dr. D. Darvish (Encino, CA). The model contain a GNE gene with the M712T mutation frequently found in HIBM patients. A possible therapy of HIBM is to restore in muscle fibers a normal GNE activity by gene therapy. This could be done by genetically modifying the patient own myoblasts before an autologous transplantation.Myoblasts were grown from normal mouse muscle biopsies and from homozygous knock-in M172T GNE mice. The level of sialylation of these myoblasts was determined by flowcytometry following labelling of the cells with FITC coupled lectins (Limax flavus lectin LFA or MAA). The level of sialylation was found to be inferior on the homozygous M712T mouse myoblasts. We then produced SIN lentiviral vector containing either the normal GNE gene or the mutated M712T GNE gene. The homozygous M712T mouse myoblasts were infected or not with a lentivirus containing the normal GNE gene or the M712T mutated gene. The cells were then selected and proliferated. The level of sialylation was increased following the viral infection with both viral vectors. The level of sialylation was increased up to 2 folds by the lentivirus containing the normal GNEgene.A 1.3 to 1.5 increased level of sialylation was also observed following lentiviral infection of myoblasts grown from a muscle biopsy of an HIBM patient also affected by the M712T mutation.In the future, intramuscular transplantation of the genetically modified myoblasts will be made in the muscles of homozygous M712T mice.Major stock holder in CellGene Inc.