부산대학교 도서관

GNE myopathy (GNEM) is a severe muscle disease caused by mutations in the UDP-GlcNAc-2-epimerase/ManNAc-6-kinase (GNE) gene, which encodes a bifunctional enzyme required for sialic acid (Sia) biosynthesis. To develop assays to demonstrate the potency of AAV gene therapy vectors in making Sia and to define the dose required for replacement of endogenous mouse Gnegene expression with human GNEin skeletal muscles. A MyoD-inducible Gne-deficient cell line, Lec3MyoDI, and a GNE-deficient human muscle cell line, were made and tested to define the potency of various AAV vectors to increase binding of Sia-specific lectins, including MAA and SNA. qPCR and qRT-PCR methods were used to quantify AAV biodistribution and GNEgene expression after intravenous delivery of AAV vectors designed with different promoters in wild-type mice. Lec3 cells showed a strong deficit in MAA binding, while GNE–/–MB135 cells did not. Overexpressing GNEin Lec3 and Lec3MyoDIcells by AAV infection stimulated MAA binding in a dose-dependent manner. Use of a constitutive promoter, CMV, showed higher induction of MAA binding than use of muscle-specific promoters (MCK, MHCK7). rAAVrh74.CMV.GNEstimulated human GNEexpression in muscles at levels equivalent to endogenous mouse Gneat a dose of 1×1013vg/kg, while AAVs with muscle-specific promoters required higher doses. AAV biodistribution in skeletal muscles trended higher when CMV was used as the promoter, and this correlated with increased sialylation of its viral capsid. Lec3 and Lec3MyoDIcells work well to assay the potency of AAV vectors in making Sia. Systemic delivery of rAAVrh74.CMV.GNEcan deliver GNEgene replacement to skeletal muscles at doses that do not overwhelm non-muscle tissues, suggesting that AAV vectors that drive constitutive organ expression could be used to treat GNEM.