Assessment of Lectin Staining Biomarkers for GNE Myopathy Gene Therapy.

INTRODUCTION/

aimsGNE myopathy (GNEM) is a rare, autosomal recessive disorder caused by mutations in the UDP-N-acetylglucosamine (GlcNAc) 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE) gene, which encodes a required enzyme for sialic acid (SA) biosynthesis. Reduced SA production is associated with progressive skeletal muscle wasting, but robust molecular biomarkers of SA deficiency and therapeutic restoration remain lacking. As gene therapy for GNEM continues to advance, there is a critical need for biomarkers that enable early, objective assessment of therapeutic response that may precede detectable clinical improvements. Here, we systematically compare a panel of glycan-binding lectins to identify sensitive and robust biomarkers of SA deficiency and restoration in GNEM.

methodsWe evaluated a panel of glycan-binding lectins for differential binding under altered sialylation using fluorescent staining and flow cytometry in a GNE-deficient cell model, followed by fluorescent lectin staining of skeletal muscle biopsies from GNEM and non-GNEM patients.

resultsSubterminal lectins SBA, PNA, and VVA showed greater sensitivity to hyposialylation than terminal lectins. By flow cytometry, SBA and PNA increased 90.38- and 50.03-fold, respectively, in GNE-deficient cells and decreased 96.06% and 91.83% following GNE restoration. In GNEM muscle, PNA showed the greatest increase (5.3-fold) and most consistent discrimination from non-GNEM muscle. DISCUSSION: PNA demonstrated the most robust and consistent performance across models, supporting its promise as a biomarker to reflect restoration of sialylation that would occur in skeletal muscle after GNEM gene therapy. Further validation in clinical samples will establish its utility as a pharmacodynamic biomarker.