Engineering sialylated N-glycans on adeno-associated virus capsids for targeted gene delivery and therapeutic applications

Abstract

Glycans with diverse biological functions have been extensively identified on enveloped viruses, whereas glycosylation on adeno-associated virus (AAV) serotypes remains poorly understood. Identifying potential glycosylation sites on AAVs could provide critical docking sites for rational engineering of AAV capsids, enabling targeted delivery of therapeutic genes. This study presents a strategy that integrates azido-monosaccharide metabolic incorporation, 1,2-diamino-4,5-methylenedioxybenzene-labeled sialic acid analysis, and mass spectrometry to identify N-glycosylation sites and glycoforms on AAVs. We identified sialylated N- oligosaccharides, particularly the conserved NNNS motif, on AAV2, AAV6, AAV7, and AAV9 capsids. These glycans play critical roles in maintaining capsid stability and enhancing resistance to neutralizing antibodies. Furthermore, we engineered an AAV vector with an azido-labeled terminal sialic acid, which was conjugated via click chemistry to cyclic Arg-Gly-Asp (RGD), a high-affinity ligand for integrin αvβ3, to generate an integrin-targeted delivery vehicle. This approach enabled the efficient delivery of c-Met-targeting shRNA in a glioma mouse model and facilitated CRISPR/Cas9-mediated SMOC2 knockout in a mouse model of kidney fibrosis using single-guide RNA (sgRNA). Our findings establish a foundation for creating editable AAV vectors through sialylated termini, thereby expanding their potential applications in basic research and therapeutic development.