Glycocalyx Restricts Adenoviral Vector Access to Apical Receptors Expressed on Respiratory Epithelium In Vitro and In Vivo: Role for Tethered Mucins as Barriers to Lumenal Infection

Abstract

Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.

Mucin impedes cytotoxic effect of 5-FU against growth of human pancreatic cancer cells: overcoming cellular barriers for therapeutic gain

“…The overexpression of MUC1 mucin has been shown to sterically hinder cell -cell and cell -matrix interactions (Wesseling et al, 1996). Moreover, the dense O-glycosylation on MUC1 can mask several cell surface receptors shown to reduce transfection efficiency (Stonebraker et al, 2004).…”

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

“…Efficient gene transfer to the mature respiratory epithelium presents a formidable challenge due to a spectrum of obstacles, including airway mucous and cilia, extracellular glycoproteins (that is glycocalyx) and pulmonary macrophages. [1][2][3][4][5][6] Another significant obstacle to viral vector gene transfer is the gradual loss of lung transduction due to constant turnover of differentiated respiratory epithelium. 7 Transduction of lung epithelial stem/progenitor cells using adeno-associated virus (AAV) can achieve sustained levels (up to 6 months) of transgene expression; however, chemical injury of the lung is necessary before vector administration.…”

Activators of viral gene expression in polarized epithelial monolayers identified by rapid-throughput drug screening

“…to adenoviral gene transfer, form polar monolayers under the growth conditions described here 22 and reproduce many features of epithelial refractoriness to adenoviral gene transfer described previously in cultured cells and murine models in vivo [22][23][24][25][26][27][28][29] (see also below). Figure 1 (upper panel) depicts the general approach and assay layout.…”