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Apical barriers to airway epithelial cell gene transfer with amphotropic retroviral vectors

Gene Therapy volume 9, pages 922–931 (2002)Cite this article

Abstract

Gene transfer to airway epithelia with amphotropic pseudotyped retroviral vectors is inefficient following apical vector application. To better understand this inefficiency, we localized the expression of Pit2, the amphotropic receptor, in polarized human airway epithelia. Pit2 was expressed on both the apical and basolateral surfaces of the cells, suggesting that factors other than receptor abundance may limit apical gene transfer efficiency. Binding studies performed with radiolabeled amphotropic MuLV suggested that the apically applied virus binds to Pit2. Hypothetical barriers to retroviral gene transfer include the apical glycocalyx and other secreted products of epithelia. In this study, we demonstrated that sialic acid, keratan sulfate and collagen type V are present on the apical surface of well-differentiated human airway epithelia. While enzyme treatment reduced the abundance of these components, the treatment also decreased the transepithelial resistance to ~35% of the controls, suggesting that the epithelial integrity was impaired. To attain an airway epithelial culture with a modified apical surface and intact epithelial integrity, we utilized 100 mM 2-deoxy-D-glucose, a glycosylation inhibitor, to prevent the glycocalyx from reforming following enzyme treatment. This approach allowed the resistance, but not the apical glycocalyx to recover. Despite this physical modification of the cell surface, the amphotropic retroviral vector failed to transduce airway epithelia following apical application. These results suggest that factors other than apical receptor abundance and the glycocalyx inhibit amphotropic retroviral gene transfer in human airway epithelia.

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Acknowledgements

The authors gratefully acknowledge the technical expertise of Phil Karp and Pary Weber of the University of Iowa Cell Culture Core in preparing the human airway epithelial cultures. We thank Dr Yubin Kang for his helpful discussions. This work was supported in part by grants from the Cystic Fibrosis Foundation (GW99G0) and NIH (R01 HL61460 and P50 HL51670, BLD and PBM). We acknowledge the support of the Morphology Core and Gene Transfer Vector Core, partially supported by the Cystic Fibrosis Foundation, NHLBI (NIH P50 HL51670), and the Center for Gene Therapy for Cystic Fibrosis and other Genetic Diseases (NIH P30 DK54759).

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Author notes

  1. G Wang

    Present address: DepartmentS of Medicine and Genetics, Louisiana State University Health Sciences Center, Gene Therapy Program, 533 Bolivar St, CSB 642, New Orleans, 70112, LA, USA

Authors and Affiliations

  1. Department of Pediatrics, Program in Gene Therapy, University of Iowa College of Medicine, Iowa City, IA, USA

    G Wang, G Williams, H Xia, M Hickey & PB McCray

  2. Department of Internal Medicine, Program in Gene Therapy, University of Iowa College of Medicine, Iowa City, IA, USA

    BL Davidson

  3. Central Microscopy Research Facility, University of Iowa College of Medicine, Iowa City, IA, USA

    J Shao

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Wang, G., Williams, G., Xia, H. et al. Apical barriers to airway epithelial cell gene transfer with amphotropic retroviral vectors. Gene Ther 9, 922–931 (2002). https://doi.org/10.1038/sj.gt.3301714

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