Influence of Cell Polarity on Retrovirus-Mediated Gene Transfer to Differentiated Human Airway Epithelia

Abstract

Gene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl− transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.

Infection of Human Airway Epithelia with H1N1, H2N2, and H3N2 Influenza A Virus Strains

“…Infection of epithelial cells by most viruses is polarized in terms of entry and release of virus particles (reviewed in (41)). We noted previously that entry of Moloney murine retrovirus vectors with amphotropic or xenotropic envelopes into human airway epithelia was restricted to the basolateral surface (21). In contrast, influenza virus infection resulted from binding to the apical plasma membrane ((32) and this study), similar to coronavirus (42,43) and measles virus (44).…”

“…In contrast, influenza virus infection resulted from binding to the apical plasma membrane ((32) and this study), similar to coronavirus (42,43) and measles virus (44). Researchers investigating viral vectors for application to the treatment of cystic fibrosis have shown that one of the major limitations to infection of airway epithelia from the apical side is receptor inaccessibility (21)(22)(23)(24). Recently, methods have been developed to transiently disrupt the tight junctions to allow envelope viruses access to all cell layers of the epithelia (45).…”

“…Millicell inserts were placed into 24-well plastic cell culture cluster (Costar, Cambridge, MA). Twenty-four hours after seeding, the mucosal medium was removed and the cells were allowed to grow at the air-liquid interface as reported previously (21). The culture medium, which consisted of a 1:1 mixture of Dulbecco's modified Eagle medium and Ham's F-12 with 2% Ultroser G (Sepracor, Inc., Marlborough, MA), 100 U of penicillin/ml, and 100 g of streptomycin/ml, was added to the wells to feed cells from the basolateral surface.…”

“…The most apical layer contains both ciliated and nonciliated cells. This model has been used to demonstrate the polarity of infection of recombinant retroviruses (21), recombinant adenoviruses (22,23), and recombinant adeno-associated viruses (24).…”

“…We first compared the abilities of X31 (H3N2), Japan (H2N2), and PR8 (H1N1) strains to infect cultures of primary human airway epithelia. This model culture system, in which human airway epithelia are cultured at the airliquid interface (18,22), has been invaluable in testing how other viruses can or cannot access intracellular compartments when applied to the mucosal surface (21,22,31). The titers of the strains tested were determined in MDCK cells, and SA-Gal specificities were confirmed on erythrocytes.…”