Adenoassociated Virus-Mediated Transfer of a Functional Water Channel into Salivary Epithelial Cells In Vitro and In Vivo

Aquaporin 1 (AQP1) is the archetypal member of a family of integral membrane proteins that function as water channels. Previously we have shown that this protein can be expressed transiently from a recombinant adenovirus (AdhAQP1) in vitro in different epithelial cell lines, and in vivo in rat submandibular glands. In the present study we have constructed a recombinant adenoassociated virus (rAAV) containing the human aquaporin 1 gene (rAAVhAQP1). rAAVhAQP1 was produced at relatively high titers. ≈10¹¹–10¹² particles/ml and ≈10⁸–10⁹ transducing units/ml. We show that the rAAVhAQP1 can transduce in vitro four epithelial cell lines of different origins, at a level sufficient to detect the recombinant hAQP1 protein by either Western blot or confocal microscopic analysis. The recombinant hAQP1 was correctly targeted to the plasma membranes in all cell lines. Function of the recombinant hAQP1 was measured as fluid flow, in response to an osmotic gradient, across a monolayer of transduced epithelial cells. The data show that even at a low level of transduction, typically ≈10% of the cells in the monolayer, transepithelial fluid movement is enhanced about threefold above basal levels. In addition, we report that rAAVhAQP1 can transduce epithelial cells in the salivary glands and liver of mice in vivo. These results suggest that rAAVs may be useful gene transfer vectors to direct the production of functional transgenes in salivary epithelial cell types.

Overview summary

Previously, we have shown several potential clinical uses for gene transfer to salivary glands, using adenoviral vectors. However, adenovirus-mediated gene transfer leads to transient transgene expression and a significant host immune response. As an alternative means to deliver genes to salivary epithelial cells, we have begun to explore the use of recombinant adenoassociated viruses (rAAVs). In this article we describe the construction of an rAAV that directs the expression of functional aquaporin 1, in vitro and in vivo.