Gene Delivery to the Airway

Model systems of differentiated airway epithelium have played a significant role in research pertaining to airway biology, pathophysiology, and gene therapy. The success of such systems is dependent on the ability to reconstitute the native cellular composition and architecture of the airway, within a setting that retains adequate flexibility for experimental manipulation. Furthermore, human airway models have provided significant advantages over other models, since the cell biology of the airway epithelium of humans can differ substantially in function and cellular composition from that of other species such as mice and rats. For example, the predominant secretory cell type in humans is the goblet cell, whereas in mice and rats it is the Clara and serous cell, respectively. Another important consideration is the marked variation observed in the tropism of recombinant viruses commonly used for gene therapy (e.g., adeno-associated virus) with recipient cells from different species. One of the most widely used human systems to date consists of polarized monolayers of primary airway epithelial cells grown on permeable membrane supports (Karp et al., 2002; Randell et al., 2011; Yamaya et al., 1992). For many studies, this system has provided adequate differentiation when cells are grown at the air-liquid interface. However, the extent of mucociliary differentiation in this experimental model is often inadequate for studies related to in vivo airway epithelial functions. To circumvent these limitations of current airway models, tracheal xenograft models have been developed to study gene transfer and airway pathophysiology in human genetic diseases (Wilson, 1997). These airway xenograft models have proved extremely useful in studying host cell–vector interactions (Engelhardt et al., 1993b; Engelhardt et al., 1992; Goldman and Wilson, 1995) with human airway epithelium, as well as pathophysiology and gene therapy of the cystic fibrosis airways (Goldman et al., 1997; Zhang et al., 1995; Zhang and Engelhardt, 1999; Zhang et al., 1998; Zhang et al., 1996), and the identification of progenitor/stem cell targets for gene therapy in the human airway (Duan et al., 1998a; Engelhardt et al., 1995). This unit describes generation of and gene transfer to several commonly used airway models. Isolation (see Basic Protocol 1) and transduction (see Basic Protocol 2) of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined (see Basic Protocol 3). Transduction of these polarized cells by recombinant adenovirus, adeno-associated virus, retrovirus, or lentivirus is also described (see Basic Protocol 4). Methods are presented for generation of human and ferret tracheal xenografts (see Basic Protocol 5) as well as both ex vivo and in vivo gene transfer to these xenografts (see Basic Protocol 6). Finally, a method for in vivo gene delivery to the lungs of rodents is included (see Basic Protocol 7). Some methods for the evaluation of transgene expression are given in the support protocols. A method for harvesting xenografts for morphological analysis is described (see Support Protocol 1). The reporter gene β-galactosidase can be detected either histochemically (Support Protocol 2) or immunocytochemically (Support Protocol 3). If green fluorescent protein (GFP) is used as a reporter gene, it can be detected fluorescently (see Support Protocol 4). Finally, histochemical detection of alkaline phosphatase gene activity is described (see Support Protocol 5). CAUTION: Radioactive, biological, and chemical substances require special handling; see appendix 2a for guidelines.