Recent identification of two receptors for the adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I ␣-2 domain allows the molecular basis of adenoviral infection to be investigated. Earlier work has shown that human airway epithelia are resistant to infection by adenovirus. Therefore, we examined the expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain the relative resistance to adenovirus infection from the apical surface. In contrast, when the virus was applied to the basolateral surface, gene transfer was much more efficient because of an interaction of adenovirus fiber with its receptors. In addition, when the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space where fiber receptors are located increase gene transfer efficiency.The mechanism of infection by type 2 and type 5 adenovirus has been extensively studied. However, most of the knowledge on adenoviral infection has been obtained from studies done on immortalized cell lines. The first steps in adenovirus infection are thought to involve primarily two proteins in the capsid, fiber and penton base (1-3). The fiber protein is important for binding to a high affinity fiber receptor. In a human oral epidermoid carcinoma cell line (KB cells), A549 cells, and HeLa cells, this receptor is thought to be present in the range of 3,000 -10,000 receptors/cell (4 -6). NIH 3T3 cells, which are resistant to adenovirus infection, have less than 100 receptors/ cell (7). After binding to the fiber receptor, penton base interaction with ␣ V ␤ 3 and ␣ V ␤ 5 integrins facilitates internalization via receptor-mediated endocytosis (2,8,9). The acidic pH the virus encounters in the endosome may trigger a conformational change that releases the virus into the cytoplasm (10 -12) and allows the adenovirus capsid to travel to the nucleus (2, 13). Then viral proteins and DNA bind to the nuclear pore complex, capsid disassembly continues, and DNA enters the nucleus accompanied by DNA-associated protein 7 (1, 2, 14). These studies have concluded that a high affinity fiber receptor is required for binding and infection and that an ␣ V ␤ integrin acts as a co-receptor.We and others (15-22) have found infection of ciliated airway epithelia by adenovirus to be inefficient. In an in vitro model of human airway epithelia, we found that unlike infections of HeLa cells adenovirus infection of ciliated airway epithelia was quite limited and that which did occur w...

Complexes of DNA and cationic lipids are promising vecuptake, whereas others had a greater effect on steps subtors for gene transfer. Most cationic lipid formulations consequent to entry. Based on those results, we tested the tain both a cationic component and a neutral co-lipid. We hypothesis that co-lipids conferring different properties found that the co-lipid could influence DNA uptake in COScould be combined to enhance gene transfer. The results 1 cells, but processes subsequent to uptake were even showed that a combination of co-lipids had a synergistic more important in determining gene expression. We effect on expression. We also found that structural analogs compared dioleoylphosphatidylethanolamine (DOPE) and of DOPE were more effective than DOPE in enhancing structural analogs of DOPE combined with cationic lipids gene transfer to mature human airway epithelia studied in and found that DNA uptake and transgene expression did vitro and to mouse lung studied in vivo. These data provide not always correlate. Transgene expression was depeninsight into the mechanism by which co-lipids influence catdent on DNA uptake into the cell, on entry of DNA into the ionic lipid-mediated gene transfer and show that optimizcytoplasm, and on release of DNA from the lipid complex.ation of the effects of co-lipids can enhance gene transfer We found that some co-lipids had a greater effect on DNA both in vitro and in vivo.

Studies in cultured cell lines have shown that adenovirus tion of hepatocytes, even though the mutation impaired infection involves binding of adenovirus fiber to its cell surinfection of HeLa cells. Hepatocytes had undetectable face receptor and binding of penton base to ␣v integrins.amounts of ␣v integrins on their cell surface and showed However, much less is known about the role of these interno specific adherence to vitronectin, the natural substrate actions in cells that are targets for adenovirus-mediated of ␣v integrins. Adenovirus with an intact penton base gene transfer. Earlier work showed that hepatocytes are enhanced infection of liver following intravenous injection, readily infected by adenovirus, making them an attractive but only by three-fold as compared with virus in which the target for gene therapy in several diseases. We found that integrin-binding motif was disrupted. These studies sugaddition of fiber protein blocked adenovirus infection of prigest that interactions between cell surface integrins and mary cultures of hepatocytes. This suggests an important penton base are not required for adenovirus infection of role for fiber and its receptor. However, mutation of the hepatocytes in vitro, but the interaction enhances infection integrin-binding motif in penton base did not inhibit infecto a small degree in vivo.

In studies of the genetic disease cystic fibrosis, recombinant adenovirus type 2 (Ad2) and Ad5 are being investigated as vectors to transfer cystic fibrosis transmembrane conductance regulator cDNA to airway epithelia. However, earlier work has shown that human airway epithelia are resistant to infection by Ad2 and Ad5. Therefore, we examined the efficiency of other adenovirus serotypes at infecting airway epithelia. We found that several serotypes of adenoviruses, in particular, wild-type Ad17, infected a greater number of cells than wild-type Ad2. The increased efficiency of wild-type Ad17 could be explained by increased fiber-dependent binding to the epithelia. Therefore, we constructed a chimeric virus, Ad2(17f)/βGal-2, which is identical to Ad2/βGal-2 with the exception of having the fiber protein of Ad17 replace Ad2 fiber. This vector retained the increased binding and efficiency of gene transfer to well-differentiated human airway epithelia. These data suggest that inclusion of Ad17 fiber into adenovirus vectors may improve the outlook for gene delivery to human airway epithelia.