Research Articles Short-Fiber Protein of Ad40 Confers Enteric Tropism and Protection Against Acidic Gastrointestinal Conditions Ester Rodrı ´guez, 1, * Carolina Romero, 2,3, * Adolfo Rı ´o, 3,4 Marta Miralles, 1 Aida Ravento ´s, 3,4 Laura Planells, 1 Joan F. Burguen ˜ o, 3 Hirofumi Hamada, 5 Jose Carlos Perales, 6 Assumpcio ´ Bosch, 1 Miguel Angel Gassull, 3,4 Ester Ferna ´ ndez, 2,3 and Miguel Chillon 1,7 Abstract The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral–fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short- fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter b-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases. Introduction I ntestinal epithelium has been proven a difficult target for gene therapy strategies despite the use of different viral and nonviral vectors. The difficulty in transducing the intes- tine can be associated, at the cellular level, to inefficient vector binding, internalization, and endocytosis. In fact, polarized epithelial cells are more permissive to basolateral than to apical transduction, suggesting different vector binding and trafficking pathways between the apical and the basolateral membranes (Duan et al., 2000; Esclatine et al., 2001; Tang et al., 2005). In addition, pre-existing immunity may neutralize viral vectors (Manno et al., 2006) and limit intestinal gene transfer. Finally, protective extracellular barriers such as mucus, pro- teases, and acidic gastric pH can reduce vector stability of orally administered vectors (Shao et al., 2006; Tang and Sibley, 2006). Initial studies with DNA plasmids and liposomes (Schmid et al., 1994; Westbrook et al., 1994; Schmid et al., 1997a) re- ported limited transduction of the intestine lasting only for a short period. More recent studies using naked DNA in mice in which colitis was induced by dextran sulfate sodium showed transduction of mucosal layer, lamina propria, and muscularis mucosae after intrarectal administration (Kanbe et al., 2006). Similarly, other nonviral vectors such as orally given chitosan–DNA nanoparticles (Chen et al., 2004), or 1 Centre de Biotecnologia Animal i Tera `pia Ge `nica (CBATEG), Departament Bioquı ´mica i Biologia Molecular, Universitat Auto ` noma Barcelona, Barcelona 08193, Spain. 2 Departament Biologia Cel.lular, Fisiologia i Immunologia, Universitat Auto ` noma Barcelona, 08193 Barcelona, Spain. 3 Centro de Investigacio ´ n Biome ´dica en Red- Enfermedades Hepa ´ticas y Digestivas (CIBER-ehd), Instituto de Salud Carlos III, Spain. 4 Institut d’Investigacio ´ en Cie `ncies de la Salut Germans Trias i Pujol, 08916 Badalona, Spain. 5 Department of Molecular Medicine, University of Sapporo, 060-8556 Sapporo, Japan. 6 Biophysics Unit, Department de Cie `ncies Fisiolo ` giques II, IDIBELL-Universitat de Barcelona, 08907 Barcelona, Spain. 7 Institucio ´ Catalana de Recerca i Estudis Avanc ¸ats (ICREA), 08010 Barcelona, Spain. *These two authors contributed equally to this work. HUMAN GENE THERAPY METHODS 24:195–204 (August 2013) ª Mary Ann Liebert, Inc. DOI: 10.1089/hgtb.2012.096 195