Gene-based vaccines have been shown to elicit protective immune responses against a number of pathogens; however, conventional gene delivery methods fail to elicit potent systemic and mucosal responses required to prevent infection by certain pathogens. With the aim of inducing more potent responses, two complementary targeting strategies were employed for the delivery of antigen genes to immunologically-relevant cells, namely mucosal cells and antigen-presenting cells (APC). This thesis explores the identification and application of ligands that target Ad vectors to APC and mucosal sites using genetic engineering and biotin-avidin coupling methods with the aim of increasing immune responses to a model transgene. For APC-targeting, biotinylated Ad (Ad-BAP) was used as a novel ligand screening platform to identify the antigen uptake receptors as the most efficient targets for increased transduction. Specifically, Ad-BAP complexed to a mannosylated ligand demonstrated efficient transduction of mouse dendritic cells and macrophages. Vaccination experiments in mice indicate this vector elicits decreased antibody responses and similar cellular responses compared to unmodified Ad. More work is required to determine the benefit of targeting APC for vaccination, because not all types of APC in mice harbor mannose receptors in situ. For targeting mucosal sites, a chimeric Ad vector (Ad-sigma1) was genetically engineered to display the mucosal-targeting sigma1 protein of reovirus. The striking structural homology between the Ad fiber protein and the reovirus sigma1 protein was exploited for the development of a functional chimeric fiber-sigma1 protein to allow virion encapsidation. Adal binds and infects cells through the reovirus receptors, junctional adhesion molecule 1 (JAM1) and cell-surface sialic acid, and not through the Ad receptor, coxsackievirus and adenovirus receptor (CAR). However, Ad-sigma1 transduction of mucosal cells in vitro shows markedly decreased efficiency as compared to unmodified Ad. Despite these defects, Ad-sigma1 elicited similar immune responses compared to unmodified Ad after mucosal vaccination of mice. These results suggest that re-engineering of the fiber-sigma1 chimera to better enable JAM1 interactions may offer promise for using Ad-sigma1 for mucosal vaccination. Further work needs to establish the relative importance of the barriers to Ad-based vaccination, namely low pH, digestive enzymes, glycocalyx, and apical receptor expression.