176. Novel Molecules That Enhance Adenovirus Transduction of the Airway Epithelium: Reinvigorating Adenovirus-Mediated Gene Therapy

Adenovirus remains the most common vector system used in gene therapy clinical trials worldwide and several adenoviral vectors show promise in phase III clinical trials. One major limitation to the efficacy of adenovirus-mediated gene therapy is low transduction due to the limited primary receptor availability in most tissues. Interventions able to boost adenovirus transduction would have significant implications for both improving transduction and lowering the viral dose below the immunogenic threshold. Most human and several key animal adenoviruses share a common receptor with group B coxsackieviruses named the coxsackievirus and adenovirus receptor (CAR). CAR has several alternatively spliced isoforms. We have recently discovered that the eight-exon isoform (CAREx8) can specifically localize to the apical membrane of polarized primary human airway epithelia where it can mediate apical adenovirus infection. Moreover, we have discovered that CAREx8 is both positively and negatively regulated by the cellular scaffolding protein MAGI-1. We hypothesized that cell permeable decoy peptides targeting the interaction between MAGI-1 and CAREx8 would increase CAREx8 protein levels and increase AdV transduction. Tat-conjugated peptide entry into polarized epithelia was confirmed by mass spectrometry and fluorescence microscopy. Relative to control, peptides increased the levels of CAREx8 at the apical surface of polarized epithelia and significantly increased AdV entry and transduction, as measured by apical surface biotinylation, qPCR, and AdV5-β-Gal transduction. Moreover, AdV5 transduction was increased by 300-500% after intranasal peptide administration in mice demonstrating in vivo activity. To determine the mechanism of action, we followed the synthesis and co-localization of CAREx8 with key molecules in the cellular ER-Golgi-vesicular trafficking pathways. We found that CAREx8 was in compartments distinct from MAGI-1 and spread throughout the apical trafficking pathway and at the apical surface of the epithelium. Removal of the peptides reversed both the amount and localization of CAREx8 and returned the susceptibility of the epithelium to baseline. Taken together, these data indicate that decoy peptides able to block the MAGI-1-CAREx8 interaction transiently increase adenovirus-mediated gene transfer and offer the potential to increase the efficacy of adenovirus-mediated gene therapy.