1. Persistent Gene Expression in Mouse Nasal Epithelia Following Baculovirus GP64 Pseudotyped FIV-Based Gene Transfer

Abstract

The use of gene transfer to treat or prevent cystic fibrosis lung disease has been limited in part by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the HA and HEF envelope proteins proved unsuccessful. Conversely, influenza D has a single envelope protein (GP64) and pseudotyping FIV with influenza D GP64 (from Thogotovirus) resulted in titers of 10e6 TU/ml. Further, influenza D GP64 conferred FIV apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D envelope glycoproteins, the result of a postulated lateral transfer of genetic material during the evolution of the two viral families. Pseudotyping FIV with GP64 from 3 species of baculovirus resulted in titers ranging from 10e7|[ndash]|10e9 TU/ml. Of note, GP64 from Autographa californica multinuclear polyhedrosis virus resulted in high titer FIV preparations (upwards of 10e9 TU/ml) and also conferred apical entry into polarized primary cultures of human airway epithelia. These titers equal those obtained with VSV-G pseudotyping. Using a luciferase reporter gene and bioluminescent imaging, we observed persistent in vivo gene transfer in the mouse nose with A. californica GP64 pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis has documented the persistence of expression in the nasal epithelia for >7 months without significant decline. We used a nuclear targeted beta-galactosidase reporter and histologicial analysis to demonstrate that surface epithelial cells were transduced in the mouse sinuses. In addition, AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than VSV-G pseudotyped FIV, as determined by both bioluminescence and histological analysis. These data suggest that AcGP64-FIV efficiently and persistently transduces nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity and have important implications for CF gene therapy.