A VSV-G Pseudotyped Last Generation Lentiviral Vector Mediates High Level and Persistent Gene Transfer in Models of Airway Epithelium In Vitro and In Vivo

The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70–80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis

Lentivirus-mediated gene transfer to the respiratory epithelium: A promising approach to gene therapy of cystic fibrosis

Gene therapy of cystic fibrosis (CF) lung disease needs highly efficient delivery and long-lasting complementation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene into the respiratory epithelium. The development of lentiviral vectors has been a recent advance in the field of gene transfer and therapy. These integrating vectors appear to be promising vehicles for gene delivery into respiratory epithelial cells by virtue of their ability to infect nondividing cells and mediate long-term persistence of transgene expression. Studies in human airway tissues and animal models have highlighted the possibility of achieving gene expression by lentiviral vectors, which outlasted the normal lifespan of the respiratory epithelium, indicating targeting of a \u2018stem cell\u2019 compartment. Modification of the paracellular permeability and pseudotyping with heterologous envelopes are the strategies currently used to overcome the paucity of specific viral receptors on the apical surface of airway epithelial cells and to reach the basolateral surface receptors. Preclinical studies on CF mice, demonstrating complementation of the CF defect, offer hope that lentivirus gene therapy can be translated into an effective treatment of CF lung disease. Besides a direct targeting of the stem/progenitor niche(s) in the CF airways, an alternative approach may envision homing of hematopoietic stem cells engineered to express the CFTR gene by lentiviral vectors. In the context of lentivirus-mediated CFTR gene transfer to the CF airways, biosafety aspects should be of primary concern. \ua9 2004 Nature Publishing Group. All rights reserved

Involvement of glycosaminoglycans in vesicular stomatitis virus G glycoprotein pseudotyped lentiviral vector-mediated gene transfer into airway epithelial cells

Background: The involvement of surface molecules in HIV-1-derived lentivirus (LV)-mediated transduction of airway epithelial cells has not been studied so far. The present study aimed to evaluate the role of glycosaminoglycans (GAGs) in gene transfer mediated by a third generation vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped LV vector in an in vitro model of polarized airway epithelial cells. Methods: Human bronchial (16HBE-S1) and tracheal (CFT1-C2) epithelial cells were grown either on plastic or on filters and transduced with the LV vector polypurine tract (PPT)-green fluoresecent protein (GFP). Zonula Occludens (ZO)-1, a marker of tight junction, and GAG localization were assessed by cytofluorimetry and confocal microscopy. Soluble GAGs and removal of cell surface GAGs were used to affect LV-mediated transduction. Results: Extensive optimization of experimental parameters (presence of polybrene during the infection, the incubation time in the presence of LV particles, period of time intercurring between infection and gene expression analysis) was carried out in plastic-adherent cells. Polybrene resulted to be cytotoxic and was not further used. In CFT1-C2 polarized cells, EGTA treatment determined a 20% decrease in transepithelial resistance, a diminished ZO-1 localization at the tight junction location and a 31% increase in GFP positive cells. Heparane sulfate was distributed evenly on the cell surface. Heparin and soluble chondroitin sulfate A and B inhibited LV-mediated transduction in a dose-dependent fashion. These results were confirmed upon enzymatic removal of GAGs from the cell surface. Conclusions: Taken together, these results show that GAGs are involved in VSV-G LV transduction of airway epithelial cells

Role of Biophysical Parameters on ex Vivo and in Vivo Gene Transfer to the Airway Epithelium by Polyethylenimine/Albumin Complexes

Efficient gene transfer to the airways by nonviral vectors is a function of different parameters, among which the size and the charge of the transfecting particles. The aim of this study was to determine the transfection efficiency of polyethylenimine (PEI)/albumin polyplexes in ex vivo and in vivo models of respiratory epithelium and to correlate it with biophysical characteristics of the particles. Complexes were obtained by adding different amounts of human serum albumin (HSA) to PEI polyplexes preformed in saline. The presence of HSA caused the formation of bigger and more negative polyplexes and increased PEI transfection efficiency in primary respiratory epithelial cells by 4-6-fold. For in vivo administration to the lung, PEI polyplexes were formed in water and optimized with respect to the N/ P ratio. PEI/pC-Luc complexes gave the highest luciferase expression at N/ P 15 when administered through the trachea. At this N/ P ratio, the size and the surface charge of albumin-containing polyplexes were not different as compared with plain PEI polyplexes. Formulation of PEI polyplexes in the presence of HSA or murine serum albumin (MSA) resulted in a 2-fold increase in luciferase expression. In mice treated with PEI or PEI/MSA polyplexes containing the nuclear beta-gal gene, X-gal staining revealed that transfected cells localized at the bronchiolar epithelium and that PEI/MSA transfected four times as many cells as PEI ( p < 0.05). Finally, double administration of PEI/MSA polyplexes resulted in a further enhancement of transfection of the lung. Our data show that serum albumin enhances PEI-mediated gene transfer to airway epithelial cells in vivo, likely facilitating the uptake of polyplexes, and indicate that this formulation would fulfill the requirement of repeated administration, as necessary in chronic lung diseases like cystic fibrosis