[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Lentiviruses (LVs) are considered one of the most promising tools for gene transfer, however, their potential to induce pro-inflammatory cytokines on delivery into the respiratory tissue remains to be established. Here we tested a third-generation vesicular stomatitis virus (VSV)-G pseudotyped LV vector in the two respiratory epithelial cell lines A549 and CFT1-C2. We observed that the VSV-G LV vector does not induce (a) activation of the nuclear factor (NF)-kappaB, which intervenes in transcription of pro-inflammatory genes; (b) expression of ICAM-1; and (c) transcription of a panel of cytokines, with the exception of a mild and transient (24h) increase of IFN-gamma mRNA. In contrast, an adenovirus-derived vector strongly activated NF-kappaB and different transcripts such as those of ICAM-1, IL-8, RANTES, IP-10, TNF-alpha, IL-6, IL-1 beta. In conclusion, this third-generation VSV-G pseudotyped LV vector does not elicit major pro-inflammatory signals in human airway epithelial cells and appears to be better suited for gene delivery strategies.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
Taken together, these results show that GAGs are involved in VSV-G LV transduction of airway epithelial cells.
No preview · Article · Dec 2008 · The Journal of Gene Medicine
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that human serum albumin (HSA) enhances PEI-mediated gene transfer into immortalized cell lines derived from various anatomical regions of the airways1. Therefore, we sought to confirm these results on ex-vivo cultures of primary respiratory cells: nasal polyps, nasal brushings and bronchial cells obtained from lung biopsies. The complexes were prepared by adding different amounts of HSA (0.2|[ndash]|20 |[mu]|g) to preformed PEI/DNA polyplexes. HSA increased PEI-mediated gene transfer in nasal outgrowths (n=5) and nasal cells obtained from brushings (n=3) by 2.6 and 4.8 times, respectively. Transfection of primary bronchial epithelial cells by HSA/PEI/DNA complexes was increased 3.2 times, relative to values obtained with PEI.It has been shown that non-viral vectors are more efficient in vivo if they are prepared in water2,3. Therefore, albumin/PEI/DNA (luciferase) complexes were prepared in water and their transfection efficiency was evaluated in the lungs of C57Bl/6 mice. We found that polyplexes with an N/P ratio of 15 produced the highest levels of luciferase expression. We also observed that the presence of albumin, both HSA and murine serum albumin (MSA), increased transfection mediated by PEI/DNA complexes. Immunohistochemical studies, employing an antibody against green fluorescent protein (GFP), further revealed that PEI mediated transfection with the GFP gene in the lung is restricted to bronchial epithelium. The same pattern of transfection was observed for PEI/ DNA/MSA complexes. To evaluate whether in vivo gene transfer is influenced by acute respiratory infection, C57Bl/6 mice were injected in the lung with the Pseudomonas aeruginosa PAO1 strain. Forty-eight hours after infection mice were transfected with PEI polyplexes containing the luciferase gene and prepared in the presence or absence of MSA. We observed that the levels of luciferase expression were higher in infected animals than in healthy animals when transfection was performed with PEI/DNA complexes, while no difference between infected and healthy animals was observed when the animals were transfected with PEI/DNA/MSA complexes.Overall, these results show that albumin increases gene transfer to the respiratory epithelium in ex-vivo and in-vivo models and suggest that the transfection efficiency of albumin-containing polyplexes is not likely to be hampered by opportunistic pathogen airway infections in CF patients.
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2006) 13, S268|[ndash]|S269; doi: 10.1016/j.ymthe.2006.08.772
694. Involvement of Glycosaminoglycans in VSV-G Pseudotyped Lentiviral Vector Mediated Gene Transfer into Airway Epithelial Cells|[ast]|
Elena Copreni1, Lucia Palmieri1, Salvatore Carrabino1, Stefano Castellani1, Luigi Naldini2 and Massimo Conese11Institute for Experimental Treatment of Cystic Fibrosis, H.S. Raffaele, Milano, Italy2TIGET, H.S. Raffaele, Milano, Italy|[ast]|This work has been supported by a grant from the Italian Cystic Fibrosis Research Foundation and by the Associazione Lombarda Fibrosi Cistica |[ndash]| ONLUS.
No preview · Article · Apr 2006 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: Although to date symptomatic therapies for cystic fibrosis (CF) - based on the eradication/control of opportunistic infections and facilitation of mucus excretion - have rapidly and significantly increased patients survival, an aetiological therapy is still far from being available. There are several ongoing studies with the aim of correcting the underlying defect of the disease ranging from gene therapy to studies on molecules which will help the mutated CFRT gene to correctly express its protein or to make it work better. Stem cell therapy is a promising technique since it does not require the use of vectors, fundamental in gene therapy. Pharmacogenomics will help developing new drugs and identifying new efficacy indicators of these drugs.
No preview · Article · Mar 2006 · Medico e Bambino
[Show abstract][Hide abstract] ABSTRACT: HIV-1 derived lentiviral (LV) vectors seem to be promising vehicles for gene transfer to the airway epithelium since they integrate in the host genome of non-dividing cells ensuring long-term expression of the transgene. Previous studies have shown that LV vectors could transduce the airway epithelium in vivo from the apical surface, only when tight junctions are disrupted or LV are pseudotyped with heterologous envelopes other than glycoprotein G of the vesicular stomatitis virus (VSV-G). Our study was aimed to investigate the efficiency of an improved VSV-G pseudotyped lentiviral vector, which contains the central polypurine tract (cPPT) and the Woodchuck post-translational regulatory elements, in human respiratory cells and in the murine lung. The vector PPT-GFP was injected intratracheally in C57BL/6 mice at the dose of 4.5×10E6 TU. After 48 hours, lungs were fixed in paraformaldehyde and cryosections stained with an antibody against GFP. Results show that PPT-GFP transduced the murine airway epithelium in the bronchial and alveolar compartments. Transgene expression was observed also after two weeks of infection with a LV dose of 1×10E8 TU. Hystological analysis showed no evidence of tissue damage in the lung, suggesting that this LV vector is not cytotoxic. Since heparan sulfate (HS) has been described to mediate infection by VSV-G-pseudotyped retroviral vectors and HIV-1, the expression of this glycosaminoglycan by human respiratory cells and its involvement in LV-mediated gene transfer were studied. Human bronchial epithelial cells (16HBE) and CF tracheal epithelial cells (CFT1) were analyzed for HS expression by flow cytometry and confocal microscopy. HS was expressed by 85-88% of cells, the expression being much stronger on the basolateral side in polarized epithelial cells. To correlate the efficiency of LV mediated transduction with HS expression, polarized CFT1 cells were infected with the lentivirus and some samples were pre-incubated with EGTA (Ethylene glycol-O,O-bis-[2-amino-ethyl]-N,N,N,N,-tetraacetic acid), which disrupts the epithelial tight junctions and allows the LV particles to access the basolateral side of the epithelium. Analysis of GFP expression by FACS indicates that the pre-conditioning increased the transduction efficiency, being the percentage of positive cells of 55%, when the infection was performed without pretreatment, and 71% when the cells were incubated with EGTA. To evaluate the interaction between glycosaminoglycans expressed on the apical surface and lentiviral particles, the LV vector was pre-incubated with heparin and then incubated with 16HBE cells. Heparin determined a dose-dependent inhibition of transduction efficiency up to 65% of untreated vector. Our study shows that 1) The PPT-GFP vector transduce murine airways and human polarized cells, without requirement of disruption of the epithelial barrier integrity and without cytotoxicy 2) HS might be a receptor involved in VSV-G-pseudotyped LV-mediated gene transfer.