Late generation lentiviral vectors: Evaluation of inflammatory potential in human airway epithelial cells

Copreni, Elena; Nicolis, Elena; Tamanini, Anna; Bezzerri, Valentino; Castellani, Stefano; Palmieri, Lucia; Giri, Maria Grazia; Vella, Antonio; Colombatti, Marco; Rizzotti, Paolo; Conese, Massimo; Cabrini, Giulio

doi:10.1016/j.virusres.2009.03.012

Cited by 7 publications

(6 citation statements)

References 54 publications

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“…The epithelium lining the bronchi/bronchioli is the target cell compartment for a therapeutic approach based on gene delivery in cystic fibrosis (CF), a chronic autosomal recessive disorder due to mutation in the CF Transmembrane Conductance Regulator (CFTR) gene [2]. LV vectors bear some fundamental characteristics which could be useful for treating the CF lung disease, such as: (1) they integrate into the host genome and determine a long-term expression of either marker or CFTR gene in animal and human xenograft models [3–7]; (2) they can be repeatedly administered without loss of efficiency [8]; and (3) they do not elicit a gross inflammatory response in vitro [9] and in vivo [4, 10]. …”

Section: Introductionmentioning

confidence: 99%

Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

Castellani

Gioia

Trotta

et al. 2010

Journal of Biomedicine and Biotechnology

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Lentiviral (LV) vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. In this study, we evaluated the transduction efficiency of a last-generation LV vector bearing Green Fluorescent Protein (GFP) gene as well as cytotoxicity and tight junction (TJ) integrity in a polarized model of airway epithelial cells. High multiplicities of infection (MOI) showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (TER) and delocalization of occludin from the TJs. To increase LV efficiency at low LV:cell ratio, we employed noncovalent association with the polycation branched 25 kDa polyethylenimine (PEI). Transduction of cells with PEI/LV particles resulted in 2.5–3.6-fold increase of percentage of GFP-positive cells only at the highest PEI:LV ratios (1×107 PEI molecules/transducing units with 50 MOI LV) as compared to plain LV. At this dose PEI/LV transduction resulted in 6.5 ± 2.4% of propidium iodide-positive cells. On the other hand, PEI/LV particles did not determine any alteration of TER and occludin localization. We conclude that PEI may be useful for improving the efficiency of gene transfer mediated by LV vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness.

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Section: Introductionmentioning

confidence: 99%

Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

Castellani

Gioia

Trotta

et al. 2010

Journal of Biomedicine and Biotechnology

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“…Limberis and colleagues [19] have demonstrated that transient expression of Green Fluorescent Protein (GFP) at day 90 in alveolar epithelium following an intratracheal injection of VSV-G-pseudotyped HIV-1-derived vector to the mouse lung is due to transgene- and gag -specific T-cell activation. However, preliminary results obtained by our group indicate that this last generation LV vector does not elicit a pro-inflammatory response in human respiratory epithelial cells in vitro [27]. Furthermore, we have observed that the LV vector does not induce a strong immune response in the lung, as demonstrated by absence of CD4+ and CD8+ lymphocyte infiltrates in the bronchi/bronchioli of mice intratracheally injected [28].…”

Section: Resultsmentioning

confidence: 98%

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

Copreni¹,

Palmieri²,

Castellani

et al. 2010

Viruses

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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.

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“…To construct a L‐hTERT/EGFP lentiviral transfer vector, we inserted the hTERT cDNA (Geron Corporation, Menlo Park, CA) into a pPS‐EF1‐LCS‐T2A‐EGFP plasmid (Addgene, Cambridge, MA) at its ligase‐free cloning site (LCS) according to the manufacturer's instructions. The VSV‐G‐pseudotyped lentiviral vectors were produced by cotransfecting 293T cells, and purified by ultracentrifugation as previously described . The titer (ifu/ml) of viral vector stocks was measured by a UltraRapid Lentiviral Titer Kit (Cell Biolabs, San Diego) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…The VSV-G-pseudotyped lentiviral vectors were produced by cotransfecting 293T cells, and purified by ultracentrifugation as previously described. 14,15 The titer (ifu/ml) of viral vector stocks was measured by a UltraRapid Lentiviral Titer Kit (Cell Biolabs, San Diego) according to the manufacturer's instructions.…”

Section: Construction Of Lentiviral Transfer Vector and Viral Preparamentioning

confidence: 99%

Prolonged expansion of human nucleus pulposus cells expressing human telomerase reverse transcriptase mediated by lentiviral vector

Wang

Ruan

et al. 2013

Journal Orthopaedic Research

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Human degenerative disc disease (DDD) is characterized by progressive loss of human nucleus pulposus (HNP) cells and extracellular matrix, in which the massive deposition are secreted by HNP cells. Cell therapy to supplement HNP cells to degenerated discs has been thought to be a promising strategy to treat DDD. However, obtaining a large quality of fully functional HNP cells has been severely hampered by limited proliferation capacity of HNP cells in vitro. Previous studies have used lipofectamine or recombinant adeno-associated viral (rAAV) vectors to deliver human telomerase reverse transcriptase (hTERT) into ovine or HNP cells to prolong the activity of nucleus pulposus cells with limited success. Here we developed a lentiviral vector bearing both hTERT and a gene encoding green fluorescence protein (L-hTERT/EGFP). This vector efficiently mediated both hTERT and EGFP into freshly isolated HNP cells. The expressions of both transgenes in L-hTERT/EGFP transduced HNP cells were detected up to day 210 post viral infection, which was twice as long as rAAV vector did. Furthermore, we observed restored telomerase activity, maintained telomere length, delayed cell senescence, and increased cell proliferation rate in those L-hTERT/EGFP transduced HNP cells. Our study suggests that lentiviral vector might be a useful gene delivery vehicle for HNP cell therapy to treat DDD. ß

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Late generation lentiviral vectors: Evaluation of inflammatory potential in human airway epithelial cells

Cited by 7 publications

References 54 publications

Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

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

Prolonged expansion of human nucleus pulposus cells expressing human telomerase reverse transcriptase mediated by lentiviral vector

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