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Virus Research 144 (2009) 8–17

Contents lists available at ScienceDirect

Virus Research

journa l homepage: www.e lsev ier .com/ locate /v i rusres

ate generation lentiviral vectors: Evaluation of inflammatory potentialn human airway epithelial cells

lena Copreni a,1, Elena Nicolis b, Anna Tamanini b, Valentino Bezzerri b, Stefano Castellani a,c,ucia Palmieri a, Maria Grazia Giri d, Antonio Vella e, Marco Colombatti e, Paolo Rizzotti b,assimo Conese a,c, Giulio Cabrini b,∗

Institute for the Experimental Treatment of Cystic Fibrosis, H.S. Raffaele, Via Olgettina, 20132 Milano, ItalyLaboratory of Molecular Pathology, Laboratory of Clinical Chemistry and Haematology, University Hospital of Verona, Piazzale Stefani 1, 37126 Verona, ItalyDepartment of Biomedical Sciences, University of Foggia, Viale L. Pinto 1, 71100 Foggia, ItalyDepartment of Medical Physics, University Hospital of Verona, Piazzale Stefani 1, 37126 Verona, ItalyDepartment of Pathology, Section of Immunology, University of Verona, Strada Le Grazie, 37134 Verona, Italy

r t i c l e i n f o

rticle history:eceived 27 March 2008eceived in revised form 10 March 2009ccepted 22 March 2009vailable online 2 April 2009

a b s t r a c t

Lentiviruses (LVs) are considered one of the most promising tools for gene transfer, however, their poten-tial 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 tworespiratory 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)-�B, which intervenes in transcription of pro-inflammatory genes;

eywords:entivirusdenovirusene therapyystic fibrosis

(b) expression of ICAM-1; and (c) transcription of a panel of cytokines, with the exception of a mildand transient (24 h) increase of IFN-� mRNA. In contrast, an adenovirus-derived vector strongly activatedNF-�B and different transcripts such as those of ICAM-1, IL-8, RANTES, IP-10, TNF-�, IL-6, IL-1�. In conclu-sion, this third-generation VSV-G pseudotyped LV vector does not elicit major pro-inflammatory signalsin human airway epithelial cells and appears to be better suited for gene delivery strategies.

ungytokines

. Introduction

Cystic fibrosis (CF) is caused by mutations in the gene encod-ng the cystic fibrosis transmembrane conductance regulator (CFTR)rotein (Riordan et al., 1989), a unique member of the ATP-Bindingassette protein family, which mediates chloride transport and reg-lates the homeostasis of other electrolytes in different exocrinergans (Schwiebert et al., 1999). More than 1500 mutations of theF gene have been identified, the most common being the deletionf the Phe508 (�F508), causing improper folding and degradationf CFTR protein. Other mutations can result in the absence or severeeduction of the functional protein, in defects of functional activa-ion or reduced channel pore conductance (Sheppard and Welsh,

999).

Gene transfer has been proposed soon after CF gene cloning asway to treat the pulmonary disease, the major determinant of

he life of CF patients, since more than 1500 mutations are dif-

∗ Corresponding author. Tel.: +39 045 812 2457; fax: +39 045 812 2840.E-mail address: giulio.cabrini@azosp.vr.it (G. Cabrini).

1 Present address: Clinical Trials Laboratory, Department of Oncology, Pharmaco-ogical Research Institute “Mario Negri”, via La Masa 19, 20156 Milano, Italy.

168-1702/$ – see front matter © 2009 Published by Elsevier B.V.oi:10.1016/j.virusres.2009.03.012

© 2009 Published by Elsevier B.V.

ficult to correct with a targeted pharmacological approach. Untilnow 29 clinical trial protocols for CF gene transfer have beenapproved and most of them have been completed (for reference seehttp://wiley.co.uk/genetherapy/clinical/). The main outcome fromthose clinical trials was that correction of the underlying bioelec-tric defect could be only partially achieved, in vivo, through thedelivery of the CFTR cDNA via viral (recombinant adenovirus andadeno-associated virus) and non-viral synthetic vectors (reviewedby Davies et al., 2001). Adenovirus vectors are known to inducea complex innate immune response (reviewed by Hartman et al.,2008). As far as CF is concerned, these results were compatible withlow efficiency of gene transfer into the epithelial cells at the sur-face of the upper or lower respiratory tract, due to the presenceof extra- and intracellular barriers (Ferrari et al., 2002). Moreover,most viral and non-viral vectors were shown to be unable to main-tain sustained CFTR expression, mainly because of the episomalnature of transferred DNA and of the inflammatory and immuneresponses (for review see Ferrari et al., 2003; Bessis et al., 2004). In

animal models, it has been shown that administration of high dosesof adenovirus (Ad) vectors resulted in only transient gene expres-sion, in vivo. Concerning first generation Ad vectors, this side effectis mainly due to the lysis of the cells transduced with the thera-peutic gene by host adaptive cytotoxic response directed towards

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eo-synthesized residual viral proteins, presented in associationith MHC class I molecules (Yang et al., 1995; Woffendin et al.,

996). A major improvement has been obtained by the design ofd vectors devoid of all viral coding sequences (helper-dependentr gutless vectors), which have been proved to give a tremendousdvantage in prolonging the duration of transgene expression inpecific organs and animal models (Morral et al., 1999). However,pplication of helper-dependent Ad vectors to different modelsas recently clarified that although they are able to circumventhe adaptive cytotoxic response they nevertheless elicit an intactnflammatory response, possibly as a result of the interactions of theector capsid with the host cell receptors (Muruve et al., 2004). Thiss not utterly surprising since it was previously reported that inacti-ated Ad particles induce pulmonary inflammation independentlyf the neo-synthesis of viral proteins (McCoy et al., 1995; Otake etl., 1998). To overcome these limitations, anti-inflammatory andmmune-suppressive approaches have been proposed (Cassivi etl., 2000; Ferrari et al., 2003), although this also indicates that theost appropriate strategy is to test alternative classes of vectors for

ene transfer in airway cells.A new approach to gene therapy of CF is to target lung air-

ay epithelium with integrating vectors, such as LV vectors, whichould offer the advantage of overcoming transient CFTR expres-

ion. LV vectors have a broad tissue tropism, including the “stemell” compartment, being able to transfer the gene of interest inoth proliferating and quiescent cells (Vigna and Naldini, 2000).elevant to CF, it was reported that LV-mediated transfer can result

n sustained transgene expression in the airway epithelium up tomonths (Goldman et al., 1997; Wang et al., 1999; Johnson et al.,

000; Kobinger et al., 2001), and that vesicular stomatitis virus GVSV-G) pseudotyped LV-mediated CFTR gene transfer could par-ially correct the electrophysiological defect in the nose of CF miceLimberis et al., 2002). Previous studies have shown that VSV-GV can efficiently transduce the airway epithelium, in vivo, whenpoorly differentiated developing airway epithelium is targeted

Goldman et al., 1997). More recently, it was reported that VSV-GV can efficiently transduce both the tracheobronchial epitheliumn human fetal tracheal xenografts developed in SCID mice, thusuggesting that this vector can target progenitor cells in the air-ay epithelium (Lim et al., 2003), and the murine intact bronchial

pithelial cell surface, in vivo, (Copreni et al., 2004).A preliminary evaluation of safety of administration of LV vec-

ors into airways showed that, in vivo, delivery of a HIV-1-derivedector in the tracheas of C57Bl/6 mice did not induce inflam-atory infiltrates post-infection (Kobinger et al., 2001), and the

ersistent expression in mice and rabbits suggested a potentialimmune tolerance” in these animal models (Wang et al., 1999;uricchio et al., 2001). However, intravenous administration of LVectors in immunocompetent mice induced the pro-inflammatoryell-adhesion molecules ICAM-1, VCAM-1, PECAM-1 and the cos-imulatory molecule B7-2 (Vandendriessche et al., 2002) andt resulted in reduced duration of expression of the transgeneFollenzi et al., 2004), whereas direct administration into liver,

uscle, and brain has been associated with milder systemic inflam-atory and immune responses (Kafri et al., 1997; Baekelandt et al.,

003). On this basis, although the preliminary results related to theirway tract appear to exclude major inflammatory and immuneide effects when LVs are delivered (Wang et al., 1999; Kobinger etl., 2001), a thorough evaluation of the safety profile of VSV-G LVectors must be pursued. The present work shows that the VSV-G LVector utilized here does not induce those pro-inflammatory signals

sually observed in human respiratory epithelial cells exposed tolassical stimuli, such as TNF-�, or other gene transfer vectors previ-usly studied, such as adenovirus-derived vectors, except for a mildnd transient induction of transcription of IFN-� in an epithelialracheal cell line.

rch 144 (2009) 8–17 9

2. Materials and methods

2.1. Cell cultures

Human respiratory epithelial cells A549 and CFT1-C2 were cul-tured following standard procedures. Briefly, the A549 alveolar typeII-derived epithelial cell line was obtained from the American TypeCulture Collection (Rockville, MD) and maintained in Dulbecco’smodified Eagles’s medium (DMEM) supplemented with 10% low-endotoxin fetal bovine serum as described (Nicolis et al., 1998).CFT1-C2 cells (derived from human tracheal epithelial cells withthe CF �F508 mutation) were a kind gift of James Yankaskas (TheUniversity of North Carolina at Chapel Hill, Chapel Hill, NC, USA)and were grown in HAM’s F12 supplemented with seven hormonesand growth substances, as previously published (Yankaskas et al.,1993).

2.2. Production of vescicular stomatitis virus-G protein (VSV-G)lentivirus (LV) and adenovirus-derived vectors

The self-inactivating pRRLsin.cPPT.CMV.Wpre-construct wasused to generate vesicular stomatitis virus (VSV)-pseudotyped LVvector stock as described (Follenzi et al., 2000) and previouslyperformed (Copreni et al., 2008) (VSV: order Mono-negavirales,family Rabdoviridae, genus Vesiculovirus, species vesicular stom-atitis virus; LV: family Retroviridae, genus Lentivirus, speciesHuman Immunodeficiency virus). Briefly, co-transfection of thefour plasmid vectors has been carried out on 293T cells by calciumphosphate precipitation. Cells have been incubated for 48 h in thepresence of 10% FBS. The supernatant containing lentiviral parti-cles has been concentrated by two rounds of ultra-centrifugationand assayed for p24 Gag antigen by ELISA. The viral titer has beendetermined by HeLa cell infection and subsequent flow cytome-try analysis, as previously described (Follenzi et al., 2000; Copreniet al., 2008). The yield of the concentrated virus was typically108–109 TU/ml and the specific activity ranged between 1.56 and4.17 × 105 TU/ng of p24. Infection doses are expressed as multiplic-ity of infection (M.O.I.), where 1 M.O.I. corresponds to 1 TU/cell.The first generation Ad-derived Ad.RSV�gal vector has been gen-erated according to Rosenfeld et al. (1992), as previously reported(Nicolis et al., 1998) (Ad: family Adenoviridae, genus Mastaden-ovirus, species Human adenovirus C type 5). Briefly, the virus waspurified by four freeze–thawing cycles, followed by two succes-sive bandings on CsCl gradients. Purified virus was dialysed against10 mM Tris–HCl (pH 8.0) and 1 mM MgCl2, aliquoted with theaddition of 10% glycerol (virus suspension buffer) and stored at−80 ◦C until use. The concentration of purified Ad was determinedby absorbance measured at 260 nm wavelength according to themethod of Mittereder et al. (1996), who assumed the conversionfactor of 1 OD unit of Ad corresponding to 1.1 × 1012 virions. Infec-tion doses are expressed as M.O.I., where 1 M.O.I. corresponds to1 PFU/cell.

2.3. Evaluation of efficiency of gene transfer

To investigate the efficiency of lentiviral transduction, A549 andCFT1-C2 respiratory epithelial cells (1 × 105) were seeded in plasticdishes and incubated with the vector in complete medium. After24 h from the infection, a preliminary evaluation of GFP expres-sion was carried out by epifluorescence. The quantitative analysisof GFP production was carried out by flow cytometry after 1 day, 1

week or 2 weeks, as previously described (Follenzi et al., 2000).Briefly, cells were washed twice with PBS, harvested by digest-ing with trypsin/EDTA and fixed in 1% paraformaldehyde. The cellswere analysed by fluorescence-activated cell sorting (FACS) with aFACS Canto apparatus (Becton-Dickinson, San Jose, CA). To evaluate

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n the same experiment the efficiency of LV-mediated gene transfernd the activation of inflammatory pathways, cells were seeded inhamber slides at 1 × 104 cells/well 24 h before the infection. TheV vector was added to the cells at 100 M.O.I. for 1 day, 1 week orweeks, in complete medium, and GFP expression was evaluated

y epifluorescence microscopy. At each time point, after evalua-ion of GFP expression, cells were fixed in 4% paraformaldehyde,ermeabilised with methanol for 5 min on ice, and kept at −20 ◦C.lternatively cells were detached and analysed directly by cytoflu-rometer. To evaluate the efficiency of Ad.RSV.�gal-mediated generansfer, cells were seeded in chamber slides at 1 × 104 cells/well4 h before the infection and the percentage of cells positive for thexpression of the reporter gene was measured by X-Gal assay asescribed previously (Lemarchand et al., 1992; Nicolis et al., 1998;elotti et al., 2001; Tamanini et al., 2003). The cells were observedith a Nikon TMD inverted microscope and those with a nuclear-

ominant blue coloration (X-Gal stain) were considered positive,hotographed and 3 fields of 100 cells for each doses of infectionere counted. The infectivity was calculated as percentage of the

nfected and uninfected cells divided by the number of infectedells.

.4. Activation of the nuclear transcription factor NF-�B

Activation of NF-�B was studied by nuclear translocation ofhe activated p65 subunit (Melotti et al., 2001; Tamanini et al.,003). Cells grown in chamber slides have been fixed with 4%araformaldehyde (w/v) in PBS for 20 min at room tempera-ure. After three washes with PBS, cells were permeabilised with

ethanol at −20 ◦C for 5 min then air dried for 1 h. Slides werencubated with 5% bovine serum albumin (BSA) in PBS for 90 mint room temperature then subjected to 1-h incubation at room tem-erature with 7 �g/ml rabbit polyclonal antibody against the NF-�B65 subunit (sc-109; Santa Cruz Biotechnology Inc., Santa Cruz, CA),ashed three times in PBS, incubated with 1:200 dilution of Texased-conjugated secondary antibody (Sigma) and examined with aikon TMD epifluorescence microscope. Alternatively, a quantita-

ive NF-�B p65 subunit capture assay was performed by TransAMF-�B p65 Transcription Factor Assay (Carlsbad, CA, USA), accord-

ng to the manufacturer’s instructions, as previously performedCarrabino et al., 2006). Briefly, nuclear extracts were added toells coated with an oligonucleotide containing a NF-�B consensus

inding site. The binding was revealed by consequent addition ofprimary antibody directed against p65 and a secondary antibody

onjugated to horseradish peroxidase and, after the addition of theubstrate, absorbance was read at 450 nm wavelength. To assesshe specificity of detection of the activated p65 subunit, nuclearxtracts from samples in which NF-�B was activated by Ad vectorere pre-incubated with a free oligonucleotide homologous to the

apture oligo (wild type oligo) or the same oligo bearing a pointutation (mutated oligo).

.5. Expression of ICAM-1 protein

Expression of ICAM-1 protein on the cell membrane of cellsxposed to the LV vector was detected by direct immunofluores-ence with a phycoerithrin (PE)-conjugated anti-human ICAM-1ntibody (CD54 PE-conjugated, BD Pharmingen, Franklin Lakes, NY,SA) and with a PE-conjugated mouse IgG1 isotype control anti-ody (BD Fast Immune), then measured by flow cytometry.

.6. Quantitation of transcripts of pro-inflammatory genes

Steady-state levels of different transcripts were quantitated afterNA Isolation, cDNA synthesis, and real-time PCR amplification.riefly, total RNA was isolated using High Pure RNA Isolation Kit

rch 144 (2009) 8–17

(Roche, Mannheim, Germany). Total RNA (2.5 �g) was reverse-transcribed to cDNA using the High Capacity cDNA Archive Kitand random primers (Applied Biosystems) in a 100-�l reaction.The cDNA (2 �l) was then amplified for 50 PCR cycles using thePlatinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen) andtemplate-specific primers in an ABI Prism 5700 sequence detectionsystem (Applied Biosystems). The sequences and concentrations ofthe forward (FP) and reverse (RP) primers were previously reportedin detail (Tamanini et al., 2006; Bezzerri et al., 2008), together withthe accession numbers and the length of the amplified fragments.Primer sets were purchased from Sigma-Genosys (The Woodlands,TX). Results collected with Sequence Detection Software (AppliedBiosystems, version 1.3) and relative quantification of gene expres-sion was performed using the comparative threshold (CT) methodas described by the manufacturer (Applied Biosystems User Bulletin2). Changes in mRNA expression levels were calculated follow-ing normalization to GAPDH, EEF1 g, G6PD and RPII as reported(Tamanini et al., 2006; Bezzerri et al., 2008). The ratios obtainedfollowing normalization are expressed as fold change over non-treated samples.

3. Results

3.1. Efficiency of gene transfer mediated by VSV-G LV andadenovirus-derived vectors in airway epithelial cells

To investigate the efficiency of lentiviral transduction, A549 andCFT1-C2 respiratory epithelial cells were seeded and incubatedwith the VSV-G LV vector formulated at different M.O.I. with com-plete cell culture medium for 1 day, 1 week or 2 weeks. Expression ofthe reporter Green Fluorescent Protein (GFP) gene was preliminar-ily evaluated by epifluorescent microscopy at 1 day (Fig. 1, panelsA and C) and by FACS analysis at 1 day, 1 week or 2 weeks. Asshown in Fig. 1, panels B and D, the GFP reporter gene expressionincreased at 1 week and 2 weeks as compared with 1 day, reachingthe efficiency of 60–70% at 1 week. At M.O.I. lower than 100 andat 1 day post-infection, GFP-positive cells were never higher than5%. To investigate the efficiency of the transduction of Ad vectorin comparison to LV, A549 cells were exposed to different doses(M.O.I. of 1, 5, 10, 100) of Ad vector for 1 day. Ad vector, as shown inFig. 1, panel E and F, was able to transfer efficiently (95%) the lacZreporter gene at M.O.I. of 100 after 1 day. M.O.I. of 100 was chosenas infecting dose for the subsequent experiments aimed to evaluatethe inflammatory response, which were performed at 1 day, 1 weekand 2 weeks.

3.2. Effect of VSV-G LV vector on activation of NF-�B

Adenovirus-derived vectors for gene transfer were shown toinduce activation of the transcription factor NF-�B. Induction ofNF-�B was related not only to the expression of residual viral genesbut also to the interaction of viral capsids with cell surface recep-tors, as demonstrated with vectors completely ablated of residualviral genes of the early regions E1 to E4 (helper-dependent or“gutless” adenovirus vectors). This was recently confirmed and clar-ified as being due to the very early interaction of adenovirus fiberwith the coxsackie–adenovirus receptor, beginning with the ini-tial phase of entry of the vector inside the epithelial respiratorycell (Tamanini et al., 2006). Here we tested the activation of NF-�B in single cells by immunocytochemistry upon exposure of A549cells to VSV-G LV vector (100 M.O.I. for 24 h), as reported in Fig. 2.

Comparing the most positive GFP-expressing cells from three rep-resentative microscopic fields (panels A–C) with the correspondingpanels (D–F), we observed that the cells which were positivelyinfected with LV vector did not show the Texas Red signal indicatingactivation of NF-�B. On the contrary, activation of NF-�B was evi-

E. Copreni et al. / Virus Research 144 (2009) 8–17 11

Fig. 1. Efficiency of transfer of the reporter gene GFP with VSV-G LV in A549 and CFT1-C2 cells. GFP-positive cells were checked both by epifluorescent microscope and flowcytometry analysis in A549 and CFT1-C2 cells transduced with 100 M.O.I. for 1 day, 1 week and 2 weeks. (A) Representative fields of A549 cells: phase contrast microscopy(bright field) and epifluorescence with GFP filter of A549 cells exposed to VSV-G LV (100 M.O.I., 1 day). (B) FACS analysis of A549 cells non-infected or infected with VSV-G LV( se cone ected4 unini

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100 M.O.I., 1 day, 1 week, 2 weeks). (C) Representative fields of CFT1-C2 cells: phaxposed to VSV-G LV (100 M.O.I., 1 day). (D) FACS analysis of CFT1-C2 cells non-inf0×. (E) Representative fields of A549 cells: �-galactosidase activity (blue color) in

nfectivity was calculated as reported in Section 2.

enced in A549 cells incubated with TNF-� (see Fig. 2H with respecto cells incubated with medium alone in Fig. 2G). As previouslyeported by other and our laboratory, first generation Ad vectorslso induced activation of NF-�B, as reported in the representativeeld shown (Fig. 2I). The activation of NF-�B was studied also by aompetitive capture assay in A549 cells. As shown in Fig. 3, VSV-GV vector did not show any activation above the constitutive levelsf NF-�B at 100 M.O.I. for 24 h, whereas Ad vector clearly activatesF-�B. In conclusion, the VSV-G LV vector added to A549 respira-

ory cells at a dose ensuring efficient infection and expression of theeporter gene did not induce activation of the transcription factorF-�B.

.3. Modulation of ICAM-1 protein expression

The InterCellular Adhesion Molecule (ICAM)-1, a gene whichontains in its promoter consensus sequences for the transcriptionactor NF-�B, has been frequently utilized as a marker of induc-ion of the innate immune response against gene transfer vectors,

trast microscopy (bright field) and epifluorescence with GFP filter of CFT1-C2 cellsor infected with VSV-G LV (100 M.O.I., 1 day, 1 week, 2 weeks). Magnification wasfected or infected cells. (F) A549 cells exposed to different doses of Ad vector, the

since it was previously shown to be upregulated upon application ofadenovirus-dependent vectors, in other and our laboratories. More-over, ICAM-1 has been found up-modulated after systemic deliveryof LV vectors in mice (Vandendriessche et al., 2002). Therefore wemeasured by cytofluorometry the expression of ICAM-1 protein inA549 cells infected with VSV-G LV vector or stimulated with TNF-�. As shown in Fig. 4, TNF-� clearly induced expression of ICAM-1well over the levels measured with medium alone, whereas theexposure of the VSV-G LV vector (100 M.O.I. for 24 h) did not induceexpression of ICAM-1 protein in A549 cells.

3.4. Modulation of expression of transcripts of chemokines andcytokines

Although NF-�B is not activated upon exposure of the humanairway respiratory cell line A549 to the VSV-G LV vector, othertranscription factors may be in principle activated by the VSV-GLV vector, leading to activation of genes involved in the innateimmune response. Therefore, the quantitation of the transcript

12 E. Copreni et al. / Virus Research 144 (2009) 8–17

Fig. 2. Effect VSV-G LV vectors on nuclear translocation of NF-�B p65 subunit. Activated NF-�B has been detected by a polyclonal antibody directed against the p65 subunitand revealed by Texas Red secondary antibody. (A–C) Expression of GFP in three representative fields of A549 cells exposed to VSV-G LV vector (100 M.O.I., 24 h) detectedwith epifluorescence microscopy (wavelengths: exc 470–490 nm, em 520–560 nm, dichrevidenced by white arrows. (D–I) Detection Texas Red signal (wavelengths: exc 546 nm, eexposed to VSV-G LV (100 M.O.I., 24 h, panels D–F correspond to panels A and B), to TNF-�I) or medium alone (panel G). Magnification was 40×.

Fig. 3. Effect of Ad and LV vectors on activation of NF-�B. A549 cells have beenexposed to Ad.RSV.�gal (Ad), a first generation adenovirus-derived vector, or tothe VSV-G LV at the multiplicity of infection of 100 M.O.I. for 24 h. Nuclear extracts(2.5 �g/each sample) have been analysed for the presence of the activated p65 sub-unit of the transcription factor NF-�B with the TransAM NF-�B p65 capture assay,according to the manufacturer’s instructions. The wild type consensus oligonu-cleotide has been used as competitor for NF-�B binding and the mutated consensusoligonucleotide as no competitor in order to monitor the specificity of the assay.Data report mean ± S.E.M., representative of three independent experiments.

oic mirror 510 nm). A549 cells expressing the reporter gene GFP at high level arem 610 nm, dichroic mirror 580 nm) indicating activation of NF-�B p65 in A549 cells

(10 ng/ml, 24 h, panel H); to Ad vector (Ad.RSV.�gal vector, 100 M.O.I., 24 h, panel

levels of a panel of chemokines involved in the recruitment of poly-morphonuclear neutrophils and mononuclear cells, such as IL-8,GRO-�, GRO-�, MIP-1�, RANTES, IP-10, together with the adhe-sion molecule ICAM-1 involved in leukocyte chemotaxis, and ofpro-inflammatory and regulatory cytokines, such as TNF-�, IL-1�,IL-6, IFN-�, TGF-�1, IL-10, was carried out using human respira-tory epithelial cells. A549 cells were firstly exposed to TNF-� as apositive control. As shown in Fig. 5, TNF-� (10 ng/ml for 24 h) sig-nificantly upregulated the transcripts of ICAM-1 (consistently withthe results obtained with ICAM-1 protein and reported in Fig. 4),and of IL-8, GRO-�, GRO-�, RANTES, IP-10, IL-1�, IL-6 and TNF-�itself. As recent data have shown that VSV-G LV vector induce typeI interferon (Brown et al., 2007; Pichlmair et al., 2007), we expandedthe panel of pro-inflammatory cytokines to type I interferon � and�. VSV-G LV vector at 100 M.O.I. for 24 h did not induce significantupregulation of the transcripts above the basal levels, as reported inFig. 5, panel C, whereas infection experiments performed in parallelwith the first generation Ad-derived vector Ad.RSV.�gal (100 M.O.I.for 24 h) significantly up-modulated from 2- to 4-fold the basal lev-els of the transcripts of ICAM-1, IL-8, RANTES, IP-10, TNF-�, IL-6,IL-1� and IFN-� in A549 cells, as previously reported by other andour laboratories, as shown in Fig. 5, panel B. The same experimentalapproach was extended to the immortalized tracheal epithelial cellline CFT1-C2, derived from a �F508 homozygous CF patient. TNF-� significantly induced upmodulation of expression of a series ofgenes of the innate immunity, such as ICAM-1, IL-8, GRO-�, GRO-�,RANTES, IP-10, IL-1�, IL-6 and TNF-� itself, as shown in Fig. 6. Theseexperiments also confirmed that the epithelial respiratory cell lineCFT1-C2 is a good responsive sensor of pro-inflammatory stimuli.The VSV-G LV vector increased only transiently (24 h) the basal lev-

els of the IFN-�, while it did not induce significant upregulationof the type I interferon � and � transcripts above the basal levels(Fig. 6, panel C), whereas the Ad.RSV.�gal vector induced a 2–4-foldupregulation of the basal levels of GRO-�, RANTES and IL-6 (Fig. 6,panel B).

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rch 144 (2009) 8–17 13

As shown in Fig. 5, panels D and E, the exposure of the humanairway respiratory cell line A549 to the VSV-G LV vector for 1 weekor 2 weeks did not induce significant upregulation of the transcriptsabove the basal levels. Similar results were obtained in the CFT1-C2cell line (Fig. 6, panels D and E).

4. Discussion

A large consensus now considers the epithelial cells of theairways not only a physical barrier towards the environmen-tal pathogens but also a key sensor system of their presence(Diamond et al., 2000). Upon interaction of pathogens with therepertoire of Toll-like receptors expressed on the apical membrane,the epithelial cells release a series of mediators early activat-ing the innate immune response (Bals and Hiemstra, 2004). Thestudy of the expression of pro-inflammatory genes in these cellsis therefore a relevant information also for understanding theinflammatory potential of vectors utilized to transfer genes into res-piratory epithelial cells. We report here that the self-inactivatingpRRLsin.cPPT.CMV.Wpre LV vector (Follenzi et al., 2000) admin-istered to human epithelial respiratory cells does not induce theclassical multiple activation of pro-inflammatory genes usuallyelicited by adenovirus-derived vectors, with the exception of a mildand transient activation of IFN-� in the CFT1-C2 tracheal epithelialcell line.

The ideal vector system should have the following characteris-tics: (1) an adequate carrying capacity; (2) to be undetectable bythe immune system; (3) to be non-inflammatory; (4) to be safe tothe patients with pre-existing lung inflammation; (5) to have anefficiency sufficient to correct the mutated phenotype; and (6) tohave long duration of expression and/or the ability to be safely re-administered. First generation vectors derived from adenovirusesinduced a strong immune response towards neo-synthesized viralproteins, encoded by the residual early E2–E4 adenovirus genomeregions and exposed on the plasma membrane in association withMHC class I molecules (Yang et al., 1995; Woffendin et al., 1996).Considering that third-generation LV vectors contain only <5%residual LV genes (Conese et al., 2007), which should preclude, denovo, expression of potentially immunogenic lentiviral proteins intransduced target cells (Vigna and Naldini, 2000), these LV vec-tors present a first major advantage over Ad-derived vectors also interms of cytotoxic immune response. Additional studies are neces-sary to address whether lentivirus-based vectors can be consideredadequate for gene therapy of CF and other airway diseases in humanbeings. First, it is not known if any immune response is generatedafter transduction of the airways with these viruses. Kobinger etal. (2001) have reported that, in vivo, delivery of a lentiviral vec-

tor pseudotyped with the envelope derived from the Zaire strain ofEbola virus in tracheas of immunocompetent mice did not induceinflammatory infiltrates at any time points post-infection. We havealso observed that a last generation LV vector does not induce astrong immune response in the lung, as demonstrated by the scarce

Fig. 4. Expression of ICAM-1 protein in A549 cells. A549 cells have been exposed toTNF-� (50 ng/ml) (B and C), the LV vector (100 M.O.I. for 24 h) (D and F) or mediumalone (A) for 24 h, then detached, incubated with phycoerithrin (PE)-conjugated mAbagainst human ICAM-1 protein or phycoerithrin-conjugated irrelevant mouse IgGand analysed with a BD cytofluorometer. (A) Medium alone, PE-conjugated anti-human ICAM-1 Ab (mean fluorescence: mean fluorescence intensity at channel 126;positive cells: 1.0%). (B) TNF-�, PE-conjugated irrelevant mouse IgG (mean fluo-rescence intensity at channel 153; positive cells 0.3%). (C) TNF-�, PE-conjugatedanti-human ICAM-1 Ab (mean fluorescence intensity at channel 2262; positive cells:87.7%). (D) LV (100 M.O.I.), PE-conj. irrelevant mouse IgG (PE mean fluorescenceintensity at channel 47). (E) LV (100 M.O.I.), PE-conj. anti-human ICAM-1 Ab (PEmean fluorescence intensity at channel 40). Representative of three independentexperiments.

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resence of CD4+ and CD8+ lymphocytes in the bronchi/bronchiolif mice intratracheally injected with the very same vector used inhis study (Copreni, Conese, unpublished observations). Moreover,ersistent expression was observed in mice and rabbits (Wang etl., 1999), which suggests ‘immunotolerance or ignorance’ of theransgene in those animal models.

To circumvent the cytotoxic immune response of first generationd-derived vectors, different improvements have been accom-lished, which led to the design and production of vectors almostompletely devoid of the early regions from E1 to E4, the so-alled Ad helper-dependent or “gutless” vectors (Kochanek, 1999;ochanek et al., 1996), resulting in prolonged duration of genexpression. However, systemic administration of an Ad helper-ependent vector in the immune-competent mouse has been foundo induce expression of TNF-�, RANTES, MIP-1�/�, MIP-2 and IP-10,uggesting the possibility of a purely capsid-dependent inflamma-ory response (Muruve et al., 2004). This opened a series of studiesimed at elucidating signals and pathways to gain further insightsn the design of new vectors. Different pro-inflammatory moleculese.g. IL-8, IP-10, RANTES, ICAM-1) were induced by replication-efective Ad vectors in different cell lines, in vitro, and/or animalodels, in vivo, even upon short time exposure (Amin et al., 1995;icolis et al., 1998; Borgland et al., 2000; Tibbles et al., 2002).

mportantly, it has been found that mitogen activated proteininases (MAPK) and the nuclear factor �B play a central role in

he very quick induction of transcription of these pro-inflammatoryenes, thus suggesting that signals dependent on initial inter-ctions of the Ad surface proteins with host cell receptors (e.g.oxsackie adenovirus receptor, heparan sulfate glycosaminoglycans

ig. 5. Effect of VSV-G LV on transcription of genes of the innate immunity in A549ells. A549 cells have been exposed to (A) TNF-� (10 ng/ml for 24 h), (B) Ad.RSV.�gal,C) VSV-G LV vectors (100 M.O.I. for 1 day), (D) VSV-G LV vectors (100 M.O.I. for 1eek) and (E) VSV-G LV vectors (100 M.O.I. for 2 weeks). Total RNA was extracted and

ranscripts were quantified by real-time RT-PCR, normalized to housekeeping genesnd expressed in relation to the amount detected in unstimulated cells, reportedith the dashed line. Statistical significance has been calculated by ANOVA, aster-

sk indicates p < 0.05. Values are mean ± S.E.M. of four independent experimentserformed in duplicate.

Fig. 5. (Continued ).

and �V integrins) and the process of vector entry and dismantlinginto the target cell are sufficient to induce soluble and adhesionmolecules promoting the inflammatory response, as it was initiallyobserved in the lung of mice and non-human primates (Nicolis etal., 1998; Tibbles et al., 2002; Melotti et al., 2001; Bowen et al.,2002; Tamanini et al., 2003). Finally, we recently demonstratedthat the early interaction of adenoviral fiber with the coxsackieadenovirus receptor, but not with heparan sulfate glycosaminogly-cans, is sufficient to induce a pro-inflammatory signaling leadingto the expression of the chemokines IL-8, GRO-�/�, RANTES andIP-10 (Tamanini et al., 2006). With respect to the possibility that LVvector can induce an innate immune response just upon interac-tion with cell surface receptors of the epithelia respiratory cell, itshould be recalled that preliminary evidence suggests that VSV-Gpseudotyped LV vectors utilize heparan sulfate glycosaminogly-cans as one of the adhesion receptors needed for initial bindingto target cells (Guibinga et al., 2002; Copreni et al., 2004; Copreniet al., 2008). This is a further advantage in the light of the pre-

vious experience gained with Ad vectors and reasonably explainsour observation that the VSV-G pseudotyped LV utilized here doesnot activate the classical pro-inflammatory transcription factor NF-�B (Fig. 2), although the complete set of receptors and the signals

s Research 144 (2009) 8–17 15

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licited during the entry processes of LV vectors are not yet eluci-ated.

The limit of this work is that is restricted to an, in vitro, approachhereas most inflammatory problems arise, in vivo, due to the

omplex interplay among viral vectors, airway epithelial cells andlood-borne cells. The low or null pro-inflammatory potential ofV vectors needs to be confirmed, in vivo. It should be stressedhat HIV-1-derived LV vectors are not able to transduce the air-ay epithelium unless preconditioning of the tight junctions with

hemical and physical agents (Wang et al., 1999; Johnson et al.,000; Borok et al., 2001). The inflammatory potential of LV vec-ors could be then underscored by this treatments. Moreover, theransient opening of tight junctions could be detrimental for sameatients, such as those with cystic fibrosis, whose airways arelled with inflammatory and bacterial products. Another strategyctively pursued to obtain transduction of the airway epithelium,n vivo, is pseudotyping with alternative envelopes (Kobinger et al.,001; Sinn et al., 2005).

In this work we have compared two viral vectors which differn different manner to transfer genes in the cells (DNA versus RNAirus) but also in different ways to access the cell nucleus (VSV-

fusion versus adenovirus receptor mediated entry). Thereforee should be cautious when LV vectors are claimed to be non-

nflammatory at all. In vivo, the route of administration, the dosef vector, the genetic background and the promoters, can make aifference in inflammatory and immune responses to the vectorBessis et al., 2004).

ig. 6. Effect of VSV-G LV on transcription of genes of the innate immunity in CFT1-2 cells. CFT1-C2 cells have been exposed to (A) TNF-� (10 ng/ml for 24 h), (B)d.RSV.�gal or (C) VSV-G LV vectors (100 M.O.I. for 1 day), (D) VSV-G LV vectors

100 M.O.I. for 1 week) and (E) VSV-G LV vectors (100 M.O.I. for 2 weeks). Total RNAas extracted and transcripts were quantified by real-time RT-PCR, normalized toousekeeping genes and expressed in relation to the amount detected in unstimu-

ated cells, reported with the dashed line. Statistical significance has been calculatedy ANOVA, asterisk indicates p < 0.05. Values are mean ± S.E.M. of four independentxperiments performed in duplicate.

Fig. 6. (Continued ).

5. Conclusions

Can we conclude that the VSV-G LV tested here are “stealth”vectors as regards the innate immune response? In this respectwe observed a minimal and transient activation of transcriptionof IFN-� only in the tracheal cell line CFT1-C2 (Fig. 6), which isreminiscent of a similar induction reported after systemic admin-istration of the vector in mice (Follenzi et al., 2004). A large partof the, in vitro, analysis of the innate immune response after addi-tion of gene transfer vectors for CF lung disease has been performedin human epithelial respiratory cells, which signal the presence ofpathogens to immune cells by expressing different soluble media-tors. The results reported here depict a rather optimistic scenarioconcerning application of LV vectors for gene delivery, highlightingat the same time the importance of biosafety studies taking intoaccount the behaviour of immune cells which are the final target of

a complex series of signals originating from epithelial respiratorycells.

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cknowledgements

We are grateful to Luigi Naldini (H. S. Raffaele, Milan, I) for pro-iding the LV vector, Transgene (SA, Strasbourg, F) for providing thed vector, to Maria Cristina Dechecchi for helpful discussions and

o Federica Quiri for excellent technical assistance. This work wasupported by grants from the Italian Cystic Fibrosis Research Foun-ation (grant FFC#04/2005 with the contribution of “DelegazioneFC del Lago di Garda” to GC) and Fondazione Cariverona – Bando005 – Malattie rare e della povertà (to GC). VB is supported bygrant from Fondazione Cariverona and from the Italian Cystic

ibrosis Research Foundation.

eferences

min, R., Wilmott, R., Scharwarz, Y., Trapnell, B.C., Stark, J., 1995. Replication-deficient adenovirus induces expression of interleukin 8 by airway epithelialcells in vitro. Hum. Gene Ther. 6, 145–153.

uricchio, A., Kobinger, G., Anand, V., Hildinger, M., O’Connor, E., Maguire, A.M., Wil-son, J.M., Bennett, J., 2001. Exchange of surface proteins impacts on viral vectorcellular specificity and transduction characteristics: the retina as a model. Hum.Mol. Genet. 10, 3075–3081.

aekelandt, V., Eggermont, K., Michiels, M., Nuttin, B., Debyser, Z., 2003. Opti-mized lentiviral vector production and purification procedure prevents immuneresponse after transduction of mouse brain. Gene Ther. 10, 1933–1940.

als, R., Hiemstra, P.S., 2004. Innate immunity in the lung: how epithelial cells fightagainst respiratory pathogens. Eur. Respir. J. 23, 327–333.

essis, N., GarciaCozar, F.J., Boissier, M.C., 2004. Immune responses to gene therapyvectors: influence on vector function and effector mechanisms. Gene Ther. 11(Suppl. 1), S10–S17.

ezzerri, V., Borgatti, M., Nicolis, E., Lampronti, I., Dechecchi, M.C., Mancini, I.,Rizzotti, P., Gambari, R., Cabrini, G., 2008. Transcription factor oligodeoxynu-cleotides to NF-�B inhibit transcription of IL-8 in bronchial cells. Am. J. Respir.Cell Mol. Biol. 39, 86–96.

orgland, S., Bowen, G., Wong, N., Libermann, T., Muruve, D., 2000. Adenovirusvector-induced expression of the C-X-C Chemokine IP-10 is mediated throughcapsid-dependent activation of NF-�B. J. Virol. 74, 3941–3947.

orok, Z., Harboe-Schmidt, J.E., Brody, S.L., You, Y., Zhou, B., Li, X., Cannon, P.M., Kim,K.J., Crandall, E.D., Kasahara, N., 2001. Vesicular stomatitis virus G-pseudotypedlentivirus vectors mediate efficient apical transduction of polarized quiescentprimary alveolar epithelial cells. J. Virol. 75, 11747–11754.

owen, G., Borgland, S., Lam, M., Libermann, T., Wong, N., Muruve, D.A., 2002. Ade-novirus vector-induced inflammation: capsid-dependent induction of the C–Cchemokine RANTES requires NF-�B. Hum. Gene Ther. 13, 367–379.

rown, B., Sitia, G., Annoni, A., Hauben, E., Sergi Sergi, L., Zingale, A., Roncarolo, M.G.,Guidotti, L.G., Naldini, L., 2007. In vivo administration of lentiviral vectors triggersa type I interferon response that restricts hepatocyte gene transfer and promotesvectors clearance. Blood 109, 2797–2805.

arrabino, S., Carpani, D., Livraghi, A., Di Cicco, M., Costantini, D., Copreni, E.,Colombo, C., Conese, M., 2006. Dysregulated interleukin-8 secretion and NF-�Bactivation in human cystic fibrosis nasal epithelial cells. J. Cyst. Fib. 5, 113–119.

assivi, S.D., Liu, M., Boehler, A., Pierre, A., Tanswell, A.K., O’Brodovich, H., Mullen, J.B.,Slutsky, A.S., Keshavjee, S.H., 2000. Transplant immunosuppression increasesand prolongs transgene expression following adenoviral-mediated transfectionof rat lungs. J. Heart Lung Transplant. 19, 984–994.

onese, M., Castellani, S., Palmieri, L., Copreni, E., 2007. Lentivirus expression vectors.In: Hefferon, K.L. (Ed.), Virus Expression Vectors. Research Signpost TransworldResearch Network, Kerala, India, pp. 81–122.

opreni, E., Penzo, M., Carrabino, S., Conese, M., 2004. Lentivirus-mediated genetransfer to the respiratory epithelium: a promising approach to gene therapy ofcystic fibrosis. Gene Ther. 11 (Suppl. 1), S67–S75.

opreni, E., Castellani, S., Palmieri, L., Penzo, M., Conese, M., 2008. Involvement ofglycosaminoglycans in vescicular stomatitis virus G glycoprotein pseudotypedlentiviral vector-mediated gene transfer into airway epithelial cells. J. Gene Med.10, 1294–1302.

avies, J.C., Geddes, D.M., Alton, E.W., 2001. Gene therapy for cystic fibrosis. J. GeneMed. 3, 409–417.

iamond, G., Legarda, D., Ryan, L.K., 2000. The innate immune response of the res-piratory epithelium. Immunol. Rev. 173, 27–38.

errari, S., Geddes, D.M., Alton, E.W., 2002. Barriers to and new approaches for genetherapy and gene delivery in cystic fibrosis. Adv. Drug Deliv. Rev. 54, 1373–1393.

errari, S., Griesenbach, U., Geddes, D.M., Alton, E., 2003. Immunological hurdles tolung gene therapy. Clin. Exp. Immunol. 132, 1–8.

ollenzi, A., Ailles, L.E., Bakovic, S., Geuna, M., Naldini, L., 2000. Gene transfer by

lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 polsequences. Nat. Genet. 25, 217–222.

ollenzi, A., Battaglia, M., Lombardo, A., Annoni, A., Roncarolo, M.G., Naldini, L.,2004. Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of humanantihemophilic factor IX in mice. Blood 103, 3700–3709.

rch 144 (2009) 8–17

Goldman, M.J., Lee, P.S., Yang, J.S., Wilson, J.M., 1997. Lentiviral vectors for genetherapy of cystic fibrosis. Hum. Gene Ther. 8, 2261–2268.

Guibinga, G.H., Miyanohara, A., Esko, J.D., Friedmann, T., 2002. Cell surface heparansulfate is a receptor for attachment of envelope protein-free retrovirus-like par-ticles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells.Mol. Ther. 5, 538–546.

Hartman, Z.C., Appledorn, D.M., Amalfitano, A., 2008. Adenovirus vector inducedinnate immune responses: impact upon efficacy and toxicity in gene therapyand vaccine applications. Virus Res. 132, 1–14.

Johnson, L.G., Olsen, J.C., Naldini, L., Boucher, R.C., 2000. Pseudotyped human lentivi-ral vector-mediated gene transfer to airway epithelia in vivo. Gene Ther. 7,568–574.

Kafri, T., Blomer, U., Peterson, D.A., Gage, F.H., Verma, I.M., 1997. Sustained expressionof genes delivered directly into liver and muscle by lentiviral vectors. Nat. Genet.17, 314–317.

Kobinger, G.P., Weiner, D.J., Yu, Q.C., Wilson, J.M., 2001. Filovirus-pseudotyped lentivi-ral vector can efficiently and stably transduce airway epithelia in vivo. Nat.Biotechnol. 19, 225–230.

Kochanek, S., Clemens, P.R., Mitani, K., Chen, H.-H., Chan, S., Caskey, C.T., 1996. Anew adenoviral vector: replacement of all viral coding sequences with 28 kb ofDNA independently expressing both full-length dystrophin and �-galactosidase.Proc. Natl. Acad. Sci. U.S.A. 93, 5731–5736.

Kochanek, S., 1999. High-capacity adenoviral vectors for gene transfer and somaticgene therapy. Hum. Gene Ther. 10, 2451–2459.

Lemarchand, P., Ari Jaffe, H., Danel, C., Cid, M.C., Kleinman, H.K., Stratford-Perricaudet, L.D., Perricaudet, M., Pavirani, A., Lecocq, J.P., Crystal, R.G., 1992.Adenovirus-mediated transfer of a recombinant human a1-antitrypsin cDNA tohuman endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 89, 6482–6486.

Lim, F.Y., Kobinger, G.P., Weiner, D.J., Radu, A., Wilson, J.M., Crombleholme, T.M.,2003. Human fetal trachea-SCID mouse xenografts: efficacy of vesicular stom-atitis virus-G pseudotyped lentiviral-mediated gene transfer. J. Pediatr. Surg. 38,834–839.

Limberis, M., Anson, D.S., Fuller, M., Parsons, D.W., 2002. Recovery of airway cys-tic fibrosis transmembrane conductance regulator function in mice with cysticfibrosis after single-dose lentivirus-mediated gene transfer. Hum. Gene Ther. 13,1961–1970.

McCoy, R.D., Davidson, B.L., Roessler, B.J., Huffnagle, G.B., Janich, S.L., Laing, T.J., Simon,R.H., 1995. Pulmonary inflammation induced by incomplete or inactivated ade-noviral particles. Hum. Gene Ther. 6, 1553–1560.

Melotti, P., Nicolis, E., Tamanini, A., Rolfini, R., Pavirani, A., Cabrini, G., 2001. Acti-vation of NF-�B mediates ICAM-1 induction in respiratory cells exposed to anadenovirus-derived vector. Gene Ther. 8, 1436–1442.

Mittereder, N., March, K.L., Trapnell, B.C., 1996. Evaluation of the concentration andbioactivity of adenovirus vectors for gene therapy. J. Virol. 70, 7498–7509.

Morral, N., O’Neal, W., Rice, K., Leland, M., Kaplan, J., Piedra, P.A., Zhou, H., Parks, R.J.,Velji, R., Aguillar-Cordova, E., Wadsworth, S., Graham, F.L., Kochanek, S., Carey,K.D., Beaudet, A.L., 1999. Administration of helper-dependent adenoviralvectorsand sequential delivery of different vector serotype for long-term liver-directedgene transfer in baboons. Proc. Natl. Acad. Sci. U.S.A. 96, 12816–12821.

Muruve, D., Cotter, M.J., Zaiss, A.K., White, L.R., Liu, Q., Chan, T., Clark, S.A., Ross,P.J., Meulenbroek, R.A., Maelandsmo, G.M., Parks, R.J., 2004. Helper-dependentadenovirus vectors elicit intact innate but attenuated adaptive host immuneresponses in vivo. J. Virol. 78, 5966–5972.

Nicolis, E., Tamanini, A., Melotti, P., Rolfini, R., Berton, G., Cassatella, M.A., Bout, A.,Pavirani, A., Cabrini, G., 1998. ICAM-1 induction in respiratory cells exposed to areplication-deficient recombinant adenovirus in vitro and in vivo. Gene Ther. 5,131–136.

Otake, K., Ennist, D.L., Harrod, K., Trapnell, B.C., 1998. Nonspecific inflamma-tion inhibits adenovirus-mediated pulmonary gene transfer and expressionindependent of specific acquired immune responses. Hum. Gene Ther. 9,2207–2222.

Pichlmair, A., Diebold, S.S., Gschmeissner, S., Takeuchi, Y., Ikeda, Y., Collins,M.K., Reis e Sousa, C., 2007. Tubulovesicular structures within vesicularstomatitis virus G protein-pseudotyped lentiviral vector preparations carryDNA and stimulates antiviral responses via Toll-like receptor 9. J. Virol. 81,539–547.

Riordan, J.R., Rommens, J.M., Kerem, B.S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielen-ski, J., Lok, S., Plavsic, N., Chou, J.L., Dumm, M.L., Iannuzzi, M.C., Collins, F.S., Tsui,L.-C., 1989. Identification of the cystic fibrosis gene: cloning and characterizationof complementary DNA. Science 245, 1066–1073.

Rosenfeld, M.A., Yoshimura, K., Trapnell, B.C., Yoneyama, K., Rosenthal, E.R.,Dalemans, W., Fukayama, M., Bargon, J., Stier, L.E., Stratford-Perricaudet, L., Per-ricaudet, M., Guggino, W.B., Pavirani, A., Lecocq, J.-P., Crystal, R.G., 1992. In vivotransfer of the human cystic fibrosis transmembrane conductance regulator geneto the airway epithelium. Cell 68, 143–155.

Schwiebert, L.M., Estell, K., Propst, S.M., 1999. Chemokine expression in CF epithe-lia: implications for the role of CFTR in RANTES expression. Am. J. Physiol. 276,C700–C710.

Sheppard, D.N., Welsh, M.J., 1999. Structure and function of the CFTR chloride chan-nel. Physiol. Rev. 79, S23–S45.

Sinn, P.L., Burnight, E.R., Hickey, M.A., Blissard, G.W., McCray Jr., P.B., 2005. Persistentgene expression in mouse nasal epithelia following feline immunodeficiencyvirus-based vector gene transfer. J. Virol. 79, 12818–12827.

Tamanini, A., Rolfini, R., Nicolis, E., Melotti, P., Cabrini, G., 2003. MAP kinases and NF-�B collaborate to induce ICAM-1 gene expression in the early phase of adenovirusinfection. Virology 307, 228–242.

s Resea

T

T

V

V

to viral antigens create barriers to lung-directed gene therapy with recombinant

E. Copreni et al. / Viru

amanini, A., Nicolis, E., Bonizzato, A., Bezzerri, V., Melotti, P., Assael, B.M., Cabrini,G., 2006. Interaction of adenovirus type 5 fiber with the coxsackievirus and ade-novirus receptor activates inflammatory response in human respiratory cells. J.Virol. 80, 11241–11254.

ibbles, L.A., Spurrell, J.C.L., Bowen, G., Liu, Q., Lam, M., Zaiss, A.K., Robbins, S.M.,Hollenberg, M.D., Wickham, T., Muruve, D.A., 2002. Activation of p38 and ERKsignaling during adenovirus vector cell entry lead to expression of the C-X-Cchemokine IP-10. J. Virol. 76, 1559–1568.

andendriessche, T., Thorrez, L., Naldini, L., Follenzi, A., Moons, L., Berneman,

Z., Collen, D., Chuah, M.K., 2002. Lentiviral vectors containing the humanimmunodeficiency virus type-1 central polypurine tract can efficiently trans-duce nondividing hepatocytes and antigen-presenting cells in vivo. Blood 100,813–822.

igna, E., Naldini, L., 2000. Lentiviral vectors: excellent tools for experimental genetransfer and promising candidates for gene therapy. J. Gene Med. 2, 308–316.

rch 144 (2009) 8–17 17

Wang, G., Slepushkin, V., Zabner, J., Keshavjee, S., Johnston, J.C., Sauter, S.L., Jolly, D.J.,Dubensky Jr., T.W., Davidson, B.L., McCray Jr., P.B., 1999. Feline immunodeficiencyvirus vectors persistently transduce nondividing airway epithelia and correct thecystic fibrosis defect. J. Clin. Invest. 104, R55–R62.

Woffendin, C., Ranga, U., Yang, Z., Xu, L., Nabel, G.J., 1996. Expression of a protectivegene-prolongs survival of T cells in human immunodeficiency virus-infectedpatients. Proc. Natl. Acad. Sci. U.S.A. 93, 2889–2894.

Yang, Y., Li, Q., Ertl, H.C.J., Wilson, J.M., 1995. Cellular and humoral immune responses

adenoviruses. J. Virol. 69, 2004–2015.Yankaskas, J.R., Haizlip, J.E., Conrad, M., Koval, D., Lazarowski, E., Paradiso, A.M.,

Rinehart Jr., C.A., Sarkadi, B., Schlegel, R., Boucher, R.C., 1993. Papilloma virusimmortalized tracheal epithelial cells retain a well-differentiated phenotype.Am. J. Physiol. 264, C1219–C1230.