Toxicity of Fiber- and Penton Base–modified Adenovirus Type 5 Vectors on Lung Development in Newborn Rats
Authors: Paul Waszak, Laure Franqueville, Marie-Laure Franco-Motoya, Manuel Rosa-Calatrava, Olivier Boucherat, Leif Lindholm, Christophe Delacourt, Pierre Boulanger
Journal: Molecular Therapy (2007)

Transient overexpression of genes involved in lung regulation might prevent alveolar developmental disorders (ADDs) in premature neonates. However, adenovirus 5 (Ad5) vectors per se, and not isolated capsid proteins, induce ADDs after tracheal administration to newborn rats. To test the hypothesis that Ad5 capsid components are mainly responsible for ADDs, we evaluated newborn rats' lung development by morphometry after tracheal administration of a panel of Ad5 vectors with mutations in the fiber or penton base. Three distinct patterns of lung response were observed on postnatal day (PD) 21: (i) emphysematous-like lesions, common to Ad5 overexposing RGD motifs; (ii) altered septation, representative of the wild-type capsid Ad5 lesion; (iii) absence of lung toxicity, shown by Ad5 vectors with fibers shortened to seven repeats. None of these patterns correlated with the degree of lung inflammation or gene transduction. In contrast, a more impaired elastogenesis associated with emphysema was preceded by a significantly increased level of activated caspase 3 on PD11. Moreover, the altered septation was associated with a persistent and significant increase in terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)–positive alveolar septal cells on PD21. Our results underline the deleterious effects of Ad-induced apoptosis, which is not only responsible for limited transgene expression but also involved in lung development disorders. Transient overexpression of genes involved in lung regulation might prevent alveolar developmental disorders (ADDs) in premature neonates. However, adenovirus 5 (Ad5) vectors per se, and not isolated capsid proteins, induce ADDs after tracheal administration to newborn rats. To test the hypothesis that Ad5 capsid components are mainly responsible for ADDs, we evaluated newborn rats' lung development by morphometry after tracheal administration of a panel of Ad5 vectors with mutations in the fiber or penton base. Three distinct patterns of lung response were observed on postnatal day (PD) 21: (i) emphysematous-like lesions, common to Ad5 overexposing RGD motifs; (ii) altered septation, representative of the wild-type capsid Ad5 lesion; (iii) absence of lung toxicity, shown by Ad5 vectors with fibers shortened to seven repeats. None of these patterns correlated with the degree of lung inflammation or gene transduction. In contrast, a more impaired elastogenesis associated with emphysema was preceded by a significantly increased level of activated caspase 3 on PD11. Moreover, the altered septation was associated with a persistent and significant increase in terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)–positive alveolar septal cells on PD21. Our results underline the deleterious effects of Ad-induced apoptosis, which is not only responsible for limited transgene expression but also involved in lung development disorders. IntroductionAdenoviral vector–mediated gene transfer was developed primarily to treat genetic deficiencies,1Driskell RA Engelhardt JF Current status of gene therapy for inherited lung diseases.Annu Rev Physiol. 2003; 65: 585-612Crossref PubMed Scopus (64) Google Scholar but gene transfer could also be used to correct temporary abnormalities associated with acquired disorders.2Eming SA Krieg T Davidson JM Gene transfer in tissue repair: status, challenges and future directions.Expert Opin Biol Ther. 2004; 4: 1373-1386Crossref PubMed Scopus (38) Google Scholar During the neonatal period,3Waddington SN Kennea NL Buckley SM Gregory LG Themis M Coutelle C Fetal and neonatal gene therapy: benefits and pitfalls.Gene Ther. 2004; 11 (Suppl. 1): S92-S97Crossref PubMed Scopus (38) Google Scholar this may constitute an attractive way to prevent acquired respiratory disorders such as the bronchopulmonary dysplasia.4Thébaud B Ladha F Michelakis E Sawicka M Thurston G Eaton F et al.Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury.Circulation. 2005; 112: 2477-2486Crossref PubMed Scopus (408) Google Scholar However, further development of this approach is limited by the interference of the adenoviral vector itself in lung development. We have previously shown that adenovirus serotype 5 (Ad5) vectors induced lung growth disorders through interactions with extracellular matrix turnover and epithelial cell proliferation in neonatal rats.5Waszak P Franco-Montoya M-L Jacob M-P Deprez I Levame M Lafuma C et al.Effect of intratracheal adenoviral vector administration on lung development in newborn rats.Hum Gene Ther. 2002; 15: 1873-1885Crossref Scopus (11) Google Scholar Immune responses were not thought to contribute significantly to recombinant adenoviral toxicity, as we found excellent immune tolerance.5Waszak P Franco-Montoya M-L Jacob M-P Deprez I Levame M Lafuma C et al.Effect of intratracheal adenoviral vector administration on lung development in newborn rats.Hum Gene Ther. 2002; 15: 1873-1885Crossref Scopus (11) Google Scholar However, several reports6NIH Report Assessment of adenoviral vector safety and toxicity: report of the National Institutes of Health Recombinant DNA Advisory Committee.Hum Gene Ther. 2002; 13: 3-13Crossref PubMed Scopus (161) Google Scholar,7Jooss K Chirmule N Immunity to adeovirus and adeno-associated viral vectors: implications for gene therapy.Gene Ther. 2003; 10: 955-963Crossref PubMed Scopus (244) Google Scholar,8Molinier-Frenkel V Lengagne R Gaden F Hong SS Choppin J Gahery-Segard H et al.Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response.J Virol. 2002; 76: 127-135Crossref PubMed Scopus (142) Google Scholar have attributed the recombinant adenoviral toxicity primarily to acute innate immune responses to viral capsid components of incoming virions. Such a link between capsid components and toxicity has not been established with respect to the abnormalities induced by Ad5 vectors in developing lung of immunologically immature animals such as newborn rats.Human Ad5, which has been widely used as a gene vector, is a non-enveloped virus whose capsid comprises 240 hexons and 12 pentons, the latter located at the 12 apices of the icosahedral capsid. The penton capsomer is composed of two components, the penton base and the fiber. The fiber comprises three structural domains referred to as the tail, shaft, and knob. The tail is linked to the penton base and contains the N-termini of the three polypeptide subunits. The shaft domain consists multiple repeats of a 15-amino-acid motif and is terminated by the knob, a globular structure that contains the C-termini of the three subunits.9Rux JJ Burnett RM Adenovirus structure.Hum Gene Ther. 2004; 15: 1167-1176Crossref PubMed Scopus (91) Google Scholar Cell entry of Ad5 vectors has been characterized in vitro as a two-step process. The first step is a high-affinity interaction between the fiber knob and the Coxsackie and adenovirus receptor (CAR),10Bergelson JM Cunningham JA Droguett G Kurt-Jones EA Krithivas A Hong JS et al.Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5.Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2606) Google Scholar which requires a flexible, long fiber shaft of ideally 22 β-repeats.11Shayakhmetov DM Lieber A Dependence of adenovirus infectivity on length of the fiber shaft domain.J Virol. 2000; 74: 10274-10286Crossref PubMed Scopus (161) Google Scholar,12Smith TA Idamakanti N Rollence ML Marshall-Neff J Kim J Mulgrew K et al.Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice.Hum Gene Ther. 2003; 14: 777-787Crossref PubMed Scopus (178) Google Scholar,13Vigne E Dedieu JF Brie A Gillardeaux A Briot D Benihoud K et al.Genetic manipulations of adenovirus type 5 fiber resulting in liver tropism attenuation.Gene Ther. 2003; 10: 153-162Crossref PubMed Scopus (50) Google Scholar,14Wu E Pache L Von Seggern DJ Mullen TM Mikyas Y Stewart PL et al.Flexibility of the adenovirus fiber is required for efficient receptor interaction.J Virol. 2003; 77: 7225-7235Crossref PubMed Scopus (118) Google Scholar Attachment to CAR is followed by the binding of penton base RGD motifs to cellular integrins (αvβ1/3/5 and α3/5β1),15Zhang Y Bergelson JM Adenovirus receptors.J Virol. 2005; 79: 12125-12131Crossref PubMed Scopus (195) Google Scholar which mediates virus internalization via clathrin-coated pits.16Wickham TJ Mathias P Cheresh DA Nemerow GR Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment.Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1944) Google Scholar Recently, in vivo studies have shown that KKTK, a heparan sulfate glycoaminoglycans–binding motif present in the shaft, is involved in Ad5-mediated liver transduction in mice12Smith TA Idamakanti N Rollence ML Marshall-Neff J Kim J Mulgrew K et al.Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice.Hum Gene Ther. 2003; 14: 777-787Crossref PubMed Scopus (178) Google Scholar,17Bayo-Puxan N Cascallo M Gros A Huch M Fillat C Alemany R Role of the putative heparan sulfate glycosaminoglycan-binding site of the adenovirus type 5 fiber shaft on liver detargeting and knob-mediated retargeting.J Gen Virol. 2006; 87: 2487-2495Crossref PubMed Scopus (66) Google Scholar and non-human primates.18Smith TA Idamakanti N Marshall-Neff J Rollence M Wright P Kaloss M et al.Receptor interactions involved in adenoviral-mediated gene delivery after systemic administration in non-human primates.Hum Gene Ther. 2003; 14: 1595-1604Crossref PubMed Scopus (102) Google ScholarAd-induced toxicity has been evaluated for various chimeric capsids or modified recombinant Ad virus particles. Modifications have included ablation of CAR binding by knob domain mutations and deletion of the penton base RGD motif with or without ablation of the KKTK motif (reviewed in ref.19Nicklin SA Wu E Nemerow GR Baker AH The influence of adenovirus fiber structure and function on vector development for gene therapy.Mol Ther. 2005; 12: 384-393Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). A fiber shaft mutation in the KKTK motif decreased both liver transduction and toxicity in non-human primates, as determined by enzymatic blood assays and pathological criteria.18Smith TA Idamakanti N Marshall-Neff J Rollence M Wright P Kaloss M et al.Receptor interactions involved in adenoviral-mediated gene delivery after systemic administration in non-human primates.Hum Gene Ther. 2003; 14: 1595-1604Crossref PubMed Scopus (102) Google Scholar A similar lower hepatotoxicity has recently been observed in mice injected intravenously with chimeric Ad5/35 vectors carrying short-shafted serotype-35 fibers.20Shayakhmetov D Li Z-Y Ni S Lieber A Analysis of adenovirus sequestration in the liver, transduction of hepatic cells, and innate toxicity after injection of fiber-modified vectors.J Virol. 2004; 78: 5368-5381Crossref PubMed Scopus (176) Google Scholar It is noteworthy that although the infectivity of a CAR binding–ablated vector was greatly reduced, biodistribution and hepatotoxicity were not systematically affected.21Alemany R Curiel DT CAR-binding ablation does not change biodistribution and toxicity of adenoviral vectors.Gene Ther. 2001; 8: 1347-1353Crossref PubMed Scopus (241) Google ScholarTo our knowledge, there are no available data on the influence of Ad capsid modifications on lung development. The main objective of this study was therefore to test the hypothesis that Ad vector capsid components are the major factors responsible for Ad5-induced lung developmental toxicity. To this end, we first analyzed the effects of isolated Ad5 major capsid components on developing lung structures after intra-tracheal administration to newborn rats. Because none of the major capsid proteins had a deleterious effect on lung development, we investigated various fiber- and penton base–modified Ad5 vectors. We then explored the mechanisms that could explain the different lung development disorders observed with the various vectors, including gene transduction, inflammation, elastogenesis, and apoptosis.ResultsLung morphometry after tracheal administration of individual Ad capsid proteinsTo evaluate the potential direct role of isolated Ad proteins on lung development, a morphometric evaluation of lung structures was performed in 21-day-old rats 18 days after tracheal administration of hexon, penton base, fiber, or penton (penton-base capsomer bound to its fiber) at doses per animal equivalent to their respective content in 6 × 1010 virus particles or mock inoculum. No significant lung growth disorders were induced by any individual protein (Supplementary Table S1). This suggested that the lung toxicity of Ad5 was due to the virion as a whole, rather than to a specific, individual viral component or components.Lung morphometry after tracheal administration of capsid-modified Ad vectorsMorphometric evaluations were performed in 21-day-old rats 18 days after tracheal administration of 1 × 1010 particles of Ad vector or mock inoculum, as described above (Table 1). We first compared the effect of the fiber shaft mutant AdβGal-KKTK* and the fiber knob mutant AdβGal-RGD4C with AdβGal, the parental clone with no modification of its capsid, which was used as the positive control and with mock inoculum (buffer solution) as the negative control.Table 1Characteristics of, and lung responses to, unmodified and capsidhypenminusmodified AdsAdβGalAdβGal-KKTK*AdβGal-RGD4CAdGFPAdGFP-R7-knobAdGFP-R7-ΔknobAdGFP-EGDReporter geneβ-galactosidaseβ-galactosidaseβ-galactosidaseGFPGFPGFPGFPCapsid mutationNoneKKTK to GAGARGD4C in HI loopNone7 repeats (R7)R7 without knobRGD to EGDInfectivity index (PFU/PP)1:31:6,0001:2301:51:2101:1,0001:6Lung toxicity patternAltered septationAltered septationEmphysematous-likeAltered septationNoneEmphysematous-likeAltered septationTransduction+++++++++00++Inflammation+++++++00+++Impaired elastogenesis++ND+++NDNoneNDNDIncreased apoptosisPD21NDPD11NDNoneNDNDAbbreviations: Ad, adenovirus; GFP, green fluorescent protein; ND, not determined; PD11, PD21, postnatal days 11 and 21, respectively; PFU, plaque-forming units; PP, physical particles. Open table in a new tab Both mutants, as well as AdβGal, induced alveolar growth disorders. Lung sections exhibited a simplified alveolar structure with fewer secondary septa and larger alveoli (Figure 1). Morphometric measurements in the AdβGal group were in agreement with our previous findings, with a significant reduction by 25% in specific alveolar surface (total or absolute alveolar surface normalized to body weight) as compared with the control group (Figure 2a). Lung sections observed in the AdβGal-KKTK* group were similar to those observed in the AdβGal group, although the disorders were less severe, with a significant reduction in specific alveolar surface by 15% (Figure 2a). These results suggested reduced septation. The AdβGal-RGD4C group differed from other groups in showing a lung distension pattern. Morphometric measurements in this group were characterized by a decrease in relative alveolar surface (alveolar surface actually measured on lung sections) (Figure 2b), associated with an increase in both specific lung volume (lung volume normalized to body weight) (Figure 2c) and specific alveolar airspace (absolute alveolar airspace normalized to body weight) (Figure 2d) as compared with the AdβGal group. Absolute and specific alveolar surface areas were also reduced in the AdβGal-RGD4C group, but without reaching significance. Data on interstitial tissue, airways, and blood vessels did not differ significantly among the groups, regardless of how they were expressed (Supplementary Table S2).Figure 2Lung morphometry after tracheal administration of adenovirus (Ad) vectors carrying long-shafted fibers. (a) Specific alveolar surface, (b) relative alveolar surface, (c) specific lung volume, and (d) specific alveolar airspace were analyzed 18 days after intra-tracheal administration to 3-day-old rats of mock inoculum or various β-galactosidase-expressing Ads, AdβGal, AdKKTK*, and AdRGD4C, at 1 × 1010 PP per animal. Each point corresponds to a single datum. Error bars show mean ± SEM. *P < 0.05 between groups sharing this symbol. **P < 0.01 between groups sharing this symbol. GFP, green fluorescent protein; PP, physical particles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next compared the effects on lung development of various Ad vectors expressing green fluorescent protein (GFP), AdGFP-R7-Δknob and AdGFP-R7-knob, carrying short-shafted (R7) fibers with or without knob deletion, and AdGFP-EGD, a penton-base RGD mutant lengthened by wild-type fiber. AdGFP, the parental vector with no modification of its capsid, and the vector buffer were used as positive and negative controls, respectively.All vectors except AdGFP-R7-knob induced alveolar growth disorders with fewer secondary septa and larger alveoli (Figure 1). Morphometric studies showed a significant 15% decrease in specific alveolar surface area in the AdGFP and AdGFP-EGD groups as compared with the control group (Figure 3a), corresponding to altered septation. As for the AdβGal-RGD4C group, lung distension was the prominent feature measured in the AdGFP-R7-Δknob group, with a significant 22% decrease in relative alveolar surface as compared with the control group (Figure 3b), associated with increases in specific lung volume (Figure 3c) and alveolar airspace (Figure 3d), but without reaching significance. Data on interstitial tissue, airways, and blood vessels did not differ significantly among the groups, regardless of how they were expressed (Supplementary Table S3).Figure 3Lung morphometry after tracheal administration of adenovirus (Ad) vectors carrying short-shafted fibers or a penton base mutation. (a) Specific alveolar surface, (b) relative alveolar surface, (c) specific lung volume and (d) specific alveolar airspace were analyzed 18 days after intra-tracheal administration to 3-day-old rats of mock inoculum or various green fluorescent protein (GFP)–expressing Ads, AdGFP, AdR7-knob, AdR7Δknob, and AdEGD, at 1 × 1010 PP. Each point corresponds to a single datum. Error bars show mean ± SEM. * or ¤P < 0.05 between groups sharing these symbols. #or §: P < 0.01 between groups sharing these symbols. PP, physical particles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ad-mediated gene transduction of newborn rat lungsTo investigate the potential relationship between Ad-induced lung development disorders and gene transduction efficiency, we quantified the expression of reporter gene, LacZ or gfp, in lung homogenates 5 days after Ad administration. β-galactosidase was found to be expressed at similar levels for AdβGal and AdβGal-RGD4C, but at tenfold lower levels for AdβGal-KKTK* (Figure 4a). With the GFP-expressing vectors, only two, AdGFP and AdGFP-EGD, had detectable GFP expression, although at 35% lower levels for AdGFP-EGD (Figure 4b). The other two, AdGFP-R7-knob and AdGFP-R7-Δknob, failed to transduce newborn rat lungs at detectable levels (Figure 4b).Figure 4Lung transduction and inflammation 5 days after tracheal administration of various adenovirus (Ad) vectors to 3-day-old rats. In (a), lung β-galactosidase and myeloperoxidase (MPO) concentrations were determined after administration of mock inoculum or Ads at 7 × 109 PP per animal. In (b), lung GFP (expressed as arbitrary units (AU)) and MPO concentrations were determined after administration of Ads at 2 × 109 PP per animal. All results are reported as means ± SEM. Numbers of animals evaluated are given in parentheses. *P < 0.05 versus AdβGal and AdRGD4C groups. **P = 0.05 versus AdGFP group. ***P = 0.05 versus control group injected with mock inoculum. PP, physical particles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lung inflammationThe degree of inflammation in treated lungs was evaluated by the concentration of myeloperoxidase (MPO) in lung parenchyma. MPO is a glycoprotein expressed in all cells of the myeloid lineage and in particular in polymorph nuclear neutrophils. MPO concentration, assayed 5 days after tracheal administration of Ad, therefore reflected the recruitment of polymorph nuclear neutrophils in lung tissue. After administration of Ad expressing βGal (Figure 4a) or GFP (Figure 4b), no significant MPO increase was observed as compared with the control group, except for the AdGFP-EGD group, where the lung MPO concentration increased by 185%. Moreover, no correlation between lung inflammation and transduction was observed, as illustrated by the AdGFP-EGD group, which exhibited a 50% higher MPO concentration despite a 35% lower transduction than the AdGFP group.Lung elastic fiber statusWe next quantified the status of parenchymal elastic fibers after administration of Ad vectors responsible for the three different patterns observed after administration: (i) AdβGal for the altered septation pattern, (ii) AdβGal-RGD4C for the emphysematous-like pattern, and (iii) AdGFP-R7-knob for unaltered lung. Morphological analysis showed a marked decrease in the number and thickness of alveolar fibers in the lungs of rats given AdβGal-RGD4C, and to a lesser degree AdβGal, compared with the control and AdGFP-R7-knob groups. The morphometric study confirmed this observation: the relative (Figure 5a) and absolute (Figure 5b) elastic fiber lengths after AdβGal-RGD4C administration were decreased significantly compared with the control and AdGFP-R7-knob groups (25–28%). In terms of specific values (Figure 5c), the decrease by 26–28% in elastic fiber length was significant for AdβGal as compared with control and AdGFP-R7-knob. The 20% decrease observed with AdβGal-RGD4C remained significant only when compared with the control group.Figure 5Lung elastic fiber lengths 18 days after tracheal administration of various adenovirus (Ad) vectors to 3-day-old rats. Morphometric data concerning (a) relative, (b) absolute, and (c) specific lung parenchyma elastic fiber lengths were analyzed after administration of a mock inoculum or AdβGal, AdGFP-R7-knob, or AdβGal-RGD4C at 1 × 1010 PP per animal. Each point corresponds to a single datum. Error bars show mean ± SEM. *P < 0.05 versus control and AdGFP-R7-knob groups. **P < 0.05 versus control group injected with mock inoculum. GFP, green fluorescent protein; PP, physical particles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lung cleaved caspase 3 contentLung cleaved caspase 3 content was quantified, before the alveolar wall physiological apoptosis period, 8 days after intra-tracheal administration of the same three vectors, AdβGal, AdβGal-RGD4C, and AdGFP-R7-knob. Although the three vectors showed an increased activated caspase 3 level, the mean 375% increase in the AdRGD4C group was the only significantly increased value compared with the control group (Figure 6a and b).Figure 6Lung cleaved caspase 3 content 8 days after tracheal administration of various adenovirus (Ad) vectors to 3-day-old rats at 1 × 1010 PP. (a) Western blot analysis. Each lane represents one animal sample. (b) Quantification of the cleaved caspase 3 signals. Results are reported as means ± SEM of the change in percentage, with the control group corresponding to 100%. *P < 0.05 versus control group. GFP, green fluorescent protein; PP, physical particles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TUNEL-positive alveolar septal cellsAfter the alveolar wall physiological apoptosis period, 18 days after intra-tracheal administration of AdβGal, AdβGal-RGD4C, and AdGFP-R7-knob, alveolar septal cells undergoing apoptosis were quantified by the in situ terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) technique. As shown in Figure 7a and b, the group injected with AdβGal was the only one to exhibit a significant increase in the percentage of apoptotic cells (threefold) compared with the control group (15.6 ± 1.3% and 5.5 ± 1.4%, respectively). In contrast to the augmentation of activated caspase 3 level at earlier times, AdβGal-RGD4C did not increase apoptosis of septal wall cells (compare Figures 6 and 7).Figure 7Terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)–positive cells in alveolar parenchyma 18 days after tracheal administration of various Ads to 3-day-old rats at 1 × 1010 PP. (a) Note the clear increase in brown positive cells (arrowheads) in the AdβGal group compared with the other groups. Stain, peroxidase; counterstain, methyl green. (b) Quantification of TUNEL-positive cells, expressed as a percentage of the total cells counted. Results are reported as means ± SEM. *P < 0.05 versus control group. Ad, adenovirus; GFP, green fluorescent protein; PP, physical particles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionWe have previously described the deleterious effects on lung growth of newborn rats induced by recombinant E1-deleted Ad null vectors.5Waszak P Franco-Montoya M-L Jacob M-P Deprez I Levame M Lafuma C et al.Effect of intratracheal adenoviral vector administration on lung development in newborn rats.Hum Gene Ther. 2002; 15: 1873-1885Crossref Scopus (11) Google Scholar The present study confirms the lung toxicity of adenovirions using AdβGal and AdGFP, two recombinant Ads with unmodified capsid and different reporter genes. However, the mechanisms responsible for the Ad-induced alveolar development disorders (ADDs) and the possible involvement of individual Ad capsid protein(s) were not investigated in our initial work. The aim of this study was therefore to test the hypothesis that Ad vector capsid components were responsible for Ad toxic effects on lung development.We first explored the effects on lung development of each of the major Ad capsid proteins tested individually: hexon, penton base, fiber, and complete penton (penton base + fiber). None of these proteins induced detectable alterations of the lung structures, suggesting that the whole adenovirion was implicated in the lung toxicity process. We then evaluated the degree of lung toxicity of Ad vectors carrying various capsid modifications in the two capsid components implicated in attachment and internalization of adenovirions: the fiber and penton base.We observed three different patterns of lung response to our vectors, as summarized in Table 1:(i) AdβGal, AdGFP, shaft mutant AdβGal-KKTK*, and penton base mutant AdGFP-EGD induced a clear alteration of alveolar septation characterized by a significant decrease in specific alveolar surface with no significant distension (Figures 1–3).(ii) Lung alterations induced by AdβGal-RGD4C and AdGFP-R7-Δknob, a short-shafted, knob-deleted mutant, consisted of lung distension or an emphysematous-like pattern, characterized by increases in specific lung volume, specific alveolar airspace, and a significant decrease in the relative alveolar surface. In this case, altered septation was likely to occur, but was probably underestimated because of the lung distension. Indeed, the reduction in alveolar surface was present but reached significance only when expressed in terms of relative value (surface density) and not when taking into account the overinflated lungs, as in absolute or specific values.(iii) AdGFP-R7-knob, a vector with a knob domain terminating its short-shafted fibers, induced no significant alteration.We speculated that the emphysematous-like toxicity pattern could have been induced primarily by the overexposure of RGD motifs, shared by both AdβGal-RGD4C and AdGFP-R7-Δknob. In the case of AdβGal-RGD4C, ectopic RGDs were displayed in the HI loop of the fiber knob, in addition to the natural RGD motifs in the penton base. In AdGFP-R7-Δknob, the deletion of the knob domain and the distal 15 shaft repeats resulted in better accessibility of the penton base RGD-loop.9Rux JJ Burnett RM Adenovirus structure.Hum Gene Ther. 2004; 15: 1167-1176Crossref PubMed Scopus (91) Google Scholar Our hypothesis was strengthened by the absence of detectable toxicity of AdGFP-R7-knob (Figure 1 and Table 1): in this particular vector, the presence of the knob domain at the extremity of the short and rigid fiber shaft is assumed to prevent, by steric hindrance, the interaction between the RGD ligands in the penton base and the host cell integrins.14Wu E Pache L Von Seggern DJ Mullen TM Mikyas Y Stewart PL et al.Flexibility of the adenovirus fiber is required for efficient receptor interaction.J Virol. 2003; 77: 7225-7235Crossref PubMed Scopus (118) Google Scholar A similar protective shield against virus–cell interactions has been proposed to explain the lack of liver toxicity of Ad5/35, a chimeric A