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11. Gene Expression Is Altered by Infection with Adenovirus and Adeno-Derived Vectors. Global Monitoring with Oligonucleotide Arrays
HOST INTERACTIONS AND CANCER THERAPY HOST INTERACTIONS AND CANCER THERAPY 11. Gene Expression Is Altered by Infection with Adenovirus and Adeno-Derived Vectors. Global Monitoring with Oligonucleotide Arrays Yuri Martina,1 Daniele Avitabile,1 Stefania Piersanti,1 Gioia Cherubini,1 Barbara Salone,1 Isabella Saggio.1 1 Genetics and Molecular Biology, University La Sapienza, Rome, Italy. In the last years researches on Adenovirus have focused mainly on its applications in the biomedical field and on its use in gene therapy protocols. Although applied research has explained some aspects of Adenoviruses biology, there are a lot of contradictory results deriving from clinical protocols. Recently the community has understood the importance of studying deeper Adenovirus biology as unique instrument to understand the biological processes derived from its infection. In this work we used the microarrays (GeneChip – Affymetrix) and the Real Time RT-PCR as techniques to investigate from a global point of view the effects on cellular transcription during infections mediated by an Adenovirus wild type (wt) type 5, an E1/E3 deleted vector derived from this virus, and a gutted adenoviral vector. Results of our analysis are concordant with the previous published data, i.e. activation of immune modulators, apoptotic genes and transcription factors, with significant differences among different viruses, especially evident at late times after infection. Besides, we succeeded in identifying a series of genes that were not known to be implicated in the infection of cells by Adenoviruses. Among these the most interesting are the Prion Protein and the receptor for the viral Semaphorin. Taken together, our results give a complete pattern of the molecular impact and toxicity on the host cell of both of wild type Adenovirus and gene therapy vectors thereof derived.
12. Tethered Mucins Restrict Adenoviral Vector Access to Apical Receptors Expressed on Respiratory Epithelial Cells Both In Vitro and In Vivo Jaclyn R. Stonebraker,1 Robert W. Lenfestey,1 Jeffrey M. Bergelson,2 Sandra Gendler,3 Richard C. Boucher,1 Wanda K. O’Neal,1 Raymond J. Pickles.1 1 CF/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Dept. of Immunologic and Infectious Diseases, Childrens Hospital of Philadelphia, Philadelphia, PA; 3Dept. of Molecular and Cellular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ. We have previously reported that inefficient adenoviral (Ad)mediated gene transfer to well-differentiated human airway epithelial (HAE) cells in vitro is due to the absence of human (h)CAR and alphaV beta5 integrins from the lumenal surface of the airway. Retargeting an Ad attachment site to the apical membrane of HAE by linking the external domain of hCAR to a glycophosphatidylinositol (GPI)-linker did not result in efficient gene transfer with lumenally-applied Ad unless the glycosylation state of the lumenal surface glycocalyx was altered. Specifically, inhibitors of O-linked glycosylation and protease were the most effective treatments at enhancing the access of Ad to apically located GPI-hCAR, suggesting that epithelial cell surface glycoproteins with O-linked glycosylation e.g., mucins, provide a barrier to efficient Ad access. To determine whether these observations in vitro are relevant to the in vivo situation, we generated transgenic mice expressing epithelial cell-specific apical membrane-targeted GPIhCAR by utilizing the keratin 18 (K18) epithelial cell-specific promoter. We evaluated nine separate K18/GPI-hCAR transgenic Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
lines and have observed apical membrane specific GPI-hCAR expression, as detected with an anti-hCAR antibody (RmcB), throughout the epithelial cells of the conducting airways including the nasal, tracheal and bronchial/bronchiolar regions. To test whether expression of GPI-hCAR in mouse airway epithelium in vivo allowed for an enhancement of Ad-mediated gene transfer, we intratracheally administered Ad expressing LacZ (5-10 ml of 1011 particles/ml) to a defined epithelium region in both GPI-hCAR transgenics (n=30) and littermate controls (n=30). Fourty-eight hours later, tracheae were removed and stained for beta-galactosidase expression. We calculated the percentage of cells infected in the region where Ad was administered using quantitative morphometrical analyses. GPIhCAR transgenics expressed LacZ in 1.7 ± 0.3 % of cells and littermate controls in 1.6 ± 0.2 % of cells, indicating no significant difference in Ad-mediated gene transfer in the absence or presence of an apical receptor. The availability of a Muc1 knockout mouse model enabled us to test our hypothesis that tethered mucins restrict Ad access. After breeding the GPI-hCAR mice with the Muc1 knockout mice and administration of AdLacZ to the tracheal epithelium, Muc1 knockout mice alone expressed transgene in 4.9 ± 0.5 % of cells, indicating an enhanced gene transfer with the absence of Muc1 alone. In mice also expressing GPI-hCAR, gene transfer efficiency was further enhanced to 7.9 ± 0.6 % of cells. These data collectively show that Muc1 tethered mucins partially restrict Ad access to the lumenal surface of the airway epithelium but suggest that other tethered mucin-types likely account for a significant portion of the glycocalyx barrier. An increased understanding of the barrier effects of airway glycocalyx structures to viruses such as Ad, may enable strategies to be developed to improve gene transfer efficiency to the lung epithelium.
13. Adenovirus Binding Triggers Activation of Cytoplasmic Dynein, the Molecular Motor That Drives Adenovirus Translocation to the Nucleus Samir Kelkar,1 Ronald G. Crystal,1 Philip L. Leopold.1 1 Weill Medical College of Cornell University, New York, NY. The importance of adenovirus (Ad)-based gene therapy vectors as therapeutic gene transfer vehicles is derived in part from the ability of the Ad capsid to accomplish efficient cell membrane penetration and translocation to the nucleus. Translocation to the nucleus requires interaction of the capsid with the molecular motor, cytoplasmic dynein, which confers movement of intracellular cargo along the microtubule cytoskeleton from the cell periphery towards the microtubule organizing center and the nucleus. Previous studies have established that cytoplasmic dynein activity can by modulated by phosphorylation of dynein subunits following activation of cell surface receptors. We hypothesized that adenovirus can potentiate the activity of the cytoplasmic dynein motor complex by altering the phosphorylation state of the cytoplasmic dynein complex in the target cell. To investigate the ability of Ad infection to alter the serine phosphorylation status of the cytoplasmic dynein complex, cell lysate was collected from naive A549 lung epithelial cells or A549 cells infected with 5x104 Ad particles per cell for 15 min. Cytoplasmic dynein was immunopurified using a monoclonal antibody against the 74.1 dynein intermediate chain, and the serine phosphorylation status of the immunopurified cytoplasmic dynein complex members evaluated by Western analysis using a monoclonal antibody against phosphoserine moieties. Cytoplasmic dynein complex peptides isolated from cells infected with Ad vectors exhibited greater serine phosphorylation than peptides isolated from naive A549 cells suggeting that Ad potentiates the activation state of dynein early during the infection process. To examine the consequences of Ad infection on dynein-microtubule interactions, cytoplasmic dynein-microtubule binding was evaluated in cell lysates prepared from Ad-infected cells. Cell lysate was collected from AdS5
12. Tethered Mucins Restrict Adenoviral Vector Access to Apical Receptors Expressed on Respiratory Epithelial Cells Both In Vitro and In Vivo Jaclyn R. Stonebraker,1 Robert W. Lenfestey,1 Jeffrey M. Bergelson,2 Sandra Gendler,3 Richard C. Boucher,1 Wanda K. O’Neal,1 Raymond J. Pickles.1 1 CF/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Dept. of Immunologic and Infectious Diseases, Childrens Hospital of Philadelphia, Philadelphia, PA; 3Dept. of Molecular and Cellular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ. We have previously reported that inefficient adenoviral (Ad)mediated gene transfer to well-differentiated human airway epithelial (HAE) cells in vitro is due to the absence of human (h)CAR and alphaV beta5 integrins from the lumenal surface of the airway. Retargeting an Ad attachment site to the apical membrane of HAE by linking the external domain of hCAR to a glycophosphatidylinositol (GPI)-linker did not result in efficient gene transfer with lumenally-applied Ad unless the glycosylation state of the lumenal surface glycocalyx was altered. Specifically, inhibitors of O-linked glycosylation and protease were the most effective treatments at enhancing the access of Ad to apically located GPI-hCAR, suggesting that epithelial cell surface glycoproteins with O-linked glycosylation e.g., mucins, provide a barrier to efficient Ad access. To determine whether these observations in vitro are relevant to the in vivo situation, we generated transgenic mice expressing epithelial cell-specific apical membrane-targeted GPIhCAR by utilizing the keratin 18 (K18) epithelial cell-specific promoter. We evaluated nine separate K18/GPI-hCAR transgenic Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
lines and have observed apical membrane specific GPI-hCAR expression, as detected with an anti-hCAR antibody (RmcB), throughout the epithelial cells of the conducting airways including the nasal, tracheal and bronchial/bronchiolar regions. To test whether expression of GPI-hCAR in mouse airway epithelium in vivo allowed for an enhancement of Ad-mediated gene transfer, we intratracheally administered Ad expressing LacZ (5-10 ml of 1011 particles/ml) to a defined epithelium region in both GPI-hCAR transgenics (n=30) and littermate controls (n=30). Fourty-eight hours later, tracheae were removed and stained for beta-galactosidase expression. We calculated the percentage of cells infected in the region where Ad was administered using quantitative morphometrical analyses. GPIhCAR transgenics expressed LacZ in 1.7 ± 0.3 % of cells and littermate controls in 1.6 ± 0.2 % of cells, indicating no significant difference in Ad-mediated gene transfer in the absence or presence of an apical receptor. The availability of a Muc1 knockout mouse model enabled us to test our hypothesis that tethered mucins restrict Ad access. After breeding the GPI-hCAR mice with the Muc1 knockout mice and administration of AdLacZ to the tracheal epithelium, Muc1 knockout mice alone expressed transgene in 4.9 ± 0.5 % of cells, indicating an enhanced gene transfer with the absence of Muc1 alone. In mice also expressing GPI-hCAR, gene transfer efficiency was further enhanced to 7.9 ± 0.6 % of cells. These data collectively show that Muc1 tethered mucins partially restrict Ad access to the lumenal surface of the airway epithelium but suggest that other tethered mucin-types likely account for a significant portion of the glycocalyx barrier. An increased understanding of the barrier effects of airway glycocalyx structures to viruses such as Ad, may enable strategies to be developed to improve gene transfer efficiency to the lung epithelium.
13. Adenovirus Binding Triggers Activation of Cytoplasmic Dynein, the Molecular Motor That Drives Adenovirus Translocation to the Nucleus Samir Kelkar,1 Ronald G. Crystal,1 Philip L. Leopold.1 1 Weill Medical College of Cornell University, New York, NY. The importance of adenovirus (Ad)-based gene therapy vectors as therapeutic gene transfer vehicles is derived in part from the ability of the Ad capsid to accomplish efficient cell membrane penetration and translocation to the nucleus. Translocation to the nucleus requires interaction of the capsid with the molecular motor, cytoplasmic dynein, which confers movement of intracellular cargo along the microtubule cytoskeleton from the cell periphery towards the microtubule organizing center and the nucleus. Previous studies have established that cytoplasmic dynein activity can by modulated by phosphorylation of dynein subunits following activation of cell surface receptors. We hypothesized that adenovirus can potentiate the activity of the cytoplasmic dynein motor complex by altering the phosphorylation state of the cytoplasmic dynein complex in the target cell. To investigate the ability of Ad infection to alter the serine phosphorylation status of the cytoplasmic dynein complex, cell lysate was collected from naive A549 lung epithelial cells or A549 cells infected with 5x104 Ad particles per cell for 15 min. Cytoplasmic dynein was immunopurified using a monoclonal antibody against the 74.1 dynein intermediate chain, and the serine phosphorylation status of the immunopurified cytoplasmic dynein complex members evaluated by Western analysis using a monoclonal antibody against phosphoserine moieties. Cytoplasmic dynein complex peptides isolated from cells infected with Ad vectors exhibited greater serine phosphorylation than peptides isolated from naive A549 cells suggeting that Ad potentiates the activation state of dynein early during the infection process. To examine the consequences of Ad infection on dynein-microtubule interactions, cytoplasmic dynein-microtubule binding was evaluated in cell lysates prepared from Ad-infected cells. Cell lysate was collected from AdS5