136 The Role of Endosomal Escape and Mitogen Activated Protein Kinases in Adenoviral Activation of the Innate Immune Response Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The Americ[.]
ADENOVIRUS AND OTHER DNA VIRUS VECTORS I not by the two flanking ITRs This would allow for encapsidation of multiple consecutive covalently linked copies of small genomes Reference list: Samulski, R J and McCarty, D M (2008) Duplexed parvovirus vectors McCarty, D M (2008) Mol Ther 16, 16481656 McCarty, D M., Monahan, P E., and Samulski, R J (2001) Gene Ther 8, 1248-1254 McCarty, D M., Fu, H., Monahan, P E., Toulson, C E., Naik, P., and Samulski, R J (2003) Gene Ther 10, 2112-2118 Urabe, M., Nakakura, T., Xin, K Q., Obara, Y., Mizukami, H., Kume, A., Kotin, R M., and Ozawa, K (2006) J Virol 80, 1874-1885 134 Targeted Integration of an rAAV Vector into the AAVS1 Site: Measurement of the Integration Rate without Selection Peter Ward,1 Christopher E Walsh.1 Department of Medicine/Tisch Cancer Institute, Mount Sinai School of Medicine, New York, NY Recently several problems have become apparent in the use of AAV as an in vivo gene delivery vehicle such as random viral integration and an immune response to the viral capsid These problems suggests the usefulness of an ex vivo method of AAV gene delivery for some applications AAV has the capacity to integrate in a site-specific manner into chromosome 19 (a site designated AAVS1) The only virally encoded protein necessary for integration into AAVS1 is the AAV Rep protein We chose to evaluate the feasibility of using a targeted integration approach that may be useful for eventual ex vivo gene transfer Cells from the liver cell line, Hep G2, were coinfected with rAAV2-GFP and wt AAV2 that was used to supply Rep in trans The percent of transduced cells was determined by FACS analysis at each passage After several passages, the percentage of positive cells decreased to a constant level Cells were sorted and individual green fluorescent cells from this sorted population were cloned and grown DNA was extracted and AAV-genomic junctions were determined by LM-PCR In cells co-infected with AAV2-GFP and wild type AAV2, 27% were transduced This fell to a constant level of 1.2 % after several weeks of growth, prior to sorting and cloning This contrasts with 18% and 0.3 % respectively, in the cells infected with rAAV2-GFP alone Of the co-infected clones, 22 out of 26 sorted clones remained fluorescent Integration analysis of 10 co-infected clones revealed that the transgene was located in the AAVS1 region of chromosome 19- in clones We also found integration in a non AAVS1 region of chromosome 19 Of interest, we found rAAV integration within an integrated wt AAV genome To enhance cell transduction and possibly integration, co-infected cells were incubated with doxorubicin, hydroxyurea, or camptothecin Although transduction values were 53, 31, and 146- fold higher respectively, compared with non-drug treated cells, we found no significant enhancement in the rate of integration A failure to achieve higher integration in these populations suggests that the efficiency of site specific integration is not limited by the number of virus genomes available for episomally based transduction In conclusion, these results suggest that by using this co-infection technique, an rAAV transgene can efficiently integrate into the AAVS1 locus and suggests that site-specific rAAV integration as an ex vivo approach is feasible Adenovirus and Other DNA Virus Vectors I 135 Microscopy Analysis of Adenovirus Serotype Interaction with its Receptor Desmoglein Zong-Yi Li,1 Hongjie Wang,1 Ines Beyer,1 Pascal Fender,2 Andre Lieber.1 Division of Medical Genetics, University of Washington, Seattle, WA; 2EMBL, Grenoble, France We identified Desmoglein (DSG2) as the primary receptor for adenovirus (Ad) serotype as well as for (Ad) serotypes 7, 11, and 14 These serotypes are important human pathogens causing respiratory and urinary tract infections So far, the mechanism of Ad3 entrance and spread in epithelial tissues has been unknown Using confocal immunofluorescence microscopy on a series of polarized human epithelial cells, including colon cancer lines T84 and CaCo as well as primary small airway epithelial cells, we found that DSG2 is localized to epithelial junctions The tight junction protein ZO-1 is localized on apical of DSG2 Other junction proteins (claudin and E-cadherin) were beneath DSG2 along the lateral cell membrane We visualized binding of Ad3 virions and subviral Ad3 penton-dodecahedra (PtDd) particles to DSG2 on polarized epithelial cells These studies suggest that Ad3 interacts with several DSG2 molecules and that Ad3-DSG2 complexes are internalized within 15 minutes Importantly, interaction of Ad3 virions or PtDds with DSG2 triggered partial dissolution of epithelial junctions, reflected in decreased staining for claudin and E-cadherin in junctions (in confocal microscopy images) and disappearance of apical desmosomes (in electron-microscopy images) A similar effect was seen with recombinant dimeric Ad3 fiber proteins (AD3K-K) Ad3, PtDd, or Ad3K-K mediated opening of junctions increased access to proteins that are trapped in junctions or localized on the basolateral membrane of epithelial cells (such as CD46 or Her2/neu-the receptor for the therapeutic monoclonal antibody Herceptin) This has practical implications Ad3K-K pretreatment of Her2/neu positive xenograft tumors in mice significantly improved the efficiency of Herceptin therapy We also hypothesize that, during Ad infection, Ad3/PtDd- triggered epithelial junction opening further increases access to DSG2 molecules that are more embedded in tight junctions thereby facilitating lateral viral spread We are currently attempting to prove this hypothesis While DSG2 is not expressed in mouse cells, its expression pattern in non-human primates appears to be similar to that in humans For studies on Ad3 pathology, we are creating human DSG2 transgenic mice and tumor cell lines So far, we have verified that Ad3 and PtDd bind to human DSG2 in transgenic cell lines Studies on the consequences of Ad3 PtDd-huDSG2 interaction on the integrity of epithelial junctions are ongoing and results will be reported Overall, our studies shed light on how the widely distributed Ad serotype infects epithelial cells 136 The Role of Endosomal Escape and Mitogen Activated Protein Kinases in Adenoviral Activation of the Innate Immune Response Jeffrey S Smith,1 Zhili Xu,1 Jie Tian,1 Donna J Palmer,2 Philip Ng,2 Andrew P Byrnes.1 Division of Cellular and Gene Therapies, Food and Drug Administration, Bethesda, MD; 2Molecular and Human Genetics, Baylor College of Medicine, Houston, TX Capsid proteins induce a strong innate immune response following systemic administration of adenovirus, and this creates a major obstacle to the therapeutic use of adenoviral vectors We investigated the role of endosomal escape and mitogen activated protein kinases (MAPKs) in the innate immune response following intravenous injection of adenoviral vectors into mice To determine the role of Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S53 ADENOVIRUS AND OTHER DNA VIRUS VECTORS I endosomal escape, we evaluated the innate immune response to ts1, a temperature sensitive Ad2 mutant that is defective in endosomal escape due to a mutation in the viral protease This was compared to the innate response produced by a replication defective helperdependent adenoviral vector (HDAd2) that is devoid of all viral genes but possesses a wt Ad2 capsid We found that ts1 was significantly deficient in stimulating the innate cytokine response compared to HDAd2 Because MAPKs are involved in intracellular pathways of cytokine induction, we investigated the role of the MAPKs ERK and p38 in the innate response to adenovirus MAPKs are activated by phosphorylation, and therefore we evaluated changes in the relative phosphorylation of these MAPKs after injection of HDAd2 and ts1 We found that the relative phosphorylation of ERK was elevated in both the liver and spleen 30 minutes after i.v administration of HDAd2, but not by ts1 Likewise, we found that HDAd2 elevated the phosphorylation of p38 in the liver, but ts1 did not To determine whether MAPKs play a role in the innate immune response to adenovirus in vivo, we treated mice with inhibitors of ERK or p38 pathways prior to injection of HDAd2 and evaluated the serum level of seven proinflamatory cytokines/chemokines (IL-12, IFN-gamma, KC, IL-6, IL1-beta, TNF-alpha and IL-10) hours after HDAd2 As a control, we examined how MAPK inhibitors affected the cytokine response to LPS Both LPS and HDAd2 produced increases in all of these cytokines/chemokines relative to buffer control animals We found that inhibition of ERK did not alter LPS-induced cytokines; however, inhibition of p38 reduced the response to LPS for all cytokines/chemokines tested except for KC In contrast, inhibition of ERK altered the level of five of the HDAd2-induced cytokines, increasing the level of IL-12, IFN-gamma and IL-6, while decreasing the level of KC and TNF-alpha Additionally, administering the p38 inhibitor prior to HDAd2 lowered the levels of only cytokines: IFNgamma, IL1-beta and IL-10 These data indicate that the involvement of MAPKs in adenovirus-induced cytokine responses is more complex than the role of MAPKs in LPS-induced responses We conclude that endosomal escape plays a critical role in triggering the innate response to systemically administered adenoviral vectors and that MAPKs play a role in this stimulation 137 The Sequential Proteolysis and Liberation of the Intracellular Domain of CD44 Modulates Adenoviral Vector Transgene Expression and Replication of Adenovirus Wesley S Bond,1,4 Cristhian J Ildefonso,1,4 Mary Y Hurwitz,2,4 Richard L Hurwitz.1,4 Interdepartmental Program in Translational Biology & Molecular Medicine, Baylor College of Medicine, Houston, TX; 2Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; Department of Pediatrics, Baylor College of Medicine, Houston, TX; 4Texas Children’s Cancer Center, Houston, TX There have been recent reports of successful applications of gene therapy to treat ocular oncologic and degenerative diseases which suggest that the intraocular environment can influence the efficiency of gene therapy viral vectors Expression of adenoviral vector-delivered transgenes has been shown to be enhanced by exposing transduced cells to bovine vitreous humor The interaction of high-molecularweight hyaluronan (HA), an abundant component of vitreous, and its receptor CD44 plays a critical role in this enhancement effect Small oligosaccharides of HA, which have been shown to interfere with high-molecular-weight HA binding, diminish CD44:HA-mediated enhancement of transgene expression CD44 undergoes a process of sequential proteolysis in the ectodomain and the transmembrane region, liberating the intracellular domain (ICD) that subsequently traffics to the nucleus Both the proteolysis of the ectodomain of CD44 by matrix metalloproteases and the subsequent intramembraneous proteolysis of CD44 by the γ-secretase complex and liberation of S54 the ICD are required for the CD44:HA-mediated enhancement of transgene expression Expression of mutant CD44 constructs lacking the protease cleavage sites as well as inhibition of metalloprotease or γ-secretase by small molecule inhibitors significantly attenuated CD44:HA-mediated enhancement of gene expression in cultured cells Expression of a CD44 ICD construct has also been shown to significantly increase transgene expression in the absence of HA Both the inhibition of HA:CD44 interaction by a CD44 blocking antibody and the inhibition of metalloprotease and γ-secretase by small molecule inhibitors was shown to significantly decrease adenoviral vector transgene expression in human conjunctiva explants In addition to expression of vector transgenes, the replication of wildtype adenovirus was decreased in the presence of metalloprotease or γ-secretase inhibitors in cultured cells These observations suggest that the CD44 signaling pathway could provide an important mechanism for controlling the expression of adenoviral vector-delivered genes in gene therapy applications as well as provide a potential therapeutic target for treatment of adenovirus infection 138 Enhanced Transduction of Fiber Modified Ad in Canine Lymphoma Cell Lines Ann Marie O’Neill,1 David T Curiel,2 Bruce F Smith.1,3 Scott Ritchey Research Center, Auburn University, Auburn, AL; Department of Radiation Oncology, Washington University School of Medicine, St Louis, MO; 3Department of Pathobiology, Auburn University, Auburn, AL Canine lymphoma represents an excellent model of human nonHodgkin’s Lymphoma - it has a similar etiology and presentation to the human disease, the model has an intact immune system, and the size of the model allows for extrapolation to humans The use of this model is further strengthened by the high similarity between dog and human genomes and the presence of evolutionarily conserved cytogenic changes in hematological malignancies Recombinant Adenoviral (Ad) vectors derived from serotypes and are among the most promising vehicles for in vivo gene delivery, based on previously demonstrated efficiency in a number of cancer gene therapy applications Entry of Ad into cells is a multi-step process, requiring binding of the native Coxsackie and Adenovirus receptor (CAR) by viral fiber knob, followed by interaction of an RGD motif in the penton base of Ad with αvβ3/5 integrins on the cell surface However, our analysis of canine lymphoma cell lines and primary lymphoma cells indicates that they are refractory to infection by Ad vectors due to the lack of the CAR and low-level expression of αvβ3 Ad5 vectors with chimeric fibers derived from non-human adenovirus serotypes were used to transfect canine lymphoma cell lines Ad5.CK1 was modified to include the canine adenovirus (CAV1) knob domain, Ad5.PK contained the porcine (Ad4) knob domain In the human context, the use of these xeno-adenoviruses may have advantages in that no previous immunity to these viruses is likely to exist and the alternative entry pathways may circumvent the CAR-integrin entry pathway in those cells that are normally refractive to Ad5 infection The vector Ad5.CK1 has been shown to alter Ad5 tropism through a CAR-independent pathway in CARdeficient human ovarian cancer cell lines and primary tissue samples The vector Ad5.PK knob domain has not yet been described but it is believed to target specific carbohydrates Canine lymphoma cell lines OSW and 17-71 were used to test the efficacy of these vectors to achieve CAR-independent transduction In OSW cells, low-level transduction was achieved with both vectors These cells were then stimulated with PMA plus ionomycin and transduction by both vectors was enhanced Following incubation with an anti-integrin antibody, transduction efficiency was reduced to or below the level seen in unstimulated cells In 17-71 cells, transduction was achieved only with Ad5.CK1 Following stimulation transduction efficiency did increase, although modestly - less than fold - with a concurrent reduction seen Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... deficient in stimulating the innate cytokine response compared to HDAd2 Because MAPKs are involved in intracellular pathways of cytokine induction, we investigated the role of the MAPKs ERK and p38 in. .. the level of five of the HDAd2-induced cytokines, increasing the level of IL-12, IFN-gamma and IL-6, while decreasing the level of KC and TNF-alpha Additionally, administering the p38 inhibitor... lowered the levels of only cytokines: IFNgamma, IL1-beta and IL-10 These data indicate that the involvement of MAPKs in adenovirus-induced cytokine responses is more complex than the role of MAPKs in