681 Ad TβRIIδcyt Treatment Decreases Liver Steatosis and Fibrosis by Decreasing mRNA Expression of CB1 Cannabinoid Receptor and TGF β and Increasing MMP 1 Molecular Therapy Volume 19, Supplement 1,[.]
ADENOVIRUS AND OTHER DNA VIRUS VECTORS III CONCLUSION: To our knowledge, this is the first study to demonstrate efficacy in a viral therapy against TNBC cells Our results support that NV1066 can infect and kill TNBC As overexpression of p-MAPK has been correlated with resistance to chemotherapy, the down-regulation of p-MAPK by NV1066 suggests a potential role for NV1066 as an adjuvant therapy in chemoresistant TNBC 679 Generation of Complex High-Capacity Adenoviral Vectors with Modified Capsids for Applications In Vitro and In Vivo Martin Hausl,1 Richard Voigtlander,1 Zsolt Ruzsics,1 Anja Ehrhardt.1 Virology, Max von Pettenkofer-Institut / LMU, Munich, Germany First-generation adenoviral vectors (FG-AdVs) are easy to produce and therefore represent the best option to analyse capsidmodifications or new expression systems On the other hand highcapacity adenoviral vectors (HC-AdVs) lacking all viral coding sequences are the most attractive option for therapeutic approaches due to reduced toxicity and immunogenicity and high capacity for sequences of interest Therefore, there is a great interest in a method for adoption and combination of vector-modifications as well as expression systems evaluated in FG-AdVs towards HC-AdVs Recently we developed a novel platform based on homologous recombination of bacterial artificial chromosomes (BACs) allowing arbitrary modifications On the basis of the plasmid pHCA-eGFP encoding a HC-AdV genome containing an eGFP expression cassette we showed smooth access to the platform exchanging regular plasmid backbone with BAC backbone Full potential of BAC technology was demonstrated utilizing a recombination pipeline based on alternating resistances for rapid incorporation of different DNA fragments into one HC-AdV BAC The resulting BAC pBHCA-2indsys encodes two expression cassettes enabling liver-specific and mifepristoneinducible expression of Renilla luciferase as well as two expression cassettes allowing doxycycline-inducible and stem cell-specific expression of eGFP-Firefly Luciferase reporter construct For generation of respective HC-AdV we currently utilize column-purified unmodified helper-virus and a fiber-modified variant, constructed by traceless exchange of the fiber with fiber chimera fib5/35 Generated HC-AdV-2indsys will be tested for functionality in hepatocytes and an oct4-expressing cell line For in vivo applications we modified hexons of the helper-virus to allow repeated administrations Therefore, we synthesized hexons with precisely exchanged hypervariable regions (HVRs) with respective sequences of other serotypes (Ad12, Ad41, Ad44, Ad48, Ad50) Additionally we generated modified hexons by complete exchanges of domains DE1 and FG1 containing sequences, which encode the HVRs from Ad4, Ad7, Ad12, Ad13, Ad41 Western blot analysis demonstrated intact interaction of modified hexons with adenoviral 100K-protein as well as efficient nuclear uptake and trimerization Subsequently we replaced the original hexon of a helper-virus BAC with an eGFP/Fluc reporter cassette incorporated in the E3 region with modified hexon sequences Although rescue of hexon modified adenoviruses is challenging, we could show successful reconstitution of of the above mentioned hexon-modified helper-viruses containing precise as well as complete exchanges of HVRs During amplification we noted a prolonged time until observing complete cytopathic effect, suggesting impaired virus assembly Currently, purified viruses are analysed in detail with respect to titers (viral particles and transducing units), biodistribution after intravenous injection in mice and potential to escape neutralizing antibodies in human sera in neutralizing antibody assays Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 680 Dual-Expression Adenoviral Vectors in Vaccine Development Xiangyang Zhou,1 Juliana C Small,1 Dongming Zhou,1 Raj K Kurupati,1 Heng Chen,1 Ang Bian,1 Hildegund C Ertl.1 The Wistar Institute, Philadelphia, PA We have previously reported on a dual-expression chimpanzee adenoviral vector (AdC7) that we developed to express nucleoprotein (NP) of influenza A virus in E1 locus under the control of CMV promoter and gag of SIV in E3 locus under the control of chicken beta actin (CB) promoter Both expression cassettes contained the CMV enhancer as well By using the vectors in vitro and in vivo, the results showed expression and immunogenicity of the transgene product encoded by the cassette in E1, to be low In order to enhance immunogenicity, the vectors were modified by removing the CMV enhancer from either cassette A vector that contained the CMV enhancer and promoter in front of the NP gene in E1 locus and SIVgag under control of CB promoter without CMV enhancer in E3 locus showed significantly improved immunogenicity for both transgene products The data suggest that duplication of even small sequences such as an enhancer has to be avoided in the design of dual expression adenoviral vectors, which could be very useful in vaccine development 681 Ad-TβRII∆cyt Treatment Decreases Liver Steatosis and Fibrosis by Decreasing mRNA Expression of CB1 Cannabinoid Receptor and TGF-β and Increasing MMP-1 Mayra G Mena,1 Ana S Sandoval,1 Jazmin Santillan,1 Juan Armendariz.1,2 Institute of Molecular Biology in Medicine and Gene Therapy, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico; 2O.P.D Hospital Civil de Guadalajara “Juan I Menchaca”, Guadalajara, Jalisco, Mexico Introduction: Transforming growth factor β (TGF-β) is the main profibrogenic cytokine; consequently strategies to block its signal pathway have been developed Gene therapy using a truncated receptor type II for TGF-β (TβRII∆cyt) had showed to reduce hepatic fibrosis; but its effect on the expression of cannabinoid receptors (CB1 and CB2) which participate in the progression of liver cirrhosis or steatosis has not been elucidated Objective: Evaluate the effects of Ad-TβRII∆cyt on fibrosis, steatosis and gene expression of profibrogenic molecules and cannabinoid receptors in rat liver Material and Methods: Cirrhosis was induced in male rats administrating carbon tetrachloride (CCl4) for weeks Cirrhotic rats were divided into groups (n=6): cirrhotic control, cirrhotic with therapeutic gene (Ad-TβRII∆cyt) and cirrhotic with irrelevant gene (Ad-GFP) At the 4th week of CCl4 intoxication, 2x1010 pi/kg of Ad-TβRII∆cyt or Ad-GFP was administered via iliac vein Sacrifice was made at day 2, and 28 after treatment Fibrosis index, steatosis, hydroxiproline content, gene expression of TGF-β, collagen α1 (I), PAI-1, MMP-1, CB1 and CB2 were analyzed, as well as, ALT and AST serum levels Results: Treatment with Ad-TβRII∆cyt decreased 22%, 28% and 35% of fibrotic tissue; while liver steatosis was reduced 47%, 43% and 42%; at day 2, and 28 respectively (p