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313 Liver Transduction and Brain Safety of an Antifibrogenic shRNA for Cannabinoid Receptor CB1 in a Model of Liver Cirrhosis Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The Americ[.]
ADENOVIRUS VECTORS AND OTHER DNA VIRUS VECTORS II 311 An Adenovirus-Sleeping Beauty Transposase Hybrid-Vector System To Treat von Willebrand Disease Manish Solanki,1 Wenli Zhang,1 Zsuzsanna Izsvak,2 Simon F De Meyer,3 Karen Vanhoorelbeke,3 Anja Ehrhardt.1 Institute of Virology and Microbiology, Private University Witten-Herdecke, Witten, North Rhine-Westphalia, Germany; Max Delbrück Center for Molecular Medicine, Berlin, Germany; Laboratory for Thrombosis Research, KU Leuven Kulak, Kortrijk, Belgium Von Willebrand factor (VWF), a multimeric plasma protein, has a crucial role in initiating primary hemostasis It binds to the damaged vessel wall and attracts platelets and platelet adhesion is followed by platelet aggregation which eventually leads to cessation of bleeding Genetic defects in the VWF gene results into the bleeding disorder von Willebrand disease (VWD) While administration of plasmaderived VWF/FVIII is an effective therapy for treatment of severe type VWD, it has clear limitations such as a short term effect, high cost and risks of contamination Monogenetic diseases, like VWD, often present an ideal target for gene therapy treatment potentially leading to a lifelong cure As the VWF cDNA is of up to 8.4kb in size, high-capacity adenovirus (HCAdV) is a desirable choice of vector because of its large transgene carrying capacity up to 36kb To achieve lifelong treatment, we have developed a hybrid-vector system which enables transgene delivery and its subsequent integration into the host genome using hyperactive Sleeping Beauty (SB) transposases HSB5 and SB100X In this study we aimed at developing HCAdV encoding VWF under the control of a liver-specific promoter flanked by SB inverted repeats and Flpe recombinase recognition sites to achieve VWF cDNA host chromosomes integration To clone the VWF in the HCAdV genome, a recombineering technology based on a bacterial artificial chromosome (BAC) carrying the HCAdV genome (B-HCAdV) was applied in combination with counter selection marker galK (galactokinase) Initially, the counter selection marker, fused with kanamycin (galK-Kan), was flanked with both ends of the VWF transgene using overlap PCR In first recombineering step the overlap PCR product was inserted into B-HCAdV followed by counter selection which replaced galKKan with VWF transgene The final clone pB-HCAdV-VWF was confirmed by diagnostic restriction enzyme digests and sequencing For HCAdV production the viral genome from pB-HCAdV-VWF was linearized and transfected into 116 producer cell line for HCAdV Reconstitution and amplification of HCAdV encoding VWF (HCAdVVWF) was successful which was monitored by eGFP expression, also expressed from HCAdV-VWF By using a similar strategy, HCAdV carrying VWF flanked by FRT sites for FLP recombination and SB transposase inverted repeats (pB-HCAdV-VWF-IR-FRT) was cloned In summary, we have successfully produced HCAdV carrying VWF gene (HCA-VWF) and we are reconstituting HCA-VWF-IRFRT that will be used for VWF genomic integration using a second vector encoding hyperactive transposase SB100X We are currently testing whether VWF protein is expressed from the vector HCAdVVWF Moreover, we are evaluating FLP mediated circularization of the VWF transgenes and transposition from HCAdV-VWF-IR-FRT in the presence of a vector encoding Flp recombinase and SB100X In the near future we will explore our novel therapeutic approach in a mouse model for curing VWD S120 312 Small Peptides Blocking SR-A and SREC-I Increase HDAd-Mediated Hepatocyte Transduction Through Inhibition of Vector Uptake By Kupffer and Sinusoidal Endothelial Cells Pasquale Piccolo,1 Pratibha Mithbaokar,1 Patrizia Annunziata,1 Nicola Brunetti-Pierri.1,2 Telethon Institute of Genetics and Medicine, Naples, Italy; Translational Medicine, Federico II University, Naples, Italy Helper-dependent adenoviral (HDAd) vectors can mediate longterm, high-level transgene expression from transduced hepatocytes with no chronic toxicity However, the vector therapeutic index is narrow because of an acute toxic response with potentially lethal consequences elicited by high vector doses Kupffer cells and liver sinusoidal endothelial cells (LSECs) are major barriers to efficient hepatocyte transduction We recently identified scavenger receptor type A (SR-A) and scavenger receptor expressed on endothelial cells type I (SREC-I) as HDAd receptors on both Kupffer cells and LSECs in vivo By phage display, we have now developed two small peptides that block SR-A (PP1) and SREC-I (PP2) mediated vector uptake and result in increased efficiency of hepatocyte transduction by HDAd Pre-incubation of J774 macrophages with either PP1 or PP2 before HDAd infection significantly reduced viral vector uptake Fluorocrome conjugated PP1 and PP2 injected intravenously into C57BL/6 mice co-localized with both CD68 and CD31 suggesting that both peptides bind SR-A and SREC-I on Kupffer cells and LSECs Compared to saline pre-treated animals, intravenous administrations in C57BL/6 mice of PP1 and PP2 peptides prior to the injection of an HDAd bearing the alpha-fetoprotein reporter gene under the control of a liver-specific promoter resulted in a significant 3.7- and 2.9-fold increase of hepatic transgene expression, respectively The enhancement of transgene expression was confirmed by increased number of beta-galactosidase positive liver cells in mice injected with HDAd-CMV-LacZ after PP1 and PP2 pre-treatment Serum IL-6 was moderately increased in mice pre-injected with both peptides prior to HDAd compared to control mice pre-treated with saline and was not associated with increased Kupffer cell mortality, as shown by hepatic CD68 staining In summary, we developed small peptides that increase significantly hepatocyte transduction efficiency, improve the HDAd therapeutic index, and have potential for clinical applications 313 Liver Transduction and Brain Safety of an Antifibrogenic shRNA for Cannabinoid Receptor CB1 in a Model of Liver Cirrhosis Adriana Díaz-Rivera,1 Juan Armendáriz-Borunda,1 Leonel GarcíaBenavides,2 Ana Sandoval-Rodríguez.1 Molecular Biology and Gene Therapy Institute, Guadalajara, Mexico; 2Institute of Experimental and Clinical Therapeutics, Guadalajara, Mexico Introduction: Pharmacological antagonist of CB1 has demonstrated anti-fibrogenic effects in different models of liver cirrhosis but, with important adverse side effects Then, gene therapy with a shRNA for CB1 delivered by hidrodinamic transfection or adenovirus may have the advantage of hepatic tropism, reducing possible side effects and increasing efficiency of transduction Also, in liver fibrosis levels of TGF-b1, Col I and α-SMA produced by activated Hepatic Stellate Cells are increased Objective: Demonstrate antifibrogenic effects of liver expression of a shRNA against CB1 and generate a replication incompetent adenovirus that transiently expresses this shRNA-CB1 Materials and methods: An shRNAs against CB1receptor was designed to align in position 1232 of the mRNA of CB1 using Block iT RNAi Designer software (Invitrogen) This shRNA was cloned into the expression plasmid pENTR™/U6 to generate pshRNA-CB1 The effectiveness of the shRNAs was evaluated by inhibition of mRNA Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ADENOVIRUS VECTORS AND OTHER DNA VIRUS VECTORS II and protein level of CB1 in an experimental model of liver cirrhosis induced by CCl4 Safety was evaluated monitoring CB1 expression in brain tissue mg/kg of pshRNA-CB1 were administrated via iliac vein using hydrodynamic injection to cirrhotic rats mRNA of TGF-b1, Col and α-SMA; AST and ALT serum levels and percentage of fibrotic tissue were evaluated Then, this sequence was used to generated an Adenovirus backbone by homologous recombination with pAd/ BLOCK-iT ™ DEST Results: Hydrodynamics-based transfection of shRNA-CB1 via iliac vein in CCl4-cirrhotic rats allows efficient delivery to the liver; silencing CB1 mRNA (76%; p>0.05) and protein (56%; p>0.05) On the other hand, brain expression of CB1 mRNA presents no difference versus control, indicating a safe administration Using this treatment, fibrogenic molecules like TGF-b1, Col I, α-SMA reduced (p