799 Development of alphaviral Vectors for the Delivery of Short Hairpin RNA Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S306 RNA VIRUS VECTORS[.]
RNA VIRUS VECTORS III 798 Electrically Mediated Plasmid Delivery to the Porcine Heart Richard Heller,1,2 Brian A Boone,3 Kenneth E Ugen,2 Sylvia I Gografe,4 Jose D Burgos,3 Margaret K Baldwin,4 Yolmari Cruz,2 Mark J Jaroszeski,5 William G Marshall.3 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA; 2Molecular Medicine, University of South Florida, Tampa, FL; 3Surgery, University of South Florida, Tampa, FL; 4Comparative Medicine, University of South Florida, Tampa, FL; 5Chemical Engineering, University of South Florida, Tampa, FL Our results indicate for the first time that regeneration of bone in a non-healing bone defect can be attained using in vivo electroporation of an osteogenic gene This bone regeneration strategy could revolutionize orthopedic treatment in patients suffering from major bone loss Moreover, our method may pave the way for the regeneration of other skeletal tissues such as cartilage and tendon 797 Ventilation or Pharmacological-Induced Inhibition of HDAC6 Can Increase Gene Transfer in Mouse Lungs Christopher D Kaufman,1 David A Dean.1 Pediatrics, University of Rochester, Rochester, NY We are interested in developing new strategies to enhance nonviral gene delivery to the lung We have shown that plasmids utilize microtubules and their associated motor proteins to traffic to the nucleus for gene expression following transfection We have further shown that application of cyclic stretch to isolated cells can increase this trafficking through reorganization of the cytoskeleton and inhibition of HDAC6 which results in increased levels of acetylated, stabilized microtubules These acetylated microtubules recruit more motor proteins and show greater DNA trafficking to the nucleus To determine if mechanical strain induced by ventilation could increase gene transfer and expression in vivo, plasmids were instilled into the mouse lung, and the mice were electroporated and then mechanically ventilated for minutes, at between 10 to 80% TLC Gene expression was measured days later When mice were ventilated at 40 to 50% TLC following electroporation-mediated gene delivery, gene expression increased by almost 4-fold This approach caused more total gene expression as well as greater distribution of gene expression in the lungs When the lungs were assayed for acetylated tubulin and HDAC6 levels, we found that levels of acetylated microtubules increased with increasing strain, as we had found in cultured cells To determine whether the increase in acetylated microtubules was responsible for the ventilation-enhanced gene transfer, we administered a small molecule inhibitor of HDAC6 to mice and evaluated gene delivery and expression in the absence of mechanical ventilation When compared to vehicle-treated mice, mice that received the HDAC6-specific inhibitor NCT-10b showed increased levels of acetylated tubulin and 3- to 4-fold increased levels of gene transfer/expression Taken together, these results indicate that modulation of HDAC6 activity in the lung, either by modest ventilation or pharmacologically, can result in increased gene delivery S306 Gene therapy is an attractive method for treating cardiovascular disease However, gene expression at therapeutic levels is often inefficient To enhance expression, delivery to the heart was performed using in vivo electroporation Delivery of two plasmids encoding either luciferase (gWizLUC) or vascular endothelial growth factor (pVEGF) was evaluated Thirteen farm pigs (neutered males, weights 25 – 30 kg.) underwent median sternotomy under general endotracheal anesthesia Plasmid (0.1 ml) was injected on the anterior wall of the left ventricle, followed by asynchronous electroporation at specific delivery parameters (field strengths and pulse widths) An injection only control was included and the injection performed after all electroporation was completed Animals were maintained for 48 hours and underwent injection site excision with a 6mm punch Several electroporation conditions were tested and the pulse conditions found to yield the highest expression were 100 V/cm field strength and 250 ms pulse width These conditions were used to deliver plasmid encoding for VEGF Myocardial VEGF expression delivered with electroporation at injection sites was also greatly enhanced versus injection of plasmid alone Increased expression was seen after 48 hours but levels dropped to background levels within days While the primary objective of increasing expression was obtained, it was observed that asynchronous pulses resulted in ventricular fibrillation in all of the pigs Although most of the pigs were successfully defibrillated, it was reasoned that the development of a synchronous (SYN) electroporation pulse delivery method in which the pulses could be delivered in synchrony with the normal heart rhythm would circumvent this problem Therefore, in a subsequent set of gene transfer experiments, electrical pulses were delivered to the heart synchronously with the QRS complex determined with the use of a surface electrocardiogram Importantly, the initial experiments indicated that delivering SYN pulses eliminated ventricular fibrillation Several electroporation parameters were evaluated Expression levels for some of the delivery conditions were comparable to those obtained in the asynchronous pulsing experiments However, the critical result was that when applying pulses in this manner the heart did not fibrillate Based on these results, gene delivery to the heart can be successfully accomplished using in vivo electroporation Further study of electroporation as a method of gene delivery to the myocardium is warranted (Supported by a research grant from the NIH (R21 HL089017) and also supported by the Office of Research, USF Health, Univ of South Florida) RNA Virus Vectors III 799 Development of alphaviral Vectors for the Delivery of Short-Hairpin RNA Menelaos Manoloukos,1 Vivian Tseveleki,2 Polyxeni Katsoupi,1 Lesley Probert,2 Teni Boulikas,1 Michael L Roberts.1 Regulon, Athens, Greece; 2Molecular Genetics Lab, Hellenic Pasteur Institute, Athens, Greece Introduction Semliki Forest Virus (SFV) vectors constitute infectious tools that are used for expression of large quantities of protein A temperature sensitive SFV vector (SFV.PD), which has Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy RNA VIRUS VECTORS III been developed previously, exhibits prolonged gene expression in targeted cells and increased safety We have achieved high efficiency of EGFP expression in cell lines and subsequent silencing of EGFP by means of double expression with the SFV.PD vector encoding short hairpin RNA against GFP Methods The SFV.PD construct was used as a vector for EGFP protein (SFV.PD-EGFP) and for an anti-EGFP short hairpin (SFV.PD.sh-EGFP) Three different cell lines, BHK21, H293T, H1299 were infected with SFV.PD-EGFP and SFV.PD.sh-EGFP Results were documented by fluorescent microscopy and FACS analysis Results Infection of cell lines with SFV.PD-EGFP resulted in strong expression of EGFP in >70 % of the cells Double infection of the cell lines with SFV.PD-EGFP and SFV pd.sh-EGFP resulted in silencing of the EGFP protein expression to a level of 20-30% of the initial intensity Infection of cell lines that stably express EGFP with SFV.PD.sh-EGFP resulted to inhibition of the EGFP protein expression both in terms of cell numbers and in the intensity of the expression Conclusions We have successfully used SFV.PD vector to mediate GFP knockdown by the overexpression of short hairpin RNA 800 Substitution of a Murine Constant Region Enhances Effector Function of a Melanoma Specific Human T-Cell Receptor Stephanie G Downey,1 Laura A Johnson,1 Richard A Morgan,1 Cyrille J Cohen,2 Zhili Zheng,1 Steven A Rosenberg,1 Steven A Feldman.1 Surgery Branch, National Cancer Institute, Bethesda, MD; Laboratory of Tumor Immunology and Immunotherapy, Bar-Ilan University, Ramat Gan, Israel Genetic modification of peripheral blood lymphocytes to express T-cell receptors (TCR) specific to known melanoma antigens (MART1 and gp100) can elicit objective tumor regression in patients with metastatic melanoma It has been demonstrated that modifications within the constant regions of a TCR can enhance surface expression and stability without altering antigen specificity In this study, we evaluated the substitution of a murine constant region into a MART1 specific TCR previously identified as DMF5 Secondarily, we evaluated the insertion of cysteine residues to promote formation of a second disulfide bond and use of an optimized linker between the sequences encoding the TCR alpha and beta chains We transduced peripheral blood lymphocytes with retroviral supernatant generated from stable packaging lines encoding melanoma-specific T-cell receptors Murinized receptors had similar transduction efficiency but significantly higher mean fluorescence intensity (MFI) and conferred significantly increased in vitro effector function in clinically relevant expansion conditions The additional cysteine and linker modifications did not further enhance expression or function We conclude that the substitution of a murine constant region increased expression and function of the DMF5 receptor and could be a tool to augment other antigen-specific receptors 801 Oncolytic Measles Virus with AntiMesothelin Antibody Single Chain Targeting Mesothelin Positive Mesotheliomas Hongtao Li,1 Kah-whye Peng,1 Stephen J Russell.1 Department of Molecular Medicine, Mayo Clinic Rochester, Rochester, MN Mesothelin (MSLN) is a glycoprotein that is over-expressed in various human tumors, including pancreatic adenocarcinomas, ovarian carcinomas and mesotheliomas The limited MSLN expression in normal tissues and high expression in cancer cells makes it a promising candidate for tumor-specific therapy Malignant pleural mesothelioma (MPM) is a devastating tumor known to be resistant to conventional therapies Malignant pleural mesothelioma Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy is mainly triggered by exposure to asbestos and the prognosis is poor with the current available treatments which include surgery, radiotherapy and chemotherapy Novel therapeutic strategies are urgently needed Live attenuated vaccine strains of measles virus (MV) have potent anti-tumor activity To minimize virus sequestration by non-target cells and collateral damage to normal tissues, the tropism and cytopathic activity of an oncolytic virus should ideally be restricted to the tumor cells being targeted After ablating recognition of the natural MV receptors CD46 and SLAM, measles viruses have been efficiently retargeted to a variety of different tumor markers including CD38 receptor, Epidermal Growth Factor Receptor (EGFR), folate receptor, IL-13 receptor In this study a single-chain antibody (ScFv) fragment against MSLN tailed with histidines was introduced into an oncolytic MV as a C-terminal extension of the hemagglutinin (H) protein The retargeted virus was successfully rescued and the replication was comparable to the unmodified MVGFP virus in Vero cells expressing a hexahistidine pseudoreceptor A western blot was carried out to confirm the chimeric hemagglutinin protein expression Flow cytometry was applied to screen the MSLN and EGFR positive mesothelioma cell lines The oncolytic Measles Virus targeting EGFR served as a positive control and MV with a single chain antibody against CD38 as a negative control Mesothelioma cell lines H2596, REN, H226, M30 and MSTO-211H were infected with the recombinant viruses at MOIs from 0.1 to 10 Display of the single chain antibody on the measles viral envelope glycoprotein redirected virus entry specifically into MSLN expressing mesothelioma cell lines REN and H226 A xenograft mouse model was established by injecting REN mesothelioma cells in peritoneal cavity In vivo experiments are going on and the neurotoxicity of the oncolytic viruses will be compared in measles replication permissive ifnarkoCD46 transgenic mice 802 Development of a Recombinant Vesicular Stomatitis Virus Vector Capable of Expressing Four Antigens for Vaccine Applications Olena Serwylo,1 Gary Wong,1,2 Kaylie Tran,1 Darwyn Kobasa,1,3 Hideki Ebihara,4 Heinz Feldmann,4 Gary P Kobinger.1,2 Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada; Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3Respiratory Viruses Viral Disease Division, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada; 4Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, Hamilton, MT Vesicular Stomatitis Virus (VSV) is an animal pathogen belonging to the family Rhabdoviridae Due to its low seroprevalence amongst humans and high immunogenicity, VSV is a potentially useful candidate for vaccine applications Furthermore, its ability to contain large foreign gene insertions without undergoing reassortment with wild-type virus makes it a flexible tool for expression of desired heterologous genes in vivo Our objective was to create a recombinant VSV vector that contains four multiple cloning sites (MCS) for the insertion of up to four foreign genes The different MCS of the new recombinant plasmid (pATX VSV∆G4) were characterized for their ability to express reporter genes Engineered pATX VSV∆G4 contained two MCS upstream of the 3’ nucleoprotein (N) gene and two MCS downstream of the matrix (M) gene In total, eight different constructs encoding the VSV glycoprotein (G) gene and various reporter gene combinations of firefly luciferase, renilla luciferase and eGFP were created in order to evaluate the expression levels of each MCS The live recombinant VSV constructs were rescued and total particle or infectious particle numbers were determined by real time RT-PCR or plaque assay respectively Expression levels of reporter genes in the different MCS were evaluated on by performing a dual S307 ... silencing of EGFP by means of double expression with the SFV.PD vector encoding short hairpin RNA against GFP Methods The SFV.PD construct was used as a vector for EGFP protein (SFV.PD-EGFP) and for. .. numbers and in the intensity of the expression Conclusions We have successfully used SFV.PD vector to mediate GFP knockdown by the overexpression of short hairpin RNA 800 Substitution of a Murine... multiple cloning sites (MCS) for the insertion of up to four foreign genes The different MCS of the new recombinant plasmid (pATX VSV∆G4) were characterized for their ability to express reporter