533 lentiviral vectors mediated long term and high efficiency expression of foreign gene in HEK 293t cells

533 Lentiviral Vectors Mediated Long Term and High Efficiency Expression of Foreign Gene in HEK 293T Cells Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene[.]

RNA VIRUS VECTORS In conclusion, our current results provide a novel chemoselection strategy using shRNAs to increase engraftment of genetically modified HSPC so that it may be possible to use this technology for the stable control of HIV 531 Designing Trans-Regulated Lentiviral Vectors for Gene Transfer in Functional Genomics and Gene Therapy Agnes Holczbauer,1 Adriana Zingone,1 Suresh Arya.2 Center for Cancer Research, National Cancer Institute, Bethesda, MD; 2Center for Cancer Research and Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD One deficiency in efficient functional genomics analysis and mounting successful gene therapy trials is the paucity of a vector system that can be regulated from within or without Knowledge of the regulation of lentivirus HIV gene expression presents a possible strategy The Tat gene of lentiviruses encodes transactivator protein that regulates viral gene expression by acting through the viral promoter We are using this observation to design a trans-regulated lentiviral vector system for gene transfer For a number of reasons, we are using vectors derived from both HIV-1 and HIV-2 that could be used to create hybrid or chimeric vectors with additional safety insurance The distinctive feature of this system as compared with the conventional vectors is that the transfer vector does not include an internal promoter to drive transgene or shRNA expression Instead, the expression is driven by the viral LTR promoter itself The viral promoter is essentially silent in most cells and needs Tat for activation This provides the means to regulate vector expression in trans The model vector must fulfill the following requirements: (i) when introduced into target cells, it must display low basal activity, (ii) if Tat is provided genetically, the target cell must be able to synthesize sufficient Tat, and if Tat is provided as a protein, it must reach the nucleus of the target cell with its integrity intact, and (iii) the vector must able to respond to Tat to activate transgene expression Because of the possible use of this system in cancer gene therapy, we tested the system in a number of cell lines from the NCI 60 cell panel that included cell derived from major solid tumors and lymphoblastoid cells We used GFP as a reporter gene and provided Tat genetically in trans For the twenty cell lines tested, by and large the model held There was low but variable level of basal GFP expression depending on the cell line, and it was elevated by Tat in all cell lines but again to variable extent (5 - 20 fold) This elevation was not observed with the unrelated puromycin vector As an alternative to providing Tat as a gene or protein, one conceivably could find a small druglike molecule by high throughput screening of chemical libraries We have developed a cell-based assay towards this goal, where transgene expression is dependent on Tat, which could be used for library screening The opinions expressed in this abstract are those of the authors and not necessarily represent views of the National Cancer Institute 532 Application of VSV-G Induced Nanovesicles (Gesicles) for the Delivery of Active Protein To Target Cells Thomas P Quinn,1 Lily Lee,1 Mei Fong,1 Michael Haugwitz,1 Hiroaki Sagawa,2 Andrew Farmer.1 Clontech Laboratories, Inc., Mountain View, CA; 2Takara Bio Inc., Otsu, Shiga, Japan Historically, phenotypic control of live cells has been achieved through transient or stable delivery of nucleic acids into primary or transformed cells However, this approach has a number of drawbacks, including low efficiency, toxicity, genome disruption at the integration site, and a potentially significant delay in phenotypic response due Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy to the requirement for transcription and translation post-delivery Another approach is to use an inducible system which allows for tight regulation of expression—but inducible systems still depend upon efficient delivery of DNA to function properly In contrast, direct intracellular delivery of recombinant proteins into live cells allows tight control over both the timing and dose of the desired polypeptide, but is limited by the need to express the protein prior to delivery (typically in E coli) This can lead to issues with protein yield, proper folding, and post-translational modifications, and likely to a reduction in activity Alternative methods of protein delivery could provide new and effective tools Here we report on a number of applications that utilize VSV-G-induced microvesicles (Gesicles)1 to deliver functional proteins directly to target cells Co-overexpression of the spike glycoprotein of VSV-G with the POI, within a mammalian packaging cell, leads to the production of VSV-G coated microvesicles containing active amounts of the POI Expanding upon previous work1, we have developed a number of Gesicles carrying the receptors Pit2, mCAT1, and CAR to alter the susceptibility of primary cells and cell lines to various viruses, including retroviral and lentiviral vectors pseudotyped with either amphotropic and ecotropic envelopes as well as type adenovirus vectors In all cases, transduction of receptor Gesicle-treated cells resulted in an increase in transduction efficiency, including rendering receptor-negative cells positive for transduction as measured by fluorescence microscopy and FACS We were also able to deliver other functional membrane proteins such as GPCRs and a cell surface marker, with both demonstrating activity in as little as hours post delivery To our knowledge, no other current delivery method is capable of delivering membrane proteins in this way Other experiments confirmed that we could deliver fluorescent proteins and other molecules to the cytoplasm All activities were found to be due to direct transfer of the POI contained within the Gesicles and not a byproduct of indirect nucleic acid transfer This work suggests that Gesicles can be considered as another efficient means to direct the phenotype of a cell of interest through the rapid and direct delivery of active protein to a chosen target cell Reference: Mangeot et al (2011) Mol Ther 19:1656-1666 533 Lentiviral Vectors-Mediated Long-Term and High Efficiency Expression of Foreign Gene in HEK 293T Cells Renhe Yan,1,2 Yingying Mao,1 Zhibing Liang,1 Yi Feng,2 Tianbai Li,3 Maomin Lu,4 Weiwang Gu,2 Jingang Zhang,4 Hongwei Li.1 School of Biotechnology, Southern Medical University, GuangZhou, GuangDong, China; 2Institute of Comparative Medicine and Center of Laboratory Animals, Southern Medical University, GuangZhou, GuangDong, China; 3College of Medicine, University of Florida, Gainesville, FL; 4Institute of Transfusion Medicine, The Academy of Military Medical Sciences, Beijing, China Objectives: Lentiviral vectors have been developed to successfully and rapidly produce decigram quantities of active recombinant proteins in human cell lines To optimize the protein production platform, the roles of different promoters, insulator, and Ubiquitous Chromatin Opening Element (UCOE) were evaluated in the efficiency and stability of foreign gene expression mediated by lentiviral vectors Methods : Five lentiviral vectors, pFIN-EF1-GFP-2AmCherH-WPRE containing EF1 promoter and HS4 insulator, pTYF-CMV(-globin intron)-EGFP containing CMV promoter and -globin intron, pTYF-CMV-EGFP containing CMV promoter, p’HR.cppt.3’1.2kb-ucoe-SFFV-GFP containing SFFV promoter and UCOE , and pTYF-EF1-EGFP with EF1 promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell) The transduced cells were passaged once every three days at a ratio of 1:10 Expression of the foreign gene, GFP, was detected using fluorescent microscopy and flow cytometry Results: GFP S205 AAV VECTORS III was stably expressed for at least weeks without significant change in fluorescent signal intensity in cultures transduced with the five different viruses At five weeks post transduction, the positive rate of GFP expression mediated by lentiviral vectors listed in Methods, are 22.73%, 54.13%, 57.63%, 6.17% and 38.73%, respectively; while the mean fluorescent signal intensities were 6392, 21486, 26597, 1436, and 9468, respectively Conclusion:The results suggest that all five vectors can stably transduce 293T cells resulting in long term expression with different efficiencies Furthermore, insulator and UCOE not cause any improvement in GFP expression The vectors containing the promoters CMV or CMV (-globin intron) which elicited the highest GFP expressions can serve to create vectors for protein production 534 Tumorigenic Effect of Transplanted Myoblasts Overexpressing Bcl-2 Based on Retroviral Vector Francisco Martinez-F,1,2 Maria Ustoa,1 Araceli Barrera-L,1 Hugo E Sandoval-Zamora,1 Bruno Guevara,1 Pablo Vizcaino-Dorado,1 Alejandro Zentella-Dehesa,3 Catalina Machuca-R.4 Molecular Biotherapeutic Program, Skin & Tissue Bank, National Institute of Rehabilitation Ministry of Health, Mexico City, Mexico; 2Dapartment of Pharmacology, School of Medicine, National Univeristy of Mexico, Mexico City, Mexico; 3Department of Bichemistry, IIB at INCMNSZ, Mexico, DF, Mexico; Laboratory of Molecular Therapy, FES-Zaragoza, National University of Mexico, Mexico City, Mexico Introduction: The Bcl-2 overexpression confers cytoprotection and enhances cell resistance in several kinds of cell and tissues Previously we found that the myoblasts transformed with a retroviral vector to overexpressing bcl-2 protein in model of muscular reparation, results in cytoarchitecture preservation and an enhancement of myotubes differentiation However, the retroviral transformation provides too a potential tumorigenic factor Objective: To evaluate the tumorigenic effect of the repaired muscle with transformed myoblast cells with a retroviral vector to overexpressing bcl-2 in a in vivo model Material and Methods: The following groups (n=4) of nude mice were transplanted with 2X106 cells Group A with normal cells (control); Group B, myoblasts transformed with a retrovirus vector based on MLV system with CMV-GFP cassette (GFP group); and Group C, myoblasts transformed with retrovirus containing Bcl-2 (under Pol I promoter) All animals were maintained in the animal care facilities, under controlled environment and normal diet After months, animals were sacrificed and studied for molecular and histological analysis Results: Animals of the control group show macroscopical rates of fibrosis and atrophy One animal shows a tumour around the mouth The GFP group shows an increased tumor growth in the site of transplant and one tumour on the shoulder The Bcl-2 group shows similar size with the contralateral leg The molecular analysis of RT-PCR shows a minor expression of Bcl-2 levels related to the GFP expression The histological analysis was similar to histological patterns obtained during the day 30 The data shows that the effect of bcl-2 could improve the control of differentiation and possibly its implication on the growth process of myoblasts Additionally, this conserved effect could be a consequence of the different promoter, suggesting that the low levels of bcl-2 in muscle improve its biological effect and a tumor phenotype negative Acknowledgments: This research project is granted by the National Council of Science and Technology of México and the Ministry of Health of Mexico Grant FOSIS/CONACYT-Salud-2011-1-161624 535 Transduction of Ferret Airways with Avian Influenza Virus Hemagglutinin Pseudotyped Equine Infectious Anemia Virus Vector Ziying Yan,1 Manij Patel,2 Xingshen Sun,1 Diana CM Lei-butters,1 Hongshu Sui,1 John C Olsen,2 John F Engelhardt.1 Department of Anatomy and Cell Biology, Center for Gene Therapy, The University of Iowa, Iowa City; 2Department of Medicine, The University of North Carolina, Chapel Hill Cystic fibrosis (CF) is an inherent disease caused by a single gene defect in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes persistent bacterial lung infections The recently developed CF ferret model rapidly acquires bacterial lung infections similar to the human disease and is an ideal model for testing lung-directed gene therapies for CF To this end, we sought to develop a lung gene therapy strategy that would allow for complementation of CFTR in newborn CF ferrets Since lung epithelial cells are rapidly dividing in the neonatal period, we hypothesize that a lentiviral vector approach would best provide long-term persistent transgene expression in this model Like humans, ferrets are very susceptible to influenza virus infection and thus we hypothesized that hemagglutinin (HA) from avian influenza A virus (subtype H7) might be ideal for mediating lentiviral infection in the airway of newborn To this end, we pseudotyped equine infectious anemia virus (EIAV) with H7-HA derived from the parental avian influenza A stain A/FPV/Rostock/8/34 The reporter vector used in this study was HA-EIAV.Sin6.1CBnZW and encoded a nuclear located lacZ gene driven by CMV enhancer and chicken -actin promoter HA-EIAV.Sin6.1CBnZW titers, as determined on 293 cells, were 2.5x108 IU/ml We first tested infectivity of this recombinant virus on polarized adult ferret primary airway epithelia (FAE) cultured at an air-liquid interface (ALI) with an inoculum of 1x106 IU at an MOI 1 IU/cell Results from these studies demonstrated 40-50% cells were transgene positive following basolateral infection However, following apical infection transduction was much less efficient with 5% LacZ positive cells In vivo airway infection was conducted by intratracheal injection of 7.5x106 IU of HA-EAIV.Sin6.1CBnZW into newborn ferret kits at 2-days of age Two-weeks after infection, tracheas and lungs were harvested and stained with X-gal Grossly, significant transgene expression was seen in all lobes and the large and small conducting airways of the lungs, but not in the trachea Histologic analysis demonstrated that HA-H7-EIAV virus efficiently transduced bronchi, bronchioles, and alveoli (ranging from 10-70% of cells in these structures) X-gal positive cells in the tracheal airway epithelia were also detected at a much lower frequency These results suggested the HA-H7 pseudotyped EIAV vector effectively transduce intra-lobar airways of the newborn ferret and may be a good gene transfer agent to test gene therapies for CF lung disease in this model AAV Vectors III 536 Multiple Molecular Alterations in Phosphodegrons 1-3 within AAV2 Capsid Demonstrates Higher Hepatic Gene Transfer Efficiency Dwaipayan Sen,1 V Kalaivani,1 Rupali Gadkari,2 G Sudha,2 N Srinivasan,2 Alok Srivastava,1,3 Giridhara R Jayandharan.1,3 Hematology, Christian Medical College, Vellore, India; Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, India; 3Center for Stem Cell Research, Christian Medical College, Vellore, India The success of AAV2 mediated hepatic gene transfer in human trials for diseases such as hemophilia has been hampered by a combination of low transduction efficiency and a robust immune response directed against these vectors We have previously shown that AAV2 is S206 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... insulator and UCOE not cause any improvement in GFP expression The vectors containing the promoters CMV or CMV (-globin intron) which elicited the highest GFP expressions can serve to create vectors. .. Council of Science and Technology of México and the Ministry of Health of Mexico Grant FOSIS/CONACYT-Salud-2011-1-161624 535 Transduction of Ferret Airways with Avian Influenza Virus Hemagglutinin... susceptible to influenza virus infection and thus we hypothesized that hemagglutinin (HA) from avian influenza A virus (subtype H7) might be ideal for mediating lentiviral infection in the airway of newborn