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760. Expression of microRNAs from Lentiviral Vectors
RNA Virus Vectors II induced pluripotent stem (iPS) cell, which required reprogramming of differentiated tissue cells by expressing multiple exogenous genes in a single cell. These vectors will also become an effective tool for large-scale protein production: the gene expression induced by the vector is quite strong as comparable as to that observed in CHO/ dhfr gene amplification system, reached up to 50 pg protein/cell/ day without the need of time-consuming amplification procedure (Nishimura, K. et al., ibid.). Altogether, these persistent SeV vectors have a number of advantageous characteristics as a tool for gene therapy, for regeneration medicine and for manufacturing protein pharmaceutics.
760. Expression of microRNAs from Lentiviral Vectors Beate K. Syttkus, Andreas Hofmann, Bodo Haas, Alexander Pfeifer. 1 Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany.
MicroRNAs (miRNAs) are recently discovered, 20-25 nt long, noncoding RNAs targeting messenger RNAs (mRNAs) in a mechanism leading to regulation on either transcriptional or translational level. In many cases, miRNAs bind to the 3´UTR of the target mRNA at multiple sites, and regulates expression at the translational level. In contrast to small interfering RNAs (siRNAs), miRNAs only require complementarity in a small seed region (7 nt) to in induce gene silencing. Therefore, a single miRNA can regulate multiple targets and miRNAs are predicted to target one third of the protein coding genes. The number of genes regulated by these small RNAs indicates the potential for development of novel drugs as well as for the diagnosis of several human diseases including cancers. To analyse the role of miRNAs in cellular function and dysfunction systems for overexpression will be necessary. As lentiviral vectors can be used for long term expression and generation of transgenic animals, we analysed different vector configurations for optimal miRNA overexpression. Here, we analysed different lentiviral vectormiRNA expression cassettes for optimal configuration of miRNA expression. We designed cytomegalovirus (CMV) promoter-driven lentiviral vectors in three configurations carrying the pri-miRNA coding region plus flanking sequences either 5´ of a GFP coding region (CMV-miRNA-GFP) or 3´ of a GFP coding region (CMVGFP-miRNA) or without GFP (CMV-miRNA). We used these vectors to transduce cells and measured the amount of miRNAs expressed via quantitative Real-Time PCR. We chose miRNA195 and miRNA 143, which have been shown to have diverse expression patterns. Cells transduced with CMV-GFP-miRNA143 showed 15 fold increased microRNA levels compared to wildtype cells and 3 fold higher expression than the CMV-miRNA construct. Similar expression patterns were observed after transductions with the three analogous constructs carrying miRNA195 . Next, we used the best expressing vector configuration, to generate vectors carrying multiple miRNAs. Presently, we are analysing these multi-miRNA vectors as compared to transduction with lentiviral constructs carrying single miRNAs. Our data indicate that lentiviral vectors are a powerful tool for overexpression of microRNAs and that the configuration in which the miRNA are cloned strongly influences the expression levels.
761. Transduction of Primary Human Monocytes Using HIV-1-Derived Vector Particles Is Enhanced by the Viral Protein Vpx Provided by SIVsmPBjDerived Virus-Like Particles Silke Schüle,1 Björn-Philipp Kloke,1 Sabine Heidmeier,1 Julia Kaiser,1 Klaus Cichutek,1 Matthias Schweizer.1 1 Division Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany.
The viral protein Vpx is essential for replication of SIVsmPBj in monocyte-derived macrophages (Fletcher et al., 1996). Recently, we described efficient transduction of primary human monocytes using SIVsmPBj-derived vector particles, which could not be achieved by HIV-1-derived vectors (which do not encode Vpx) (Mühlebach et al., Mol. Ther.12, 2005). We demonstrated that the Vpx protein is the sole accessory protein in PBj-derived vectors needed for efficient transduction of monocytes (Wolfrum et al., Virology 364, 2007). To check the possibility of providing the Vpx function to HIV vectors, we generated here Vpr/Vpx fusion proteins. Vpr was hypothesized to be required for fusion protein incorporation into HIV-1 vector particles, Vpx was supposed to provide the gene transfer function for monocytes. Incorporation of the Vpr/Vpx fusion proteins by SIVsmPBj vectors lacking all accesssory genes enabled transduction of monocytes, as expected. Also, all Vpr/Vpx fusion proteins were found to be incorporated into HIV-1 vector particles. However, HIV-1 vector particles incorporating the Vpr/Vpx proteins did not lead to detectable transduction of primary human monocytes. This indicated that for monocyte transduction the Vpx protein had to be delivered in the background of SIVsmPBj vector particles. To confirm this hypothesis, Vpx proteins were provided by pre-incubation of monocytes with non-transducing virus-like particles (VLPs) derived from SIVsmPBj or HIV-1. When VLPs derived from SIVsmPBj were used, gene transfer into monocytes pre-treated with Vpx- or Vpr/Vpx-containing PBj-VLPs was achieved using HIV-1 vector particles. In contrast, delivery of the Vpr/Vpx via HIV-1-VLPs did not mediate transduction of primary monocytes by HIV-1 vector particles. This confirmed the hypothesis that monocyte transduction by SIVsmPBj-derived vectors requires specific Gag or Pol functions in addition to PBj-Vpx. To determine Vpx functions required for monocyte transduction, we generated various Vpx constructs containing mutations in highly conserved amino acid residues. The localisation pattern of the new constructs was analysed by Confocal Laser-Scanning-Microscopy. VLPs were generated containing the mutant Vpx variants to identify amino acid residues that are essential for monocyte transduction by SIVsmPBj or HIV-1 vectors. The proper function of Vpx was impaired by all mutations, which was reflected by a decreased capability of monocyte transduction. We identified the tyrosine at position 69 as an essential amino acid for Vpx function, since transduction of Vpx- ala 69-VLP pretreated monocytes by SIVsmmPBj or HIV-1 vectors was completely abolished.
762. The Impact of Architecture and Dynamics on the Outcome of Tumor Virotherapy
Carlos Reis,1 Jorge M. Pacheco,1 David Dingli.2 1 CFTC and Department of Physics, University of Lisbon, Lisbon, Portugal; 2Department of Molecular Medicine, Mayo Clinic, Rochester, MN. Introduction: Replication competent viruses based on the Edmonston vaccine strain of measles virus (MV-Edm) have potent and selective activity against various types of tumor in vitro but the responses in vivo are more variable. Some tumors are eliminated consistently while others persist despite evidence of ongoing viral propagation. In order to understand these disparate results, we have developed models for the spatial growth of a tumor population followed by infection with a replicating virus that can spread by cell
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
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760. Expression of microRNAs from Lentiviral Vectors Beate K. Syttkus, Andreas Hofmann, Bodo Haas, Alexander Pfeifer. 1 Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany.
MicroRNAs (miRNAs) are recently discovered, 20-25 nt long, noncoding RNAs targeting messenger RNAs (mRNAs) in a mechanism leading to regulation on either transcriptional or translational level. In many cases, miRNAs bind to the 3´UTR of the target mRNA at multiple sites, and regulates expression at the translational level. In contrast to small interfering RNAs (siRNAs), miRNAs only require complementarity in a small seed region (7 nt) to in induce gene silencing. Therefore, a single miRNA can regulate multiple targets and miRNAs are predicted to target one third of the protein coding genes. The number of genes regulated by these small RNAs indicates the potential for development of novel drugs as well as for the diagnosis of several human diseases including cancers. To analyse the role of miRNAs in cellular function and dysfunction systems for overexpression will be necessary. As lentiviral vectors can be used for long term expression and generation of transgenic animals, we analysed different vector configurations for optimal miRNA overexpression. Here, we analysed different lentiviral vectormiRNA expression cassettes for optimal configuration of miRNA expression. We designed cytomegalovirus (CMV) promoter-driven lentiviral vectors in three configurations carrying the pri-miRNA coding region plus flanking sequences either 5´ of a GFP coding region (CMV-miRNA-GFP) or 3´ of a GFP coding region (CMVGFP-miRNA) or without GFP (CMV-miRNA). We used these vectors to transduce cells and measured the amount of miRNAs expressed via quantitative Real-Time PCR. We chose miRNA195 and miRNA 143, which have been shown to have diverse expression patterns. Cells transduced with CMV-GFP-miRNA143 showed 15 fold increased microRNA levels compared to wildtype cells and 3 fold higher expression than the CMV-miRNA construct. Similar expression patterns were observed after transductions with the three analogous constructs carrying miRNA195 . Next, we used the best expressing vector configuration, to generate vectors carrying multiple miRNAs. Presently, we are analysing these multi-miRNA vectors as compared to transduction with lentiviral constructs carrying single miRNAs. Our data indicate that lentiviral vectors are a powerful tool for overexpression of microRNAs and that the configuration in which the miRNA are cloned strongly influences the expression levels.
761. Transduction of Primary Human Monocytes Using HIV-1-Derived Vector Particles Is Enhanced by the Viral Protein Vpx Provided by SIVsmPBjDerived Virus-Like Particles Silke Schüle,1 Björn-Philipp Kloke,1 Sabine Heidmeier,1 Julia Kaiser,1 Klaus Cichutek,1 Matthias Schweizer.1 1 Division Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany.
The viral protein Vpx is essential for replication of SIVsmPBj in monocyte-derived macrophages (Fletcher et al., 1996). Recently, we described efficient transduction of primary human monocytes using SIVsmPBj-derived vector particles, which could not be achieved by HIV-1-derived vectors (which do not encode Vpx) (Mühlebach et al., Mol. Ther.12, 2005). We demonstrated that the Vpx protein is the sole accessory protein in PBj-derived vectors needed for efficient transduction of monocytes (Wolfrum et al., Virology 364, 2007). To check the possibility of providing the Vpx function to HIV vectors, we generated here Vpr/Vpx fusion proteins. Vpr was hypothesized to be required for fusion protein incorporation into HIV-1 vector particles, Vpx was supposed to provide the gene transfer function for monocytes. Incorporation of the Vpr/Vpx fusion proteins by SIVsmPBj vectors lacking all accesssory genes enabled transduction of monocytes, as expected. Also, all Vpr/Vpx fusion proteins were found to be incorporated into HIV-1 vector particles. However, HIV-1 vector particles incorporating the Vpr/Vpx proteins did not lead to detectable transduction of primary human monocytes. This indicated that for monocyte transduction the Vpx protein had to be delivered in the background of SIVsmPBj vector particles. To confirm this hypothesis, Vpx proteins were provided by pre-incubation of monocytes with non-transducing virus-like particles (VLPs) derived from SIVsmPBj or HIV-1. When VLPs derived from SIVsmPBj were used, gene transfer into monocytes pre-treated with Vpx- or Vpr/Vpx-containing PBj-VLPs was achieved using HIV-1 vector particles. In contrast, delivery of the Vpr/Vpx via HIV-1-VLPs did not mediate transduction of primary monocytes by HIV-1 vector particles. This confirmed the hypothesis that monocyte transduction by SIVsmPBj-derived vectors requires specific Gag or Pol functions in addition to PBj-Vpx. To determine Vpx functions required for monocyte transduction, we generated various Vpx constructs containing mutations in highly conserved amino acid residues. The localisation pattern of the new constructs was analysed by Confocal Laser-Scanning-Microscopy. VLPs were generated containing the mutant Vpx variants to identify amino acid residues that are essential for monocyte transduction by SIVsmPBj or HIV-1 vectors. The proper function of Vpx was impaired by all mutations, which was reflected by a decreased capability of monocyte transduction. We identified the tyrosine at position 69 as an essential amino acid for Vpx function, since transduction of Vpx- ala 69-VLP pretreated monocytes by SIVsmmPBj or HIV-1 vectors was completely abolished.
762. The Impact of Architecture and Dynamics on the Outcome of Tumor Virotherapy
Carlos Reis,1 Jorge M. Pacheco,1 David Dingli.2 1 CFTC and Department of Physics, University of Lisbon, Lisbon, Portugal; 2Department of Molecular Medicine, Mayo Clinic, Rochester, MN. Introduction: Replication competent viruses based on the Edmonston vaccine strain of measles virus (MV-Edm) have potent and selective activity against various types of tumor in vitro but the responses in vivo are more variable. Some tumors are eliminated consistently while others persist despite evidence of ongoing viral propagation. In order to understand these disparate results, we have developed models for the spatial growth of a tumor population followed by infection with a replicating virus that can spread by cell
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
S285