- Email: [email protected]
671. Construction and Assessment of Short-Hairpin RNA Eukaryotic Expression Vector Targeting TGF-β1 Labled by GFP
Oligonucleotide Therapies I prevent generation of escape variants since no natural isolate exists with a propensity to harbor all the possible mutations.
669. A Novel Lab-Scale Method for Encapsulation of siRNA in Cationic Liposomes Kevin Buyens,1 Stefaan C. De Smedt,1 Niek N. Sanders.1 Faculty of Pharmaceutical Sciences, Ghent University, Gent, Belgium.
1
Introduction A lot of efforts are currently made in the development of nano-scaled carrier systems that can guide siRNA molecules to their target cells after intravenous injection. One of the main issues in this research is the integrity of the siRNA containing nanoparticles in the blood stream. The integrity of the nanoparticles comprises both the particle size and the stable encapsulation of the siRNA. PEGylation is widely used for avoiding aggregation of nanoparticles in the blood stream. Furthermore, we believe that encapsulation of siRNA inside PEGylated liposomes or in between lipid multilayers is necessary to avoid disassembly of siRNA-liposome complexes by blood components. Objective The goal of this work is to develop a method to produce cationic PEGylated liposomes which fully encapsulate siRNA, and do not display aggregation when injected in the blood stream. Moreover, the method aims to be applicable on lab scale in order to avoid the use of large amounts of costly materials (both lipids and siRNA) for preliminary research in liposomal siRNA formulations. Method Classic preparation of siRNA-containing liposomes is done by mixing cationic liposomes with siRNA or vice versa. The liposomes are obtained by rehydrating a lipid film with a buffer solution followed by sonification or extrusion. In the newly described method the lipid films are directly hydrated with a siRNA solution and extruded. Subsequently, the surface bound siRNA is removed by addition of an excess of polyanions and the siRNA containing liposomes are purified by gel permeation chromatography. Results Classical mixtures of non-pegylated cationic liposomes with siRNA give fairly stable siRNA encapsulation, even when exposed to serum, but they tend to form aggregates. Classical mixtures of pegylated cationic liposomes with siRNA do not display much aggregate formation, but release their siRNA when brought into serum. Preparation of the same mixuteres via the described alternative method results in a good encapsulation of the siRNA in both non-pegylated and the pegylated liposomes. Furthermore, when siRNA is encapsulated in pegylated liposomes, no aggregation was observed in serum. Conclusion Non-pegylated liposomes can yield stable siRNA encapsulation, whereas pegylated liposomes can prevent aggregation. The newly developed method allows to combine both requirements, which results in a platform for the preparation of PEGylated liposomes containing encapsulated siRNA that do not aggregate. More importantly, this method can be done on a small scale, which makes it very useful for in vitro research.
670. Regulatable RNA Interference of VEGF Receptor 1 and Receptor 2
Roseanne Girnary,1 Mikko Turunen,1 Seppo Yla-Herttuala.1,2 1 Ark Therapeutics Group PLC, Microkatu 1S, Kuopio, Finland; 2 Department of Biotechnology and Molecular Medicine, AI Virtanen Institute, Kuopio, Finland. Vascular endothelial growth factor (VEGF)-A binds and activates VEGF receptors 1 (VEGFR1) and 2 (VEGFR2). These receptors play a large role in the progression of several human diseases. They are more abundantly expressed in tumour vasculature, rheumatoid arthritis and ocular disorders such as diabetic retinopathy and are thus important targets in the treatment of human disease. Specific down-regulation of these genes may effectively control VEGFVEGFR signalling with high efficiency and limited side-effects. In this study, shRNAs against VEGFR1 or VEGFR2 were cloned into the S250
backbone of the primary transcript of the human miR-30 microRNA within a lentiviral vector which contains all the necessary elements to achieve inducible, stable knockdown of VEGFR1 or VEGFR2. Upon addition of doxycycline, RNA and protein levels revealed conditional knockdown of both these genes in murine endothelial cell lines. In addition, co-expression of a transgene was possible. The potential lentiviral vector based shRNAs identified from the in vitro data are to be applied to in vivo tumour models in order to achieve a desired therapeutic effect.
671. Construction and Assessment of ShortHairpin RNA Eukaryotic Expression Vector Targeting TGF-β1 Labled by GFP Yaling Han, Na Li, Jian Kang, Yanmei Qi. 1 Department of Cardiology, Shenyang Northern Hospital, Shenyang, China.
[Background]: Transforming growth factor (TGF-β1), a potent multifunctional cytokine, is thought to play a key role in modulating vascular development, although its specific effects on smooth muscle cells (SMCs) and their precursors during development remain unclear. RNA interference (RNAi) has emerged as a powerful tool in gene function research. Three short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-β1 were designed for further research on the effects of TGF-β1 on vasculogenesis and angiogenesis. [Methods]: Based on Gateway cloning technology, three pairs of siRNA target sequences coding from the mRNA of TGF-β1 gene provided by GenBank were designed and three pairs of nucleotides labeled by specific enzyme sites were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector using T4 ligation enzyme, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labeled by GFP through the LR recombination reaction. Meanwhile, an negative control vector named pDS_T0 were contructed. The three resulting TGF-β1 shRNA expression vectors were named pDS_Ta, pDS_Tb, and pDS_Tc, respectively. After sequencing successfully, the vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-β1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RTPCR) and Western Blot were used to assess the silencing efficiency in mRNA and protein expression level. Furthermore, the BrdU incorporation assay and Flow Cytometry were used to detect the differences in proliferation between cells transfected with pDS_Ta, pDS_Tb and pDS_Tc. [Results]: RT-PCR and Western Blot showed that pDS_Tc downregulated TGF-β1 mRNA and protein expression markedly in NIH/3T3 cells. And the BrdU incorporation assay and Flow Cytometry showed that the proliferation ability of NIH/3T3 cells transfected with pDS_Tc was markedly decreased compared to cells transfected with pDS_T0. [Conclusion]: ShRNA eukaryotic expression vectors targeting TGF-β1 were successfully constructed which can be used for further investigation on the mechanism through which TGF-β1 regulates vasculogenesis and angiogenesis.
672. Selection of an Internalization-Competent RNA Aptamer Specific for the HER2 Receptor
Kristina W. Thiel,1 Ryan M. Whitaker,1 Xiu Ying Liu,1 James O. McNamara,1 Paloma H. Giangrande.1 1 Internal Medicine, University of Iowa, Iowa City, IA.
The application of siRNA technology to cancer treatment necessitates an efficient technique to specifically deliver the siRNA to cancer cells. Recently, our group reported that RNA aptamers can be used to selectively transport siRNAs to prostate cancer cells. RNA aptamers are structured RNA ligands that bind to target proteins with an affinity similar to that of antibodies. Due to their small size and chemical nature, RNA aptamers are more cost effective to Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
669. A Novel Lab-Scale Method for Encapsulation of siRNA in Cationic Liposomes Kevin Buyens,1 Stefaan C. De Smedt,1 Niek N. Sanders.1 Faculty of Pharmaceutical Sciences, Ghent University, Gent, Belgium.
1
Introduction A lot of efforts are currently made in the development of nano-scaled carrier systems that can guide siRNA molecules to their target cells after intravenous injection. One of the main issues in this research is the integrity of the siRNA containing nanoparticles in the blood stream. The integrity of the nanoparticles comprises both the particle size and the stable encapsulation of the siRNA. PEGylation is widely used for avoiding aggregation of nanoparticles in the blood stream. Furthermore, we believe that encapsulation of siRNA inside PEGylated liposomes or in between lipid multilayers is necessary to avoid disassembly of siRNA-liposome complexes by blood components. Objective The goal of this work is to develop a method to produce cationic PEGylated liposomes which fully encapsulate siRNA, and do not display aggregation when injected in the blood stream. Moreover, the method aims to be applicable on lab scale in order to avoid the use of large amounts of costly materials (both lipids and siRNA) for preliminary research in liposomal siRNA formulations. Method Classic preparation of siRNA-containing liposomes is done by mixing cationic liposomes with siRNA or vice versa. The liposomes are obtained by rehydrating a lipid film with a buffer solution followed by sonification or extrusion. In the newly described method the lipid films are directly hydrated with a siRNA solution and extruded. Subsequently, the surface bound siRNA is removed by addition of an excess of polyanions and the siRNA containing liposomes are purified by gel permeation chromatography. Results Classical mixtures of non-pegylated cationic liposomes with siRNA give fairly stable siRNA encapsulation, even when exposed to serum, but they tend to form aggregates. Classical mixtures of pegylated cationic liposomes with siRNA do not display much aggregate formation, but release their siRNA when brought into serum. Preparation of the same mixuteres via the described alternative method results in a good encapsulation of the siRNA in both non-pegylated and the pegylated liposomes. Furthermore, when siRNA is encapsulated in pegylated liposomes, no aggregation was observed in serum. Conclusion Non-pegylated liposomes can yield stable siRNA encapsulation, whereas pegylated liposomes can prevent aggregation. The newly developed method allows to combine both requirements, which results in a platform for the preparation of PEGylated liposomes containing encapsulated siRNA that do not aggregate. More importantly, this method can be done on a small scale, which makes it very useful for in vitro research.
670. Regulatable RNA Interference of VEGF Receptor 1 and Receptor 2
Roseanne Girnary,1 Mikko Turunen,1 Seppo Yla-Herttuala.1,2 1 Ark Therapeutics Group PLC, Microkatu 1S, Kuopio, Finland; 2 Department of Biotechnology and Molecular Medicine, AI Virtanen Institute, Kuopio, Finland. Vascular endothelial growth factor (VEGF)-A binds and activates VEGF receptors 1 (VEGFR1) and 2 (VEGFR2). These receptors play a large role in the progression of several human diseases. They are more abundantly expressed in tumour vasculature, rheumatoid arthritis and ocular disorders such as diabetic retinopathy and are thus important targets in the treatment of human disease. Specific down-regulation of these genes may effectively control VEGFVEGFR signalling with high efficiency and limited side-effects. In this study, shRNAs against VEGFR1 or VEGFR2 were cloned into the S250
backbone of the primary transcript of the human miR-30 microRNA within a lentiviral vector which contains all the necessary elements to achieve inducible, stable knockdown of VEGFR1 or VEGFR2. Upon addition of doxycycline, RNA and protein levels revealed conditional knockdown of both these genes in murine endothelial cell lines. In addition, co-expression of a transgene was possible. The potential lentiviral vector based shRNAs identified from the in vitro data are to be applied to in vivo tumour models in order to achieve a desired therapeutic effect.
671. Construction and Assessment of ShortHairpin RNA Eukaryotic Expression Vector Targeting TGF-β1 Labled by GFP Yaling Han, Na Li, Jian Kang, Yanmei Qi. 1 Department of Cardiology, Shenyang Northern Hospital, Shenyang, China.
[Background]: Transforming growth factor (TGF-β1), a potent multifunctional cytokine, is thought to play a key role in modulating vascular development, although its specific effects on smooth muscle cells (SMCs) and their precursors during development remain unclear. RNA interference (RNAi) has emerged as a powerful tool in gene function research. Three short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-β1 were designed for further research on the effects of TGF-β1 on vasculogenesis and angiogenesis. [Methods]: Based on Gateway cloning technology, three pairs of siRNA target sequences coding from the mRNA of TGF-β1 gene provided by GenBank were designed and three pairs of nucleotides labeled by specific enzyme sites were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector using T4 ligation enzyme, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labeled by GFP through the LR recombination reaction. Meanwhile, an negative control vector named pDS_T0 were contructed. The three resulting TGF-β1 shRNA expression vectors were named pDS_Ta, pDS_Tb, and pDS_Tc, respectively. After sequencing successfully, the vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-β1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RTPCR) and Western Blot were used to assess the silencing efficiency in mRNA and protein expression level. Furthermore, the BrdU incorporation assay and Flow Cytometry were used to detect the differences in proliferation between cells transfected with pDS_Ta, pDS_Tb and pDS_Tc. [Results]: RT-PCR and Western Blot showed that pDS_Tc downregulated TGF-β1 mRNA and protein expression markedly in NIH/3T3 cells. And the BrdU incorporation assay and Flow Cytometry showed that the proliferation ability of NIH/3T3 cells transfected with pDS_Tc was markedly decreased compared to cells transfected with pDS_T0. [Conclusion]: ShRNA eukaryotic expression vectors targeting TGF-β1 were successfully constructed which can be used for further investigation on the mechanism through which TGF-β1 regulates vasculogenesis and angiogenesis.
672. Selection of an Internalization-Competent RNA Aptamer Specific for the HER2 Receptor
Kristina W. Thiel,1 Ryan M. Whitaker,1 Xiu Ying Liu,1 James O. McNamara,1 Paloma H. Giangrande.1 1 Internal Medicine, University of Iowa, Iowa City, IA.
The application of siRNA technology to cancer treatment necessitates an efficient technique to specifically deliver the siRNA to cancer cells. Recently, our group reported that RNA aptamers can be used to selectively transport siRNAs to prostate cancer cells. RNA aptamers are structured RNA ligands that bind to target proteins with an affinity similar to that of antibodies. Due to their small size and chemical nature, RNA aptamers are more cost effective to Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy