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1022. Non Viral Systems for Enhanced siRNA Delivery
RNAI DELIVERY, EXON SKIPPING AND GENE CORRECTION response to matrix metalloproteinase.³ In the present study, to further improve the silencing activity of MEND, we modified R8-MEND with GALA (R8/GALA-MEND). In addition, we compared the silencing activity of R8/GALA-MEND with that of conventional MEND composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a cationic lipid widely used as a lipoplex component, with or without PPD. We first examined the optimal concentration of R8 and GALA modified on the lipid envelope of R8/GALA-MEND. Then, we investigated the effect of modification of R8/GALA-MEND with PPD or non-cleavable conventional PEG-lipids on the silencing activity. When we modified R8/GALA-MEND with a conventional PEG-lipid, its knockdown efficiency was decreased dramatically. In contrast, PPD-modified R8/GALA-MEND showed nearly equal silencing effect to PEG-unmodified R8/GALA-MEND. Finally, we compared a R8/GALA-MEND with DOTAP-based MEND. R8/ GALA-MEND shows a much greater silencing ability than DOTAPbased MEND, even if modified with PPD. These results indicate that R8/GALA-MEND is a promising carrier for siRNA delivery to tumor cells. Reference: 1) Nakamura Y., Kogure K., Futaki S., Harashima H., J. Control. Release, 119, 360-367 (2007). 2) Sasaki K., Kogure K., Chaki S., Nakamura Y., Moriguchi R., Hamada H., Danev R., Nagayama K., Futaki S., Harashima H., Anal. Bioanal. Chem., 391, 2717-2727 (2008). 3) Hatakeyama H., Akita H., Kogure K., Oishi M., Nagasaki Y., Kihira Y., Ueno M., Kobayashi H., Kikuchi H., Harashima H., Gene Ther., 14, 68-77 (2007).
1022. Non Viral Systems for Enhanced siRNA Delivery
Anne-Laure Bolcato-Bellemin,1 Marie-Elise Bonnet,1 Patrick Neuberg,1 Jean-Paul Behr,2 Patrick Erbacher. 1 Polyplus-Transfection, Strasbourg, France; 2Laboratoire de Chimie Génétique, Université Louis Pasteur, Illkirch, France.
With the recent discovery of RNA interference, siRNA has become a gene silencing tool for gene therapy. However, RNAi shares with gene therapy the lack of efficient in vivo delivery techniques. Non viral delivery vehicles, such as cationic polymers or cationic lipids can be used for siRNA complexation and delivery. The purpose of our work is to develop and improve delivery reagent for in vivo delivery of siRNA. To date, linear polyethylenimine (L-PEI) has being widely used for in vivo delivery of nucleic acid due to its efficiency, versatility, low toxicity. However, the strength and the stability of the complexes formed between PEI and a gene or a siRNA is different. The small rigid two-turn double helix of the siRNA does not condense with linear PEI as weel as with long double stranded DNA. Thus with the goal of improving strength and stability of siRNA / L-PEI complexes, we have designed sticky siRNAs (ssiRNAs) mimicking the structure of long double-stranded DNA in order to increase delivery of siRNA and silencing efficiency. Our modified siRNA have the ability to reversibly oligomerize using 3’ complementary and extended overhangs and to mimic DNA structure. We have shown that ssiRNAs are significantly more effective than siRNAs when using PEI as a delivery reagent both in vitro and in vivo. Moreover, we have shown the absence of major induction of pro-inflammatory cytokines and hepatic enzymes in the blood after systemic injection in mice of siRNAs or ssiRNAs delivered with L-PEI. In parallel we have developed cationic amphiphile systems for delivery of siRNA in vitro and in vivo. Such delivery systems are able to efficiently silence gene at picomolar concentration of siRNA in vitro and showed promising results in different in vivo models. Taken together, our data highlight the potency of our synthetic cationic molecules as non-viral delivery reagents of choice for siRNA-based therapy.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
1023. How To Make siRNA Lipoplexes Efficient? Add a DNA Cargo
Hervé Rhinn,1 Céline Largeau,1 Pascal Bigey,1 René Lai Kuen,2 Daniel Scherman,1 Virginie Escriou.1 1 Unité de Pharmacologie Chimique et Génétique, U640 Inserm UMR8151 CNRS ENSCP Paris Descartes, Paris, France; 2SCICM, INSERM-CNRS-IRD-Université Paris Descartes, Paris, France. RNAi represents a promising strategy for the treatment of various disorders. Important tasks however still remain for RNAi in vivo applications, such as stability of siRNA after injection and optimization of the targeting to the candidate cells. The half-life of unmodified siRNA in vivo is short due to rapid degradation by endogenous nucleases and efficient elimination from the blood flow. In this context, lipofection appears an attractive way to deliver siRNA in vivo. We recently reported an efficient formulation of siRNA into lipoplexes that was able to deliver siRNA targeting TNF-α following intravenous injection and to restore immunological balance in the mouse collagen-induced arthritis model. Since this efficient formulation included the pre association of siRNA with a DNA carrier, we decided to extensively characterised siRNA lipoplexes with or without DNA carrier, in order to better understand the DNA carrier enhancing effect. We showed that addition of DNA carrier in siRNA lipoplexes allowed us to obtain specific gene extinction in vitro, detectable at mRNA and protein level using reduced siRNA concentration. This procedure has also the advantage to be applicable to other lipid vectors, like lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA carrier using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, notably in the amount of lipid required to obtain neutrality of the complex or complete complexation of nucleic acid and in stability of the complex towards incubation with heparan sulfate. Taken together these results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to cationic lipid/ liposome complexes more efficient to transfer siRNA for low siRNA concentration and in the presence of serum. This increased efficiency could come from a modified stability of the complexes towards cellular components.
1024. Scaffold-Based Gene Silencing Approach to Regenerative Medicine
Haoqing Cao,1 Chou Chai,2 Sing Yian Chew.1 1 Chemical & Biomedical Engineering, Nanyang Technological University, Singapore, Singapore; 2Duke-NUS Graduate Medical School, Singapore, Singapore. Fibrous scaffolds represent a novel class of materials in regenerative medicine. To expand upon this promising platform, we adopt a novel method for enhanced tissue regeneration: incorporation of small-interfering-RNA (siRNA) for scaffold-based gene-silencing applications. In this study, poly(ε-caprolactone) nanofibers (PCL, ϕ∼372 +/-50nm) encapsulating siRNA was fabricated with varying loading levels (0-27wt%) of poly(ethylene-glycol) (PEG). Encapsulation of FAM-labelled oligonucleotide demonstrated a uniform dispersion of siRNA in aggregate form throughout the nanofibers. In vitro analyses indicated a sustained release of siRNA ( ≥ 2-17.7%) for at least 23 days under physiological conditions as detected by RiboGreen assay. Close examination of fibre architecture under SEM suggested a diffusion process. Using gel electrophoresis, we confirmed the intact structure of the siRNA (21bp) after the period of sustained release and after prolonged nanofiber-encapsulation for at least 49 days under physiological conditions. Specific genesilencing of both endogenous (GAPDH) and exogenous (eGFP) gene in eGFP-HEK293 cells indicated the preservation of siRNA bioactivity throughout the period of sustained release. Altogether, our results indicate that such gene-silencing nanofibrous scaffolds S389
1022. Non Viral Systems for Enhanced siRNA Delivery
Anne-Laure Bolcato-Bellemin,1 Marie-Elise Bonnet,1 Patrick Neuberg,1 Jean-Paul Behr,2 Patrick Erbacher. 1 Polyplus-Transfection, Strasbourg, France; 2Laboratoire de Chimie Génétique, Université Louis Pasteur, Illkirch, France.
With the recent discovery of RNA interference, siRNA has become a gene silencing tool for gene therapy. However, RNAi shares with gene therapy the lack of efficient in vivo delivery techniques. Non viral delivery vehicles, such as cationic polymers or cationic lipids can be used for siRNA complexation and delivery. The purpose of our work is to develop and improve delivery reagent for in vivo delivery of siRNA. To date, linear polyethylenimine (L-PEI) has being widely used for in vivo delivery of nucleic acid due to its efficiency, versatility, low toxicity. However, the strength and the stability of the complexes formed between PEI and a gene or a siRNA is different. The small rigid two-turn double helix of the siRNA does not condense with linear PEI as weel as with long double stranded DNA. Thus with the goal of improving strength and stability of siRNA / L-PEI complexes, we have designed sticky siRNAs (ssiRNAs) mimicking the structure of long double-stranded DNA in order to increase delivery of siRNA and silencing efficiency. Our modified siRNA have the ability to reversibly oligomerize using 3’ complementary and extended overhangs and to mimic DNA structure. We have shown that ssiRNAs are significantly more effective than siRNAs when using PEI as a delivery reagent both in vitro and in vivo. Moreover, we have shown the absence of major induction of pro-inflammatory cytokines and hepatic enzymes in the blood after systemic injection in mice of siRNAs or ssiRNAs delivered with L-PEI. In parallel we have developed cationic amphiphile systems for delivery of siRNA in vitro and in vivo. Such delivery systems are able to efficiently silence gene at picomolar concentration of siRNA in vitro and showed promising results in different in vivo models. Taken together, our data highlight the potency of our synthetic cationic molecules as non-viral delivery reagents of choice for siRNA-based therapy.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
1023. How To Make siRNA Lipoplexes Efficient? Add a DNA Cargo
Hervé Rhinn,1 Céline Largeau,1 Pascal Bigey,1 René Lai Kuen,2 Daniel Scherman,1 Virginie Escriou.1 1 Unité de Pharmacologie Chimique et Génétique, U640 Inserm UMR8151 CNRS ENSCP Paris Descartes, Paris, France; 2SCICM, INSERM-CNRS-IRD-Université Paris Descartes, Paris, France. RNAi represents a promising strategy for the treatment of various disorders. Important tasks however still remain for RNAi in vivo applications, such as stability of siRNA after injection and optimization of the targeting to the candidate cells. The half-life of unmodified siRNA in vivo is short due to rapid degradation by endogenous nucleases and efficient elimination from the blood flow. In this context, lipofection appears an attractive way to deliver siRNA in vivo. We recently reported an efficient formulation of siRNA into lipoplexes that was able to deliver siRNA targeting TNF-α following intravenous injection and to restore immunological balance in the mouse collagen-induced arthritis model. Since this efficient formulation included the pre association of siRNA with a DNA carrier, we decided to extensively characterised siRNA lipoplexes with or without DNA carrier, in order to better understand the DNA carrier enhancing effect. We showed that addition of DNA carrier in siRNA lipoplexes allowed us to obtain specific gene extinction in vitro, detectable at mRNA and protein level using reduced siRNA concentration. This procedure has also the advantage to be applicable to other lipid vectors, like lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA carrier using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, notably in the amount of lipid required to obtain neutrality of the complex or complete complexation of nucleic acid and in stability of the complex towards incubation with heparan sulfate. Taken together these results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to cationic lipid/ liposome complexes more efficient to transfer siRNA for low siRNA concentration and in the presence of serum. This increased efficiency could come from a modified stability of the complexes towards cellular components.
1024. Scaffold-Based Gene Silencing Approach to Regenerative Medicine
Haoqing Cao,1 Chou Chai,2 Sing Yian Chew.1 1 Chemical & Biomedical Engineering, Nanyang Technological University, Singapore, Singapore; 2Duke-NUS Graduate Medical School, Singapore, Singapore. Fibrous scaffolds represent a novel class of materials in regenerative medicine. To expand upon this promising platform, we adopt a novel method for enhanced tissue regeneration: incorporation of small-interfering-RNA (siRNA) for scaffold-based gene-silencing applications. In this study, poly(ε-caprolactone) nanofibers (PCL, ϕ∼372 +/-50nm) encapsulating siRNA was fabricated with varying loading levels (0-27wt%) of poly(ethylene-glycol) (PEG). Encapsulation of FAM-labelled oligonucleotide demonstrated a uniform dispersion of siRNA in aggregate form throughout the nanofibers. In vitro analyses indicated a sustained release of siRNA ( ≥ 2-17.7%) for at least 23 days under physiological conditions as detected by RiboGreen assay. Close examination of fibre architecture under SEM suggested a diffusion process. Using gel electrophoresis, we confirmed the intact structure of the siRNA (21bp) after the period of sustained release and after prolonged nanofiber-encapsulation for at least 49 days under physiological conditions. Specific genesilencing of both endogenous (GAPDH) and exogenous (eGFP) gene in eGFP-HEK293 cells indicated the preservation of siRNA bioactivity throughout the period of sustained release. Altogether, our results indicate that such gene-silencing nanofibrous scaffolds S389