A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium

For the application of RNA interference (RNAi) in vivo the functional delivery of short interfering RNAs (siRNAs) is still the major obstacle. Therefore, delivery technologies need to be established for the systemic application of RNAi in vivo. Here we report uptake, biodistribution and in vivo efficacy of siRNA molecules formulated into siRNA-lipoplexes. The applied formulation is based on complex formation of positively charged liposomes, a mixture of cationic and fusogenic lipids complexed with the negatively charged siRNA. We determined by fluorescence microscopy the temporal and spatial distribution of fluorescently labeled siRNA-lipoplexes, the body clearance and endothelial cell type specific uptake after single intravenous injection. Furthermore, by using siRNA molecules for targeting endothelia-specifically expressed genes, such as CD31 and Tie2, we were able to demonstrate downregulation of the corresponding mRNA and protein in vivo. Taken together, we show the applicability of this non-viral delivery technology for inducing RNAi in the vasculature of mice after systemic application.

[1]  R. Shao,et al.  Silencing of hdm2 oncogene by siRNA inhibits p53-dependent human breast cancer , 2004, Cancer Gene Therapy.

[2]  Chryso Kanthou,et al.  Disrupting tumour blood vessels , 2005, Nature Reviews Cancer.

[3]  C. Férec,et al.  Contribution of plasmid DNA to hepatotoxicity after systemic administration of lipoplexes. , 2001, Human gene therapy.

[4]  A. Klippel,et al.  Activation of Phosphatidylinositol 3-Kinase Is Sufficient for Cell Cycle Entry and Promotes Cellular Changes Characteristic of Oncogenic Transformation , 1998, Molecular and Cellular Biology.

[5]  F. Szoka,et al.  Mechanism of oligonucleotide release from cationic liposomes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Assam El-Osta,et al.  RNA interference and potential therapeutic applications of short interfering RNAs , 2005, Cancer Gene Therapy.

[7]  Keith Bowman,et al.  Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs , 2005, Nature Biotechnology.

[8]  M. Eccles,et al.  Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells , 2004, FEBS letters.

[9]  Judy Lieberman,et al.  Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors , 2005, Nature Biotechnology.

[10]  Matthias John,et al.  Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs , 2004, Nature.

[11]  Hong Yang,et al.  Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene , 2005, Nature Medicine.

[12]  Y. Barenholz,et al.  Lipoplex-induced hemagglutination: potential involvement in intravenous gene delivery , 2002, Gene Therapy.

[13]  F. Szoka,et al.  Cationic lipids are essential for gene delivery mediated by intravenous administration of lipoplexes , 1999, Gene Therapy.

[14]  J. Bennett,et al.  Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. , 2003, Molecular vision.

[15]  S. Barik,et al.  Inhibition of respiratory viruses by nasally administered siRNA , 2005, Nature Medicine.

[16]  F. Leenders,et al.  PKN3 is required for malignant prostate cell growth downstream of activated PI 3‐kinase , 2004, The EMBO journal.

[17]  M. Dellian,et al.  Effect of the surface charge of liposomes on their uptake by angiogenic tumor vessels , 2003, International journal of cancer.

[18]  S. Uprichard.  The therapeutic potential of RNA interference , 2005, FEBS Letters.

[19]  A. Judge,et al.  Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA , 2005, Nature Biotechnology.

[20]  P. Carmeliet.  Angiogenesis in health and disease , 2003, Nature Medicine.

[21]  F. He,et al.  Cationic lipids enhance siRNA-mediated interferon response in mice. , 2005, Biochemical and biophysical research communications.

[22]  Qing Ge,et al.  Inhibition of influenza virus production in virus-infected mice by RNA interference. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[23]  C. L. Johnson,et al.  Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice. , 2000, Human gene therapy.

[24]  P. Chien,et al.  Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo , 2005, Cancer Gene Therapy.

[25]  D. Hanahan,et al.  Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice. , 1998, The Journal of clinical investigation.

[26]  S. Kimura,et al.  Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer. , 2005, The Journal of clinical investigation.

[27]  Zhenghong Yuan,et al.  Toll‐like receptor‐9 induced by physical trauma mediates release of cytokines following exposure to CpG motif in mouse skin , 2003, Immunology.

[28]  R. Schiffelers,et al.  Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. , 2004, Nucleic acids research.

[29]  Peter Carmeliet,et al.  Manipulating angiogenesis in medicine , 2004, Journal of internal medicine.

[30]  F. Natt,et al.  siRNA relieves chronic neuropathic pain. , 2004, Nucleic acids research.

[31]  Patty J. Lee,et al.  Small Interfering RNA Targeting Heme Oxygenase-1 Enhances Ischemia-Reperfusion-induced Lung Apoptosis* , 2004, Journal of Biological Chemistry.

[32]  Mouldy Sioud,et al.  Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[33]  Theresa M. Allen,et al.  Pharmacokinetics of long-circulating liposomes , 1995 .

[34]  S. Chae,et al.  Requirement for sphingosine 1-phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference. , 2004, The Journal of clinical investigation.

[35]  Cell Penetrating Peptides , 2000 .

[36]  Raymond M Schiffelers,et al.  Inhibition of ocular angiogenesis by siRNA targeting vascular endothelial growth factor pathway genes: therapeutic strategy for herpetic stromal keratitis. , 2004, The American journal of pathology.

[37]  T. Rana,et al.  siRNA function in RNAi: a chemical modification analysis. , 2003, RNA.

[38]  J. Northrop,et al.  Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Flavell,et al.  Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3 , 2001, Nature.

[40]  Y. Yuzawa,et al.  A Small Interfering RNA Targeting Vascular Endothelial Growth Factor as Cancer Therapeutics , 2004, Cancer Research.

[41]  T. Rana,et al.  RNAi in human cells: basic structural and functional features of small interfering RNA. , 2002, Molecular cell.

[42]  M. Hirano,et al.  The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity. , 1994, Molecular and cellular biology.

[43]  G. Thurston.  Role of Angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis , 2003, Cell and Tissue Research.

[44]  Eric Vives,et al.  Cell-penetrating Peptides , 2003, The Journal of Biological Chemistry.

[45]  T. Ochiya,et al.  Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[46]  A. Klippel,et al.  Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells. , 2003, Nucleic acids research.

[47]  M. Gait,et al.  Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides , 2005, Nucleic acids research.

[48]  Z. Paroo,et al.  Biodistribution of phosphodiester and phosphorothioate siRNA. , 2004, Bioorganic & medicinal chemistry letters.

[49]  T. Irimura,et al.  Antitumor Activity of Small Interfering RNA/Cationic Liposome Complex in Mouse Models of Cancer , 2004, Clinical Cancer Research.

[50]  E. Lesnik,et al.  Pharmacokinetic properties of 2'-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats. , 2001, The Journal of pharmacology and experimental therapeutics.

[51]  Pamela F. Jones,et al.  Isolation of Angiopoietin-1, a Ligand for the TIE2 Receptor, by Secretion-Trap Expression Cloning , 1996, Cell.

[52]  A. Aigner,et al.  RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo , 2005, Gene Therapy.

[53]  P. Carmeliet,et al.  Angiogenesis in cancer and other diseases , 2000, Nature.

[54]  Mark E. Davis,et al.  Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. , 2005, Cancer research.

[55]  Anil K Sood,et al.  Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. , 2005, Cancer research.

[56]  J. Lieberman,et al.  Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[57]  S. Akira,et al.  Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7 , 2005, Nature Medicine.