Abstract
Post-transcriptional gene silencing mediated by double-stranded RNA represents an evolutionarily conserved cellular mechanism. Small dsRNAs, such as microRNAs (miRNAs), are part of the main regulatory mechanisms of gene expression in cells. The possibilities of harnessing this intrinsic natural mechanism of gene silencing for therapeutic applications was opened up by the discovery by Tom Tuschl’s team a few years ago that chemically synthesized small 21-mers of double-stranded RNA (small interfering RNA, siRNA) could inhibit gene expression without induction of cellular antiviral-like responses. siRNAs are especially of interest for cancer therapeutics because they allow specific inhibition of mutated oncogenes and other genes that aid and abet the growth of cancer cells. However, recent insights make it clear that siRNA faces some major hurdles before it can be used as a drug. Some of these problems are similar to those associated with classic antisense approaches, such as lack of delivery to specific tissues (other than the liver) or tumors, while other problems are more specific for siRNA, such as stability of the RNA molecules in circulation, off-target effects, interference with the endogenous miRNA machinery, and immune responses toward dsRNA. Chemical modifications of siRNA may help prevent these unwanted side effects. Initial studies show that minimal modifications with locked nucleic acids (LNA) help to reduce most of the unwanted side effects. In this chapter we will explore the limitations and possibilities of LNA-modified siRNA that may be used in future therapeutic applications.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., and Mello C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.
Meister G. and Tuschl T. (2004) Mechanisms of gene silencing by double-stranded RNA.Nature 431, 343–349.
Jackson R.J. and Standart N. (2007) How do microRNAs regulate gene expression? Sci. Stke.367, rel; DOI: 10.1126/stke.3672007rel.
Berezikov E., Thuemmler F., van Laake L.W., Kondova I., Bontrop R., Cuppen E., and Plasterk R.H. (2006) Diversity of microRNAs in human and chimpanzee brain.Nat. Genet. 38, 1375–1377.
Elbashir S.M., Harborth J., Lendeckel W., Yalcin A., Weber K., and Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 411, 494–498.
Crooke S.T. (1999) Molecular mechanisms of action of antisense drugs.Biochim. Biophys. Acta 1489, 31–44.
Mahato R.I., Cheng K., and Guntaka R.V. (2005) Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA.Expert Opin. Drug Deliv. 2, 3–28.
Amarzguioui M., Holen T., Babaie E., and Prydz H. (2003) Tolerance for mutations and chemical modifications in a siRNA.Nucleic Acids Res. 31, 589–595.
Braasch D.A., Jensen S., Liu Y., Kaur K., Arar K., White M.A., and Corey D.R. (2003) RNA interference in mammalian cells by chemically-modified RNA.Biochemistry 42, 7967–7975.
Czauderna F., Fechtner M., Dames S., Aygun H., Klippel A., Pronk G.J., Giese K., and Kaufmann J. (2003) Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.Nucleic Acids Res. 31, 2705–2716.
Harborth J., Elbashir S.M., Vandenburgh K., Manninga H., Scaringe S.A., Weber K., and Tuschl T. (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing.Antisense Nucleic Acid Drug Dev. 13, 83–105.
Fluiter K., ten Asbroek A.L., de Wissel M.B., Jakobs M.E., Wissenbach M., Olsson H., Olsen O., Oerum H., and Baas F. (2003) In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides.Nucleic Acids Res. 31, 953–962.
Elmen J., Thonberg H., Ljungberg K., Frieden M., Westergaard M., Xu Y., Wahren B., Liang Z., Orum H., Koch T., and Wahlestedt C. (2005) Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality.Nucleic Acids Res. 33, 439–447.
Mook O.R., Baas F., de Wissel M.B., and Fluiter K. (2007) Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo.Mol. Cancer Ther. 6, 833–843.
Martinez J. and Tuschl T. (2004) RISC is a 5′ phosphomonoester-producing RNA endonuclease Genes Dev. 18, 975–980.
Leuschner P.J., Ameres S.L., Kueng S., and Martinez J. (2006) Cleavage of the siRNA passenger strand during RISC assembly in human cells.EMBO Rep. 7, 314–320.
Jackson A.L., Burchard J., Leake D., Reynolds A., Schelter J., Guo J., Johnson J.M., Lim L., Karpilow J., Nichols K., Marshall W., Khvorova A., and Linsley P.S. (2006) Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing.RNA 12, 1197–205.
Boutla A., Delidakis C., Livadaras I., Tsagris M., and Tabler M. (2001) Short 5′-phosphorylated double-stranded RNAs induce RNA interference in Drosophila Curr. Biol. 11, 1776–1780.
Harborth J., Elbashir S.M., Vandenburgh K., Manninga H., Scaringe S.A., Weber K., and Tuschl T. (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing.Antisense Nucleic Acid Drug Dev. 13, 83–105.
Saxena S., Jonsson Z.O., and Dutta A. (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells.J. Biol. Chem. 278, 44312–44319.
Jackson A.L., Bartz S.R., Schelter J., Kobayashi S.V., Burchard J., Mao M., Li B., Cavet G., and Linsley P.S. (2003) Expression profiling reveals off target gene regulation by RNAi.Nat. Biotechnol. 21, 635–637.
Sledz C.A., Holko M., de Veer M.J., Silverman R.H., and Williams B.R. (2003) Activation of the interferon system by short-interfering RNAs.Nat. Cell Biol. 5, 834–839.
Bridge A.J., Pebernard S., Ducraux A., Nicoulaz A.L., and Iggo R. (2003) Induction of an interferon response by RNAi vectors in mammalian cells.Nat. Genet. 34, 263–264.
Jackson A.L., Burchard J., Schelter J., Chau B.N., Cleary M., Lim L., and Linsley P.S. (2006) Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity.RNA 12, 1179–1187.
Lewis B.P., Burge C.B., and Bartel D.P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell 120, 15–20.
Lewis B.P., Shih I.H., Jones-Rhoades M.W., Bartel D.P., and Burge C.B. (2003) Prediction of mammalian microRNA targets.Cell 115, 787–798.
Birmingham A., Anderson E.M., Reynolds A., Ilsley-Tyree D., Leake D., Fedorov Y., Baskerville S., Maksimova E., Robinson K., Karpilow J., Marshall W.S., and Khvorova A. (2006) 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets Nat. Methods 3, 199–204.
Lim L.P., Lau N.C., Garrett-Engele P., Grimson A., Schelter J.M., Castle J., Bartel D.P., Linsley P.S., and Johnson J.M. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.Nature 433, 769–773.
Lin X., Ruan X., Anderson M.G., McDowell J.A., Kroeger P.E., Fesik S.W., and Shen Y. (2005) siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.Nucleic Acids Res. 33, 4527–4535.
Xie X., Lu J., Kulbokas E.J., Golub T.R., Mootha V., Lindblad-Toh K., Lander E.S., and Kellis M. (2005) Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals Nature 434, 338–345.
Brennecke J., Stark A., Russell R.B., Cohen S.M. (2005) Principles of microRNA-target recognition.PLoS Biol. 3, e85.
Amarzguioui M., Holen T., Babaie E., and Prydz H. (2003) Tolerance for mutations and chemical modifications in a siRNA.Nucleic Acids Res. 31, 589–595.
Bramsen J.B., Laursen M.B., Damgaard C.K., Lena S.W., Babu B.R., Wengel J., and Kjems J. (2007) Improved silencing properties using small internally segmented interfering RNAs.Nucleic Acids Res. 35, 5886–5897
Matranga C., Tomari Y., Shin C., Bartel D.P., and Zamore P.D. (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.Cell 123, 607–620.
Grimm D., Streetz K.L., Jopling C.L., Storm T.A., Pandey K., Davis C.R., Marion P., Salazar F., and Kay M.A. (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.Nature 441, 537–541.
Schlee M., Hornung V., and Hartmann G. (2006) siRNA and isRNA: Two edges of one sword.Mol. Ther. 14, 463–470.
Judge A.D., Sood V., Shaw J.R., Fang D., McClintock K., and MacLachlan I. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.Nat. Biotechnol. 23, 457–462.
Alexopoulou L., Holt A.C., Medzhitov R., and Flavell R.A. (2001) Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature 413, 732–738.
Sioud M. (2005) Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization.J. Mol. Biol. 348, 1079–1090.
Marques J.T. and Williams B.R. (2005) Activation of the mammalian immune system by siRNAs.Nat. Biotechnol. 23, 1399–1405.
Hornung V., Guenthner-Biller M., Bourquin C., Ablasser A., Schlee M., Uematsu S., Noronha A., Manoharan M., Akira S., de Fougerolles A., Endres S., and Hartmann G. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.Nat. Med. 11, 263–270.
Judge A.D., Bola G., Lee A.C., and MacLachlan I. (2006) Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.Mol. Ther. 13, 494–505.
Robbins M., Judge A., Liang L., McClintock K., Yaworski E., and Maclachlan I. (2007) 2′-O-methyl-modified RNAs Act as TLR7 Antagonists Mol. Ther. 15, 1663–1669.
Urban-Klein B., Werth S., Abuharbeid S., Czubayko F., and Aigner A. (2005) RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo.Gene Ther. 12, 461–466.
van de Water F.M., Boerman O.C., Wouterse A.C., Peters J.G., Russel F.G., and Masereeuw R. (2006) Intravenously administered short interfering RNA accumulates in the kidney and selectively suppresses gene function in renal proximal tubules.Drug Metab. Dispos. 34, 1393–1397.
Liua N., Dingb H., Vanderheydena J., Zhua Z., and Zhang Y. (2007) Radiolabeling small RNA with technetium-99m for visualizing cellular delivery and mouse biodistribution.Surgery 142, 262–269.
Schiffelers R.M., Ansari A., Xu J., Zhou Q., Tang Q., Storm G., Molema G., Lu P.Y., Scaria P.V., Woodle M.C. (2004) Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle.Nucleic Acids Res. 32, e149.
Ma Z., Li J., He F., Wilson A., Pitt B., and Li S. (2005) Cationic lipids enhance siRNA-mediated interferon response in mice.Biochem. Biophys. Res. Commun. 330, 755–759.
Liu X., Howard K.A., Dong M., Andersen M.O., Rahbek U.L., Johnsen M.G., Hansen O.C., Besenbacher F., and Kjems J. (2007) The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing.Biomaterials 28, 1280–1288.
Soutschek J., Akinc A., Bramlage B., Charisse K., Constien R., Donoghue M., Elbashir S., Geick A., Hadwiger P., Harborth J., John M., Kesavan V., Lavine G., Pandey R.K., Racie T., Rajeev K.G., Rohl I., Toudjarska I., Wang G., Wuschko S., Bumcrot D., Koteliansky V., Limmer S., Manoharan M., and Vornlocher H.P. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature 432, 173–178.
Krutzfeldt J., Rajewsky N., Braich R., Rajeev K.G., Tuschl T., Manoharan M., and Stoffel M. (2005) Silencing of microRNAs in vivo with ‘antagomirs’.Nature 438, 685–689.
Song E., Zhu P., Lee S.K., Chowdhury D., Kussman S., Dykxhoorn D.M., Feng Y., Palliser D., Weiner D.B., Shankar P., Marasco W.A., and Lieberman J. (2005) Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat. Biotechnol. 23, 709–717.
Fluiter K., Frieden M., Vreijling J., Rosenbohm C., De Wissel M.B., Christensen S.M., Koch T., Orum H., and Baas F. (2005) On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide.Chembiochem 6, 1104–1109.
Sorensen M.D., Petersen M., and Wengel J. (2003) Functionalized LNA (locked nucleic acid): high-affinity hybridization of oligonucleotides containing N-acylated and N-alkylated 2′-amino-LNA monomers Chem. Commun. (Camb) 17, 2130–2131.
Larson S.D., Jackson L.N., Chen L.A., Rychahou P.G., and Evers B.M. (2007) Effectiveness of siRNA uptake in target tissues by various delivery methods.Surgery 142, 262–269.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Fluiter , K., Mook , O., Baas , F. (2009). The Therapeutic Potential of LNA-modified siRNAs: Reduction of Off-target Effects by Chemical Modification of the siRNA Sequence. In: Sioud, M. (eds) siRNA and miRNA Gene Silencing. Methods in Molecular Biology, vol 487. Humana Press. https://doi.org/10.1007/978-1-60327-547-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-60327-547-7_9
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-546-0
Online ISBN: 978-1-60327-547-7
eBook Packages: Springer Protocols