Targeted Delivery of Antisense Oligonucleotides and siRNAs into Mammalian Cells

  • Mouldy Sioud Email author
Part of the Methods in Molecular Biology book series (MIMB, volume 487)


RNA interference (RNAi) is a natural mechanism for gene silencing that can be harnessed for the development of RNA-based drugs. Although synthetic small interfering RNA (siRNAs) can be delivered in vitro to virtually all cell types using lipid-based transfection agents or electroporation, efficient strategies for achieving either systemic or targeted delivery remains one of the major in vivo challenges. Among the targeting strategies, receptor-targeted delivery provides an innovative strategy to selectively direct therapeutics to cancer cells. Receptor-binding peptides can be incorporated into gene-delivery vesicles or directly conjugated to siRNAs in the hope of promoting their localization in target cells expressing the cognate receptors. This chapter discusses the current status of siRNA-targeting strategies using either peptides identified through iterative screening of random peptide phage libraries or naturally occurring peptides. Also, transcriptional targeting strategies and detailed protocols for the selection of cancer cell-binding peptide from random peptide phage libraries are described.


RNAi siRNA random peptide libraries hormone peptides peptide analogues endocytose cell surface receptors 



We thank Dr Anne Dybwad for critical reading of the manuscript and the group members for their contribution to this work.


  1. 1.
    Garanger, E., Boturyn, D., and Dumy, P. (2007) Tumor targeting with RGD peptide ligands-design of new molecular conjugates for imaging and therapy. Anticancer Agents Med. Chem. 7, 552–558.PubMedGoogle Scholar
  2. 2.
    Shadidi, M. and Sioud, M. (2003) Selective targeting of cancer cells using synthetic peptides. Drug Resist Updat. 6, 363–371.PubMedCrossRefGoogle Scholar
  3. 3.
    Aina, O.H., Sroka, T.C., Chen, M.L., et al. (2002) Therapeutic cancer targeting peptides. Biopolymers 66, 184–199.PubMedCrossRefGoogle Scholar
  4. 4.
    Sioud, M. (2005) On the delivery of small interfering RNAs into mammalian cells. Expert Opin. Drug Deliv. 2, 639–651.PubMedCrossRefGoogle Scholar
  5. 5.
    Shadidi, M. and Sioud, M. (2003) Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells. FASEB J. 17, 256–258.PubMedGoogle Scholar
  6. 6.
    Patel, D.S., Dessalew, N., Iqbal, P., . et al. (2007) Structure-based approaches in the design of GSK-3 selective inhibitors. Curr. Protein Pept. Sci. 8, 352–364.PubMedCrossRefGoogle Scholar
  7. 7.
    Sioud, M., Førre, Ø., and Dybwad, A. (1996) Selection of ligands for polyclonal antibodies from random peptide libraries: potential identification of (auto)antigens that may trigger B and T cell responses in autoimmune diseases. Clin. Immunol. Immunopathol. 79, 105–114.PubMedCrossRefGoogle Scholar
  8. 8.
    Romanov, V.I. (2003) Phage display selection and evaluation of cancer drug targets. Curr. Cancer Drug Targets. 3, 119–129.PubMedCrossRefGoogle Scholar
  9. 9.
    Falciani, C., Lozzi, L., Pini, A., . et al. (2005) Bioactive peptides from libraries. Chem. Biol. 12, 417–426.PubMedCrossRefGoogle Scholar
  10. 10.
    Houghten, R.A., Pinilla, C., Blondelle, S.E., . et al. (1991) Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354, 84–86.PubMedCrossRefGoogle Scholar
  11. 11.
    Fukuda, M.N., Ohyama, C., Lowitz, K., et al. (2000) A peptide mimic of E-selectin ligand inhibits sialyl Lewis X-dependent lung colonization of tumor cells. Cancer Res. 60, 450–456.PubMedGoogle Scholar
  12. 12.
    Lee, J.H., Engler, J.A., Collawn, J.F., et al. (2001) Receptor mediated uptake of peptides that bind the human transferrin receptor. Eur. J. Biochem. 268, 2004–2012.PubMedCrossRefGoogle Scholar
  13. 13.
    Campa, M.J., Serlin, S.B., and Patz, E.F. (2002) Development of novel tumor imaging agents with phage-display combinatorial peptide libraries. Acad. Radiol. 9, 927–932.PubMedCrossRefGoogle Scholar
  14. 14.
    Alaoui-Jamali, M.A. and Qiang, H. (2003) The interface between ErbB and non-ErbB receptors in tumor invasion: clinical implications and opportunities for target discovery. Drug Resist. Updat. 6, 95–107.PubMedCrossRefGoogle Scholar
  15. 15.
    Urbanelli, L., Ronchini, C., Fontana, L., et al. (2001) Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage. J. Mol. Biol. 313, 965–976.PubMedCrossRefGoogle Scholar
  16. 16.
    Karasseva, N.G., Glinsky, V.V., Chen, N.X., et al. (2002) Identification and characterization of peptides that bind human ErbB-2 selected from a bacteriophage display library. J. Protein Chem. 21, 287–296.PubMedCrossRefGoogle Scholar
  17. 17.
    Folkman, J. (2002) Role of angiogenesis in tumor growth and metastasis. Semin. Oncol. 29, 15–18.PubMedGoogle Scholar
  18. 18.
    Pasqualini, R. and Ruoslahti, E. (1996) Organ targeting in vivo using phage display peptide libraries. Nature 380, 364–366.PubMedCrossRefGoogle Scholar
  19. 19.
    Pasqualini, R., Koivunen, E., and Ruoslahti, E. (1997) Alpha v integrins as receptors for tumor targeting by circulating ligands. Nat. Biotechnol. 15, 542–546.PubMedCrossRefGoogle Scholar
  20. 20.
    Pasqualini, R., Koivunen, E., Kain, R., et al. (2000) Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res. 60, 272–722.Google Scholar
  21. 21.
    Arap, W., Pasqualini, R., and Ruoslahti, E. (1998) Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377–380.PubMedCrossRefGoogle Scholar
  22. 22.
    de Groot, F.M., Broxterman, H.J., Adams, H.P., et al. (2002) Design, synthesis, and biological evaluation of a dual tumor-specific motive containing integrin-targeted plasmin-cleavable doxorubicin prodrug. Mol. Cancer Ther. 1, 901–911.PubMedGoogle Scholar
  23. 23.
    Cheng, J.Q., Jiang, X., Fraser, M., et al. (2002) Role of X-linked inhibitor of apoptosis proteins in chemoresistance in ovarian cancer: possible involvement of the phosphoinositide-3 kinase/Akt pathway. Drug Resist. Updat. 5, 131–146.PubMedCrossRefGoogle Scholar
  24. 24.
    Su, Z.F., Liu, G., Gupta, S., et al. (2002) In vitro and in vivo evaluation of a Technetium-99m-labeled cyclic RGD peptide as a specific marker of alpha(v)beta(3) integrin for tumor imaging. Bioconjug. Chem. 13, 561–570.PubMedCrossRefGoogle Scholar
  25. 25.
    Wang, X.F., Birringer, M., Dong, L.F., et al. (2007) A peptide conjugate of vitamin E succinate targets breast cancer cells with high ErbB2 expression. Cancer Res. 67, 3337–3344.PubMedCrossRefGoogle Scholar
  26. 26.
    Song, E., Zhu, P., Lee, S.K., et al. (2005) Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23, 709–717.PubMedCrossRefGoogle Scholar
  27. 27.
    Peer, D., Zhu, P., Carman, C.V., et al. (2007) Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1. Proc. Natl. Acad. Sci. U S A 104, 4095–4100.PubMedCrossRefGoogle Scholar
  28. 28.
    McNamara, J.O., Andrechek, E.R., Wang, Y., et al. (2006) Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat. Biotechnol. 24, 1005–1015.PubMedCrossRefGoogle Scholar
  29. 29.
    Körner, M. and Reubi, J.C. (2007) NPY receptors in human cancer: A review of current knowledge. Peptides 28, 419–425.PubMedCrossRefGoogle Scholar
  30. 30.
    Riccabona, G. and Decristoforo, C. (2003) Peptide targeted imaging of cancer. Cancer Biother. Radiopharm. 18, 675–687.PubMedCrossRefGoogle Scholar
  31. 31.
    Hoffman, T.J., Quinn, T.P., and Volkert, W.A. (2001) Radiometallated receptor-avid peptide conjugates for specific in vivo. targeting of cancer cells Nucl. Med. Biol. 28, 527–539.PubMedCrossRefGoogle Scholar
  32. 32.
    Dharap, S.S. and Minko, T. (2003) Targeted proapoptotic LHRH-BH3 peptide. Pharm. Res. 20, 889–896.PubMedCrossRefGoogle Scholar
  33. 33.
    Shir, A. and Levitzki, A. (2001) Gene therapy for glioblastoma: Future perspective for delivery systems and molecular targets. Cell Mol. Neurobiol. 21, 645–656.PubMedCrossRefGoogle Scholar
  34. 34.
    Dharap, S.S., Wang, Y., Chandna, P., et al. (2005) Tumour-specific targeting of an anticancer drug delivery system by LHRH peptide PNAS 102,12962–12967PubMedCrossRefGoogle Scholar
  35. 35.
    Huang, P.S. and Oliff, A. (2001) Drug-targeting strategies in cancer therapy. Curr. Opin. Genet. Dev. 11, 104–110.PubMedCrossRefGoogle Scholar
  36. 36.
    Muratovska, A. and Eccles, M.R. (2004) Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells. FEBS lett. 558, 63–68.PubMedCrossRefGoogle Scholar
  37. 37.
    Crombez, L., Charnet, A., Morris, M.C., et al. (2007) A non-covalent peptide-based strategy for siRNA delivery. Biochem. Soc. Trans. 35, 44–46PubMedCrossRefGoogle Scholar
  38. 38.
    Hannon, G.J. and Rossi, J.J. (2004) Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378.PubMedCrossRefGoogle Scholar
  39. 39.
    Grimm, D., Streetz, K.L., Jopling, C.L., et al. (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441, 537–541.PubMedCrossRefGoogle Scholar
  40. 40.
    Jackson, A.L., Bartz, S.R., Schelter, J., et al. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637.PubMedCrossRefGoogle Scholar
  41. 41.
    Lin, X., Ruan, X., Anderson, M.G., et al. (2005) siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527–4535.PubMedCrossRefGoogle Scholar
  42. 42.
    McIntyre, G.J. and Fanning, G.C. (2006) Design and cloning strategies for constructing shRNA expression vectors. BMC Biotechnol. 6, 1.PubMedCrossRefGoogle Scholar
  43. 43.
    Van de Wetering, M., Oving, I., Muncan, V., et al. (2003) Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609–615.PubMedCrossRefGoogle Scholar
  44. 44.
    Saukkonen, K. and Hemminki, A. (2004) Tissue-specific promoters for cancer gene therapy. Expert Opin. Biol. Ther. 4, 683–696.PubMedCrossRefGoogle Scholar
  45. 45.
    Altieri, D.C. (2003) Validating survivin as a cancer therapeutic target. Nat. Rev. Cancer 3, 46–54.PubMedCrossRefGoogle Scholar
  46. 46.
    Huynh, T., Wälchli, S., and Sioud, M. (2006) Transcriptional targeting of small interfering RNAs into cancer cells. Biochem. Biophys. Res. Commun. 350, 854–859.PubMedCrossRefGoogle Scholar
  47. 47.
    Song, J., Pang, S., Lu, Y., et al. (2004) Gene silencing in androgen-responsive prostate cancer cells from the tissue-specific prostate-specific antigen promoter. Cancer Res. 64, 7661–7663.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Institute for Cancer Research, Department of ImmunologyMolecular Medicine Group, M. Sioud (馓)MontebelloNorway

Personalised recommendations