Antisense Oligonucleotides for in Vivo Studies of Angiotensin Receptors

  • M. Ian Phillips
  • Philipp Ambühl
  • Robert Gyurko
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 396)


Synthetic antisense (AS) oligodeoxynucleotides inhibit genetic expression by sequence-specific hybridization to mRNA that renders the mRNA inactive for translation. We have been using AS oligos to lower blood pressure by inhibiting angiotensin receptors and angiotensinogen in freely moving, whole animals. This recent application of antisense technology to in vivo studies, opens a new way of approaching physiological problems with the precision of molecular biology. The possibility of blocking specific gene expression by AS inhibition without multiple, non-specific side effects has potential for therapeutic uses in many diseases. Antisense inhibition is an extremely attractive pharmacological and investigative approach since it offers base-to-base specificity to the target protein and versatility appropriate to the complexity of the genetic code. However, there are a number of issues to be considered before using antisense in any experimental or clinical setting. These include (1) selection of target sequence, (2) the mechanism of cellular uptake, (3) stability of antisense oligos in cells and body fluids, (4) possible intracellular sites of action and (5) effectiveness, in terms of specificity, and duration of action.


Cellular Uptake Antisense Oligonucleotide Receptor mRNA Antisense Inhibition Anti Sense Oligonucleotide 
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  1. 1.
    Stull, R.A., Taylor, L.A. and Szoka, F.C., Predicting antisense oligonucleotide inhibitory efficacy: a computational approach using histograms and thermodynamic indices, Nucl.Acids Res., 20(13) (1992) 3501–3508.PubMedCrossRefGoogle Scholar
  2. 2.
    Cowsert, L.M., Fox, M.C., Zon, G. and Mirabelli, C.K., in vitro evaluation of phosphorothioate oligonu-cleotides targeted to the E2 mRNA of papillomavirus: potential treatment for genital warts, Antimicrob. Agents Chemother. 37(2) (1993) 171–177.PubMedCrossRefGoogle Scholar
  3. 3.
    Wakita, T. and Wands, J.R., Specific inhibition of hepatitis C virus expression by antisense oligonu-cleotides. In vitro model for selection of target sequence, J. Bol. Chem., 269(19) (1994) 14205–14210.Google Scholar
  4. 4.
    Lima, W.F., Monia, B.P., Ecker, D.J. and Freier, S.M., Implication of RNA structure on antisense oligonucleotide hybridization kinetics, Biochemistry, 31(48) (1992) 12055–12061.PubMedCrossRefGoogle Scholar
  5. 5.
    Rittner, K and Sczakiel, G., Identification and analysis of antisense RNA target regions of the human immunodeficiency virus type 1, Nucl.Acid Res., 19(7) (1991) 1421–1426.CrossRefGoogle Scholar
  6. 6.
    Jaroszevski, J.W., Syi, J.L., Ghosh, M., Ghosh, K. and Cohen, J.S., Targeting of antisense DNA: comparison of activity of anti-rabbit beta-globin oligodeoxyribonucleoside phosphorothioates with computer predictions of mRNA folding, Antisense Res.Dev., 3(4) (1993) 339–348.Google Scholar
  7. 7.
    Fakler, B., Herlitze, S., Amthor, B., Zenner, H.P. and Ruppersberg, J.P., Short antisense oligonucleotide-mediated inhibition is strongly dependent on oligo length and concentration but almost independent of location of the target sequence, J. Biol. Chem., 269(23) (1994) 16187–16194.PubMedGoogle Scholar
  8. 8.
    Puskas, L.G. and Bottka, S., Prokaryotic test system for evaluation of oligonucleotide-affected antisense inhibition, Anal.Biochem., 222(2) (1994) 305–309.PubMedCrossRefGoogle Scholar
  9. 9.
    Singer, M. and Berg, P. In: Genes and Genomes, University Science Books, 54-59.Google Scholar
  10. 10.
    Iversen, P.L., Zhu, S., Meyer, A. and Zon, G., Cellular uptake and subcdellular distribution of phosphorothioate oligonucleotides into cultured cells, Antisense Res.Dev., 2(3) (1992) 211–222.PubMedGoogle Scholar
  11. 11.
    Colige, A., Sokolov, B.P., Nugent, P., Baserge, R. and Prockop, D.J., Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells, Biochemistry, 32(1) (1993) 7–11.PubMedCrossRefGoogle Scholar
  12. 12.
    Helene, C., The anti-gene strategy: control of gene expression by triplex-forming-oligonucleotides, Anticancer Drug Res., 6(6) (1991) 569–584.Google Scholar
  13. 13.
    Bennett, C.F., Condon, T.P., Grimm, S., Chan, H. and Chiang, M.Y., Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides, J. Immunol, 152(7) (1994) 3530–3540.PubMedGoogle Scholar
  14. 14.
    Bacon, T.A. and Wickstrom, E., Walking along human c-myc mRNA with antisense oligonucleotides: maximum efficacy at the 5′ cap region, Oncogene Res., 6(1) (1991) 13–19.PubMedGoogle Scholar
  15. 15.
    Campbell, J.M, Bacon, T.A. and Wickstrom, E., Oligodeoxynucleotide phosphorothioate stability in subcellular extracts, culture media, ser and cerebrospinal fluid, J. Biochem. Biophys. Methods 20(3) (1990) 259–267.PubMedCrossRefGoogle Scholar
  16. 16.
    Wahlestedt, C., Antisense oligonucleotide strategies in neuropharmacology, TiPS, 15 (1994) 42–46.PubMedGoogle Scholar
  17. 17.
    Gyurko, R., Wielbo, D., Phillips, M.I., Antisense inhibition of AT1 receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin, Regul. Pept. 49(2) (1993) 167–74.PubMedCrossRefGoogle Scholar
  18. 18.
    Gyurko, R., Li, B., Sumners, C. and Phillips, M.I., Antisense inhibition of angiotensin type-1 receptor expression in NG108-15 cells, Soc. for Neurosci. (1994) 220.7Google Scholar
  19. 19.
    Li, B. and Phillips, M.I., Cellular uptake of oligonucleotides in bovine adrenal cells (in press) (1995).Google Scholar
  20. 20.
    Yu, C., Brussaard, A.B., Yang, X., Listerud, M. and Role, L.W., Uptake of antisense oligonucleotides and functional block of acetylcholine receptor subunit gene expression in primary embryonic neurons, Dev-Genet 14(4) (1993) 296–304.PubMedCrossRefGoogle Scholar
  21. 21.
    Marti, G., Egan, W. Noguichi, P. Zon, G., Matsukura, M. and Broder, S., Oligodeoxyribonucleotide phosphorothioate fluxes and localization in hematopoietic cells, Antisense Res. Rev. 2(1) (1992) 27–39.Google Scholar
  22. 22.
    Clarenc, J.P., LeBleu, B. and Leonetti, J.P., Characterization of the nuclear binding sites of oligodeoxyri-bonucleotides and their analogs, J. Biol. Chem. 268(8) (1993) 5600–5604.PubMedGoogle Scholar
  23. 23.
    Sixou, S., Szoka, F.C., Jr., Green, G.A., Giusti, B., Zong, G. and Chin, D.J., Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET), Nuclei Acids Res. 22(4) (1994) 662–668.CrossRefGoogle Scholar
  24. 24.
    Temsamani, J., Kubert, M., Tang, J., Padmapriya, A., Agrawal, S., Cellular uptake of oligodeoxynucleotide phosphorothioates and their analogs, Antisense Res. Dev. 4(1) (1994) 35–42.PubMedGoogle Scholar
  25. 25.
    Wahlestedt, C., Golanov, E., Yamamoto, S., Yee, F., Ericson, H., Yoo, H., Inturrisi, C.E., Reis, D.J., Antisense oligonucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischemic infarctions, Nature 363(6426) (1993) 260–263.PubMedCrossRefGoogle Scholar
  26. 26.
    Rougee, M., Faucon, B., Mergny, J.L., Barcelo, F., Giovannangeli, C., Garestier, T., Helene, C., Kinetics and thermodynamics of triple-helix formation: effects of ionic strength and mismatches, Biochemistry 31 (1992) 9269–9278.PubMedCrossRefGoogle Scholar
  27. 27.
    Crooke, R.M., In vitro toxicology and pharmacokinetics of antisense oligonucleotides, Anticancer Drug Des. 6(6) (1991) 609–646.Google Scholar
  28. 28.
    Phillips, M.I., Wielbo, D. and Gyurko, R., Antisense inhibition of hypertension: A new strategy for renin-angiotensin candidate genes, Kidney Intl. 46 (1994) 1554–1556.CrossRefGoogle Scholar
  29. 29.
    Wielbo, D., Sernia, C., Gyurko, R. and Phillips, M.I., Antisense inhibition of hypertension in the spontaneously hypertensive rat, Hypertension 25 (1994) 314–319.CrossRefGoogle Scholar
  30. 30.
    Phillips, M.I., Functions of angiotensin II in the central nervous system, Ann. Rev. Physiol. 49 (1985) 413–415.CrossRefGoogle Scholar
  31. 31.
    Sakai, R.R., He, P.F., Yang, X.D., Ma., L.Y., Guo, Y.F., Reilly, J.J., Moga, C.N. and Fluharty, S.J., Intracerebroventricular administration of AT1 receptor antisense oligonucleotide inhibits the behavioral actions of angiotensin II, J. Neurochem. 62 (1994) 2053–2056.PubMedCrossRefGoogle Scholar
  32. 32.
    Meng, H-B., Wielbo, D., Gyurko, R. and Phillips, Mi.I. AT1 receptor mRNA antisense oligonucleotide inhibits central angiotensin induced thirst and Vasopressin, Regul. Pept. 54 (1994) 543–551.PubMedCrossRefGoogle Scholar
  33. 33.
    Ambühl, P., Gyurko, R. and Phillips, M.I., A decrease in angiotensin receptor binding in rat brain nuclei by antisense oligonucleotides to the angiotensin AT1 receptor (in press) (1995).Google Scholar
  34. 34.
    Kakar, S.S., Riel, K.K. and Neill, J.D., Differential expression of angiotensin II receptor subtype mRNAs (AT-1A and AT-1B) in the brain, Biochem. Biophys. Res. Commun., 185 (1992) 688–692.PubMedCrossRefGoogle Scholar
  35. 35.
    Lenkei, Z., Corvol, P. and Llorens-Cortes, C., The angiotensin receptor subtype AT1A predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control, Mol. Brain Res., 30 (1995) 53–60.PubMedCrossRefGoogle Scholar
  36. 36.
    Morishita, R., Gibbons, G.H., Kaneda, Y., Ogihara, T. and Dzau, V.J., Pharmacokinetics of antisense oligodeoxyribonucleotides (cyclin B1 and CDC 2 kinase) in the vessel wall in vivo: enhanced therapeutic utility for restenosis by HVJ-liposome delivery, Gene 149(1) (1994) 13–19.PubMedCrossRefGoogle Scholar
  37. 37.
    Kaneda, Y., Iwai, K. and Uchida, T., Increased expression of DNA cointroduced with nuclear protein in adult rat liver, Science 243 (1989) 375–378.PubMedCrossRefGoogle Scholar
  38. 38.
    Stein, C.A. and Krieg, A.M., Problems in interpretation of data derived from in vitro and in vivo use of antisense oligodeoxynucleotides (Editorial), Antisense Res. Dev. 4 (1994) 67–69.PubMedGoogle Scholar
  39. 39.
    Muzyczka, N., Use of adeno-associated virus as a general transduction vector for mammalian cells, Curr. Top Microbiol. Immunol. 158 (1992) 97–129.PubMedCrossRefGoogle Scholar
  40. 40.
    Ponnazhagan, S., Nallari, M.L. Srivastava, A., Suppression of human alpha-globin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors, J. Exp. Med. 179(2) (1994) 733–738.PubMedCrossRefGoogle Scholar
  41. 41.
    Chatterjee, S., Johnson, P.R., Wong, K.K., Jr., Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector, Science 258 (1992) 1485–1488.PubMedCrossRefGoogle Scholar
  42. 42.
    Caulfield, M., Lavender, P., Farrell, M., Munroe, P., Lawson, M., Turner, P., Clark, A., Linkage of angiotensinogen gene to essential hypertension, New Eng. J. Med. 330 (1994) 1629–1633.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • M. Ian Phillips
    • 1
  • Philipp Ambühl
    • 1
  • Robert Gyurko
    • 1
  1. 1.Department of Physiology, College of MedicineUniversity of FloridaGainesvilleUSA

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