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Cellular penetration and antisense activity by a phenoxazine-substituted heptanucleotide

Nature Biotechnology volume 17, pages 48–52 (1999)Cite this article

Abstract

One of the major barriers to the development of antisense therapeutics has been their poor bioavailability. Numerous oligonucleotide modifications have been synthesized and evaluated for enhanced cellular permeation with limited success. Phenoxazine, a tricyclic 2´ deoxycytidine analog, was designed to improve stacking interactions between heterocycles of oligonucleotide/RNA hybrids and to enhance cellular uptake. However, the bioactivity and cellular permeation properties of phenoxazine-modified oligonucleotides were unknown. Incorporation of four phenoxazine bases into a previously optimized C-5 propyne pyrimidine modified 7-mer phosphorothioate oligonucleotide targeting SV40 large T antigen enhanced in vitro binding affinity for its RNA target and redirected RNAse H-mediated cleavage as compared with the 7-mer C-5 propynyl phosphorothioate oligonucleotide (S-ON). The phenoxazine/C-5 propynyl U 7-mer S-ON showed dose-dependent, sequence-specific, and target-selective antisense activity following microinjection into cells. Incubation of the phenoxazine/C-5 propynyl U S-ON with a variety of tissue culture cells, in the absence of any cationic lipid, revealed unaided cellular penetration, nuclear accumulation, and subsequent antisense activity. The unique permeation properties and gene-specific antisense activity of the 7-mer phenoxazine/C-5 propynyl U S-ON paves the way for developing potent, cost-effective, self-permeable antisense therapeutics.

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Figure 1: Structure and chemical modifications of oligonucleotides.
Figure 2: Phenoxazine-modified phosphorothioate oligonucleotides (S-ONs) are RNAse H competent and redirect cleavage.
Figure 3: The phenoxazine/C-5 propynyl U S-ON demonstrates sequence-specific antisense activity and unaided cellular permeation.

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References

  1. Wagner, R.W. 1995. The state of the art in antisense research. Nat. Med. 1: 1116–1118.

    Article  Google Scholar 

  2. Stein, C.A. 1997. Controversies in the cellular pharmacology of oligodeoxynucleotides. Antisense Nucleic Acid Drug Dev. 7: 207–209.

    Article  Google Scholar 

  3. Stein, C.A. and Krieg, A.M. 1994. Problems in interpretation of data derived from in vitro and in vivo use of the antisense oligonucleotides. Antisense Res. and Dev. 4: 67–69.

    Article  Google Scholar 

  4. Nestle, F.O., Mitra, R.S., Bennett, C.F., Chan, H., and Nickoloff, B.J. 1994. Cationic lipid is not required for uptake and selective inhibitory activity of ICAM–1 phosphorothioate antisense oligonucleotides in keratinocytes. J. Invest. Dermatol. 103: 569– 575.

    Article  Google Scholar 

  5. Giachetti, C. and Chin, D.J. 1996. Increased oligonucleotide permeability in keratinocytes of artificial skin correlates with differentiation and altered membrane function. J. Invest. Dermatol. 107: 256–262.

    Article  Google Scholar 

  6. Wagner, R.W., Matteucci, M.D., Lewis, J.G., Gutierrez, A.J., Moulds, C., and Froehler, B.C. 1993. Antisense gene inhibition by oligonucleotide containing propyne pyrimidines. Science 260: 1510–1513.

    Article  Google Scholar 

  7. Bergan, R., Connell, Y., Fahmy, B., and Neckers, L. 1993 . Electroporation enhances c–myc antisense oligodeoxynucleotide efficacy. Nucleic Acids Res. 21: 3567– 3573.

    Article  Google Scholar 

  8. Spiller, D.G. and Tidd, D.M. 1995. Nuclear delivery of oligodeoxynucleotide through reversible permeabilization of human leukemia cells with streptolysin O. Antisense Res. Dev. 5: 13–21.

    Article  Google Scholar 

  9. Partridge, M., Vincent, A., Matthews, P., Puma, J., Stein, D., and Summerton, J. 1996. A simple method for delivering morpholino antisense oligonucleotides into the cytoplasm of cells. Antisense Nucleic Acid Drug Dev. 6: 169– 175.

    Article  Google Scholar 

  10. Pichon, C., Freulon, I., Midoux, P., Mayer, R., Monsigny, M., and Roche, A.–C. 1997. Cytosolic and nuclear delivery of oligonucleotides mediated by an amphiphilic anionic peptide. Antisense Nucleic Acid Drug Dev. 7: 335–343.

    Article  Google Scholar 

  11. Lewis, J.G., Lin, K.Y., Kothavale, A., Flanagan, W.M., Matteucci, M.D., DePrince, R.B. et al. 1996. A serum–resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. Proc. Natl. Acad. Sci. USA 93: 3176– 3181.

    Article  Google Scholar 

  12. Zelphati, O. and Francis C. Szoka, J. 1996. Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharm. Res. 13: 1367–1372.

    Article  Google Scholar 

  13. Marcusson, E.G., Bhat, B., Manoharan, M., Bennett, C.F., and Dean, N.M. 1998. Phosphorothioate oligodeoxyribonucleotides disassociate from cationic lipids before entering the nucleus. Nucleic Acids Res. 26: 2016–2023.

    Article  Google Scholar 

  14. Matteucci, M. 1997. Oligonucleotide analogues: an overview. Ciba Found. Symp. 209: 5–14.

    Google Scholar 

  15. Krieg, A.M., Tonkinson, J., Matson, S., Zhao, Q., Saxon, M., Zhang, L.–M. et al. 1993. Modification of antisense phosphodiester oligodeoxynucleotides by a 5´ cholesteryl moiety increases cellular association and improves efficacy. Proc. Natl. Acad. Sci. USA 90: 1048–1052.

    Article  Google Scholar 

  16. Lu, X.–M., Fischman, A.J., Jyawook, S.L., Hendricks, K., Tompkins, R.G., and Yarmush, M.L. 1994. Antisense DNA delivery in vivo: liver targeting by receptor–mediated uptake. J. Nucl. Med. 35: 269–275.

    Google Scholar 

  17. Kang, Y.–S., Boado, R.J., and Pardridge, W.M. 1995. Pharmacokinetics and organ clearance of a 3´–biotinylated, internally [32P]–labeled phosphodoester oligodeoxynucleotide coupled to a neutral avidin/monoclonal antibody conjugate. Drug Metab. Disp. 23: 55–59.

    Google Scholar 

  18. Hughes, J.A., Bennett, C.F., Cook, P.D., Guinosso, C.J., Mirabelli, C.K., and Juliano, R.L. 1994. Lipid membrane permeability of 2´–modified derivatives of phosphorothioate oligonucleotides. J. Pharm. Sci. 83: 597– 600.

    Article  Google Scholar 

  19. Wagner, R.W., Matteucci, M.D., Grant, D., Huang, T., and Froehler, B.C. 1996. Potent and selective inhibition of gene expression by an antisense heptanucleotide. Nat. Biotechnol. 14: 840– 844.

    Article  Google Scholar 

  20. Wagner, R.W. and Flanagan, W.M. 1997. Antisense technology and prospects for therapy of infectious disease and cancer. Mol. Med. Today 3: 31–38.

    Article  Google Scholar 

  21. Loke, S.L., Stein, C.A., Zhang, X.H., Mori, K., Nakanishi, M., Subasinghe, C. et al. 1989. Characterization of oligonucleotide transport into living cells. Proc. Natl. Acad. Sci. USA 86: 3474– 3478.

    Article  Google Scholar 

  22. Miller, P.S., Yano, J., Yano, E., Carroll, C., Jayaraman, K., and Ts'o, P.O.P. 1979. Synthesis and characterization of dideoxyribonucleoside methyl phosphonates. Biochemistry 18: 5134 –5143.

    Article  Google Scholar 

  23. Jones, R.J., Lin, K.–Y., Milligan, J.F., Wadwani, S., and Matteucci, M.D. 1993. Synthesis and binding properties of pyrimidine oligodeoxynucleoside analogs containing neutral phosphodiester replacements: the formacetal and 3´–thioformacetal internucleoside linkages. J. Org. Chem. 58: 2983–2991.

    Article  Google Scholar 

  24. Vasseur, J.–J., Debart, F., Sanghvi, Y.S., and Cook, P.D. 1992. Oligonucleosides: Synthesis of a novel methylhydroxylamine–linked nucleoside dimer and its incorporation into antisense sequences. J. Am. Chem. Soc. 114: 4006–4007.

    Article  Google Scholar 

  25. Nielsen, P.E., Egholm, M., and Buchardt, O. 1994. A DNA mimic with a peptide backbone. Bioconjug. Chem. 5: 3– 7.

    Article  Google Scholar 

  26. Lin, K.–Y., Jones, R.J., and Matteucci, M. 1995. Synthesis and incorporation into oligodeoxynucleotides which have enhanced binding to complementary RNA. J. Am. Chem. Soc. 117: 3873– 3874.

    Article  Google Scholar 

  27. Lima, W.F. and Crooke, S.T. 1997. Binding affinity and specificity of Escherichia coli RNAse H1: Impact of the kinetics of catalysis of antisense oligonucleotide–RNA hybrids. Biochemistry 36: 390–398.

    Article  Google Scholar 

  28. Moulds, C., Lewis, J.G., Froehler, B.C., Grant, D., Huang, T., Milligan, J.F. et al. 1995. Site and mechanism of antisense inhibition by C–5 propyne oligonucleotides. Biochemistry 34: 5044–5053.

    Article  Google Scholar 

  29. Flores–Rozas, H. and Hurwitz, J. 1993 . Characterization of a new RNA helicase from nuclear extracts of HeLa cells which translocates in the 5´ to 3´ direction. J. Biol. Chem. 268: 21372–21383.

    Google Scholar 

  30. Monia, B.P., Johnston, J.F., Greiger, T., Muller, M., and Fabbro, D. 1996. Antitumor activity of a phosphorothioate antisense oligonucleotide targeted against c–raf kinase. Nat. Med. 2: 668– 675.

    Article  Google Scholar 

  31. McKay, R. and Dean, M.N. 1994. Inhibition of protein kinase C–alpha expression in mice after systematic administration of phosphorothioate. Proc. Natl. Acad. Sci. USA 91: 1762–1766.

    Google Scholar 

  32. Stepkowski, S.M., Tu, Y., Condon, T.P., and Bennett, C.F. 1994 . Blocking of heart allograft rejection by intercellular adhesion molecule–1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities. J. Immunol. 5336– 5346.

  33. Plenat, F. 1996. Animal models of antisense oligonucleotides: lessons for use in humans. Mol. Med. Today 2: 250 –257.

    Article  Google Scholar 

  34. Flanagan, W.M. 1998. Antisense comes of age. Cancer and Metastasis Reviews 17: 169–176.

    Article  Google Scholar 

  35. Srinivasan, S.K. and Iversen, P. 1995 . Review of in vivo pharmacokinetics and toxicology of phosphorothioate oligonucleotides. J. Clin. Lab. Anal. 9: 129–137.

    Article  Google Scholar 

  36. Bijsterbosch, M.K., Manoharan, M., Rump, E.T., Vrueh, R.L.A.D., Veghel, R.V., Tivel, K.L. et al. 1997. In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells. Nucleic Acids Res. 25: 3290–3296.

    Article  Google Scholar 

  37. Agrawal, S., Temsamani, J., Galbraith, W., and Tang, J. 1995. Pharmacokinetics of antisense oligonucleotides. Drug Disp. 28: 7–16.

    Google Scholar 

  38. Crooke, S.T., Graham, M.J., Zuckerman, J.E., Brooks, D., Conklin, B.S., Cummins, L.L. et al. 1996. Pharmacokinetic properties of several novel oligonucleotide analogs in mice. J. Pharmacol. Exp. Ther. 923–937.

  39. Geary, R.S., Leeds, J.M., Henry, S.P., Monteith, D.K., and Levin, A.A. 1997. Antisense oligonucleotide inhibitors for the treatment of cancer: pharmacokinetic properties of phosphorothioate oligodeoxynucleotides. Anti–cancer Drug Des. 12: 383–393.

    Google Scholar 

  40. Delong, R.K., Nolting, A., Fisher, M., Chen, Q., Wickstrom, E., Kligshteyn, M. et al. 1997. Comparative pharmacokinetics, tissue distribution, and tumor accumulation of phosphorothioate, phosphorodithioate, and methylphosphonate oligonucleotides in nude mice. Antisense Nucleic Acid Drug Dev. 7: 71–77.

    Article  Google Scholar 

  41. Fisher, T.L., Terhorst, T., Cao, X., and Wagner, R.W. 1993 . Intracellular disposition and metabolism of fluorescently labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res. 21: 3857– 3865.

    Article  Google Scholar 

  42. Froehler, B.C., Wadwani, S., Terhorst, T.J., and Gerrard, S.R. 1992. Oligodeoxynucleotides containing C–5 propyne analogs of 2´–deoxyuridine and 2´–deoxycytidine. Tetrahedron Lett. 33: 5307–5310.

    Article  Google Scholar 

  43. Froehler, B.C., Jones, R.J., Cao, X., and Terhorst, T.J. 1993 . Oligonucleotides derived from 5–(1–propynyl)–2´–O–allyl–uridine and 5–(1–propynyl)–2´–allyl–cytidine: Synthesis and RNA duplex formation. Tetrahedron Lett. 34: 1003–1006.

    Article  Google Scholar 

  44. Flanagan, W.M., Su, L.L., and Wagner, R.W. 1996. Elucidation of gene function using C–5 propyne antisense oligonucleotides. Nat. Biotechnol. 14: 1139–1145.

    Article  Google Scholar 

  45. Gutierrez, A.J., Matteucci, M.D., Grant, D., Matsumura, S., Wagner, R.W., and Froehler, B.C. 1997. Antisense gene inhibition by C–5–substituted deoxyuridine–containing oligodeoxynucleotides. Biochemistry 36: 743–748 .

    Article  Google Scholar 

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Author notes

  1. Richard W. Wagner

    Present address: Phylos, Inc., Lexington, MA, 02421

  2. W. Michael Flanagan and Richard W. Wagner: Both authors contributed equally to this work.

Authors and Affiliations

  1. Gilead Sciences, Inc., Foster City, 94404, CA

    W. Michael Flanagan, Richard W. Wagner, Deborah Grant, Kuei-Ying Lin & Mark D. Matteucci

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  1. W. Michael Flanagan
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  2. Richard W. Wagner
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Correspondence to W. Michael Flanagan or Mark D. Matteucci.

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Flanagan, W., Wagner, R., Grant, D. et al. Cellular penetration and antisense activity by a phenoxazine-substituted heptanucleotide. Nat Biotechnol 17, 48–52 (1999). https://doi.org/10.1038/5220

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