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Delivery of Phosphorodiamidate Morpholino Antisense Oligomers in Cancer Cells

  • Gayathri R. DeviEmail author
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 542)

Summary

Phosphorodiamidate morpholino oligomers (PMO), which have a neutral chemistry, are extensively being used as tools for selective inhibition of gene expression in cell culture models and are currently in human clinical trials. PMO oligomers possess a unique structure, in which the deoxyribose moiety of DNA is replaced with a six-membered morpholine ring and the charged phosphodiester internucleoside linkages are replaced with neutral phosphorodiamidate linkages. PMO internalization in uptake-permissive cells has been observed to be specific, saturable, and energy-dependent, suggesting a receptor-mediated uptake mechanism. Understanding PMO transport should facilitate the design of more effective synthetic antisense oligomers as therapeutic agents.

Keywords

Apoptosis delivery fluorescence immunoblots morpholino oligonucleotides phosphorodiamidate PMO 

Notes

Acknowledgments

I thank Katherine Aird and Rami Ghanayem for editorial and technical assistance and Dr. Patrick Iversen (Avi BioPharma Inc.). Some of the work presented here was supported by Department of Defense (DOD) grants DAMD17-01-1-0017 (GRD) and W81XWH-07-1-0392 (GRD).

References

  1. 1.
    Nasevicius A, Ekker SC. (2000) Effective targeted gene “knockdown” in zebrafish. Nat Genet 26:216–220.PubMedCrossRefGoogle Scholar
  2. 2.
    Sahu NK, Shilakari G, Nayak A, Kohli DV. (2007) Antisense technology: a selective tool for gene expression regulation and gene targeting. Curr Pharm Biotechnol 8:291–304.PubMedCrossRefGoogle Scholar
  3. 3.
    Tillman LG, Geary RS, Hardee GE. (2008) Oral delivery of antisense oligonucleotides in man. J Pharm Sci 97:225–236.PubMedCrossRefGoogle Scholar
  4. 4.
    Stein CA, Benimetskaya L, Mani S. (2005) Antisense strategies for oncogene inactivation. Semin Oncol 32:563–572.PubMedCrossRefGoogle Scholar
  5. 5.
    Ghosh C, Stein D, Weller D, Iversen P. (2000) Evaluation of antisense mechanisms of action. Methods Enzymol 313:135–143.PubMedCrossRefGoogle Scholar
  6. 6.
    Arora V, Devi GR, Iversen PL. (2004) Neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics. Curr Pharm Biotechnol 5:431–439.PubMedCrossRefGoogle Scholar
  7. 7.
    Summerton J. (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1489:141–158.PubMedGoogle Scholar
  8. 8.
    Summerton J, Weller D. (1997) Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 7:187–195.PubMedCrossRefGoogle Scholar
  9. 9.
    Giles RV, Spiller DG, Clark RE, Tidd DM. (1999) Antisense morpholino oligonucleotide analog induces missplicing of C-myc mRNA. Antisense Nucleic Acid Drug Dev 9:213–220.PubMedCrossRefGoogle Scholar
  10. 10.
    Aartsma-Rus A, van Ommen GJ. (2007) Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications. RNA 13:1609–1624.PubMedCrossRefGoogle Scholar
  11. 11.
    Stein CA. (1997) Controversies in the cellular pharmacology of oligodeoxynucleotides. Antisense Nucleic Acid Drug Dev 7:207–209.PubMedCrossRefGoogle Scholar
  12. 12.
    Hans SS, Hans BA, Dhillon R, Dmuchowski C, Glover J. (1998) Effect of dopamine on renal function after arteriography in patients with pre-existing renal insufficiency. Am Surg 64:432–436.PubMedGoogle Scholar
  13. 13.
    Biessen EA, Vietsch H, Kuiper J, Bijsterbosch MK, Berkel TJ. (1998) Liver uptake of phosphodiester oligodeoxynucleotides is mediated by scavenger receptors. Mol Pharmacol 53:262–269.PubMedGoogle Scholar
  14. 14.
    Yakubov LA, Deeva EA, Zarytova VF, Ivanova EM, Ryte AS, Yurchenko LV,. (1989) Mechanism of oligonucleotide uptake by cells: involvement of specific receptors? Proc Natl Acad Sci USA 86:6454–6458.PubMedCrossRefGoogle Scholar
  15. 15.
    Loke SL, Stein CA, Zhang XH, Mori K, Nakanishi M, Subasinghe C,. (1989) Characterization of oligonucleotide transport into living cells. Proc Natl Acad Sci USA 86:3474–3478.PubMedCrossRefGoogle Scholar
  16. 16.
    Benimetskaya L, Loike JD, Khaled Z, Loike G, Silverstein SC, Cao L,. (1997) Mac-1 (CD11b/CD18) is an oligodeoxynucleotide-binding protein. Nat Med 4:414–420.CrossRefGoogle Scholar
  17. 17.
    Laktionov PP, Dazard JE, Vives E, Rykova EY, Piette J, Vlassov VV,. (1999) Characterization of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes. Nucleic Acids Res 27:2315–2324.PubMedCrossRefGoogle Scholar
  18. 18.
    de Diesbach P, N'Kuli F, Delmee M, Courtoy PJ. (2003) Infection by Mycoplasma hyorhinis strongly enhances uptake of antisense oligonucleotides: a reassessment of receptor-mediated endocytosis in the HepG2 cell line. Nucleic Acids Res 31:886–892.PubMedCrossRefGoogle Scholar
  19. 19.
    Gray GD, Basu S, Wickstrom E. (1997) Transformed and immortalized cellular uptake of oligodeoxynucleoside phosphorothioates, 3′-alkylamino oligodeoxynucleotides, 2′-O-methyl oligoribonucleotides, oligodeoxynucleoside methylphosphonates, and peptide nucleic acids. Biochem Pharmacol 53:1465–1476.PubMedCrossRefGoogle Scholar
  20. 20.
    Levis JT, Butler WO, Tseng BY, Ts'o PO. (1995) Cellular uptake of oligodeoxyribonucleoside methylphosphonates. Antisense Res Dev 5:251–259.PubMedGoogle Scholar
  21. 21.
    Delong RK, Miller PS. (1996) Inhibition of human collagenase activity by antisense oligonucleoside methylphosphonates. Antisense Nucleic Acid Drug Dev 6:273–280.PubMedCrossRefGoogle Scholar
  22. 22.
    Partridge M, Vincent A, Matthews P, Puma J, Stein D, Summerton J. (1996) A simple method for delivering morpholino antisense oligos into the cytoplasm of cells. Antisense Nucleic Acid Drug Dev 6:169–175.PubMedCrossRefGoogle Scholar
  23. 23.
    Ghosh C, Iversen PL. (2000) Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers. Antisense Nucleic Acid Drug Dev 10:263–274.PubMedCrossRefGoogle Scholar
  24. 24.
    Morcos PA. (2001) Achieving efficient delivery of morpholino oligos in cultured cells. Genesis 30:94–102.PubMedCrossRefGoogle Scholar
  25. 25.
    Hudziak RM, Summerton J, Weller DD, Iversen PL. (2000) Antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc. Antisense Nucleic Acid Drug Dev 10:163–176.PubMedCrossRefGoogle Scholar
  26. 26.
    Arora V, Iversen PL. (2000) Antisense oligonucleotides targeted to the p53 gene modulate liver regeneration in vivo. Drug Metab Dispos 28:131–138.PubMedGoogle Scholar
  27. 27.
    Arora V, Iversen PL. (2001) Redirection of drug metabolism using antisense technology. Curr Opin Mol Ther 3:249–257.PubMedGoogle Scholar
  28. 28.
    Arora V, Knapp DC, Reddy MT, Weller DD, Iversen PL. (2002) Bioavailability and efficacy of antisense morpholino oligomers targeted to c-myc and cytochrome P-450 3A2 following oral administration in rats. J Pharm Sci 91:1009–1018.PubMedCrossRefGoogle Scholar
  29. 29.
    Arora V, Cate ML, Ghosh C, Iversen PL. (2002) Phosphorodiamidate morpholino antisense oligomers inhibit expression of human cytochrome P450 3A4 and alter selected drug metabolism. Drug Metab Dispos 7:757–762.CrossRefGoogle Scholar
  30. 30.
    Arora V, Hannah TL, Iversen PL, Brand RM. (2002) Transdermal use of phosphorodiamidate morpholino oligomer AVI-4472 inhibits cytochrome P450 3A2 activity in male rats. Pharm Res 10:465–1470.Google Scholar
  31. 31.
    Devi GR, Oldenkamp JR, London CA, Iversen PL. (2002) Inhibition of human chorionic gonadotropin beta-subunit modulates the mitogenic effect of c-myc in human prostate cancer cells. Prostate 53:200–210.PubMedCrossRefGoogle Scholar
  32. 32.
    London CA, Sekhon HS, Arora V, Stein DA, Iversen PL, Devi GR. (2003) A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell invasion and tumorigenicity. Cancer Gene Ther 10:823–832.PubMedCrossRefGoogle Scholar
  33. 33.
    Iversen PL, Arora V, Acker AJ, Mason DH, Devi GR. (2003) Efficacy of antisense morpholino oligomer targeted to c-myc in prostate cancer xenograft murine model and a Phase I safety study in humans. Clin Cancer Res 9:2510–2519.PubMedGoogle Scholar
  34. 34.
    Amantana A, London CA, Iversen PL, Devi GR. (2004) X-linked inhibitor of apoptosis protein inhibition induces apoptosis and enhances chemotherapy sensitivity in human prostate cancer cells. Mol Cancer Ther 3:699–707.PubMedGoogle Scholar
  35. 35.
    Sekhon HS, London CA, Sekhon M, Iversen PL, Devi GR. (2007) c-MYC antisense phosphosphorodiamidate morpholino oligomer inhibits lung metastasis in a murine tumor model. Lung Cancer Dec 18 (Epub ahead of publication).Google Scholar
  36. 36.
    Suwanmanee T, Sierakowska H, Lacerra G, Svasti S, Kirby S, Walsh CE,. (2002) Restoration of human beta-globin gene expression in murine and human IVS2–654 thalassemic erythroid cells by free uptake of antisense oligonucleotides. Mol Pharmacol 62:545–553.PubMedCrossRefGoogle Scholar
  37. 37.
    Suwanmanee T, Sierakowska H, Fucharoen S, Kole R. (2002) Repair of a splicing defect in erythroid cells from patients with beta-thalassemia/HbE disorder. Mol Ther 6:718–726.PubMedCrossRefGoogle Scholar
  38. 38.
    Kang SH, Cho MJ, Kole R. (1998) Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development.Biochemistry 37:6235–6239.PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Surgery and Duke Comprehensive Cancer Ctr, Box 2606Duke University Medical CenterDurhamUSA

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