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. 1999 Jun 15;340(Pt 3):783–792.

Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo.

E A Biessen 1, H Vietsch 1, E T Rump 1, K Fluiter 1, J Kuiper 1, M K Bijsterbosch 1, T J van Berkel 1
PMCID: PMC1220312  PMID: 10359665

Abstract

Anti-sense oligodeoxynucleotides (ODNs) hold great promise for correcting the biosynthesis of clinically relevant proteins. The potential of ODNs for modulating liver-specific genes might be increased by preventing untimely elimination and by improving the local bioavailability of ODNs in the target tissue. In the present study we have assessed whether the local ODN concentration can be enhanced by the targeted delivery of ODNs through conjugation to a ligand for the parenchymal liver cell-specific asialoglycoprotein receptor. A capped ODN (miscellaneous 20-mer sequence) was derivatized with a ligand with high affinity for this receptor, N2-[N2-(N2,N6-bis¿N-[p-(beta-d-galactopyranosyloxy) anilino] thiocarbamyl¿-L-lysyl)-N6-(N-¿p-[beta-D -galactopyranosyloxy] anilino¿ thiocarbamyl)-L-lysyl]-N6-[N- (p-¿beta-D-galactopyranosyloxy¿anilino)thiocarbamyl]-L-lysine (L3G4) (Kd 6.5+/-0.2 nM, mean+/-S.D.). Both the uptake studies in vitro and the confocal laser scan microscopy studies demonstrated that L3G4-ODN was far more efficiently bound to and taken up by parenchymal liver cells than underivatized ODN. Studies in vivo in rats showed that hepatic uptake could be greatly enhanced from 19+/-1% to 77+/-6% of the injected dose after glycoconjugation. Importantly, specific ODN accumulation of ODN into parenchymal liver cells was improved almost 60-fold after derivatization with L3G4, and could be attributed to the asialoglycoprotein receptor. In conclusion, the scavenger receptor-mediated elimination pathway for miscellaneous ODN sequences can be circumvented by direct conjugation to a synthetic tag for the asialoglycoprotein receptor. In this manner a crucial requisite is met towards the application of ODNs in vivo to modulate the biosynthesis of parenchymal liver cell-specific genes such as those for apolipoprotein (a), cholesterol ester transfer protein and viral proteins.

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Selected References

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  1. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  2. Bennett C. F., Chiang M. Y., Chan H., Shoemaker J. E., Mirabelli C. K. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol Pharmacol. 1992 Jun;41(6):1023–1033. [PubMed] [Google Scholar]
  3. Biessen E. A., Beuting D. M., Vietsch H., Bijsterbosch M. K., Van Berkel T. J. Specific targeting of the antiviral drug 5-iodo 2'-deoxyuridine to the parenchymal liver cell using lactosylated poly-L-lysine. J Hepatol. 1994 Nov;21(5):806–815. doi: 10.1016/s0168-8278(94)80243-2. [DOI] [PubMed] [Google Scholar]
  4. Biessen E. A., Vietsch H., Kuiper J., Bijsterbosch M. K., Berkel T. J. Liver uptake of phosphodiester oligodeoxynucleotides is mediated by scavenger receptors. Mol Pharmacol. 1998 Feb;53(2):262–269. doi: 10.1124/mol.53.2.262. [DOI] [PubMed] [Google Scholar]
  5. Biessen E. A., van Teijlingen M., Vietsch H., Barrett-Bergshoeff M. M., Bijsterbosch M. K., Rijken D. C., van Berkel T. J., Kuiper J. Antagonists of the mannose receptor and the LDL receptor-related protein dramatically delay the clearance of tissue plasminogen activator. Circulation. 1997 Jan 7;95(1):46–52. doi: 10.1161/01.cir.95.1.46. [DOI] [PubMed] [Google Scholar]
  6. Bijsterbosch M. K., Manoharan M., Rump E. T., De Vrueh R. L., van Veghel R., Tivel K. L., Biessen E. A., Bennett C. F., Cook P. D., van Berkel T. J. In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells. Nucleic Acids Res. 1997 Aug 15;25(16):3290–3296. doi: 10.1093/nar/25.16.3290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bonfils E., Depierreux C., Midoux P., Thuong N. T., Monsigny M., Roche A. C. Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates. Nucleic Acids Res. 1992 Sep 11;20(17):4621–4629. doi: 10.1093/nar/20.17.4621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bunnell B. A., Askari F. K., Wilson J. M. Targeted delivery of antisense oligonucleotides by molecular conjugates. Somat Cell Mol Genet. 1992 Nov;18(6):559–569. doi: 10.1007/BF01232652. [DOI] [PubMed] [Google Scholar]
  9. Chiu M. H., Tamura T., Wadhwa M. S., Rice K. G. In vivo targeting function of N-linked oligosaccharides with terminating galactose and N-acetylgalactosamine residues. J Biol Chem. 1994 Jun 10;269(23):16195–16202. [PubMed] [Google Scholar]
  10. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haensler J., Szoka F. C., Jr Synthesis and characterization of a trigalactosylated bisacridine compound to target DNA to hepatocytes. Bioconjug Chem. 1993 Jan-Feb;4(1):85–93. doi: 10.1021/bc00019a012. [DOI] [PubMed] [Google Scholar]
  12. Hamilton H. W., Steinbaugh B. A., Stewart B. H., Chan O. H., Schmid H. L., Schroeder R., Ryan M. J., Keiser J., Taylor M. D., Blankley C. J. Evaluation of physicochemical parameters important to the oral bioavailability of peptide-like compounds: implications for the synthesis of renin inhibitors. J Med Chem. 1995 Apr 28;38(9):1446–1455. doi: 10.1021/jm00009a005. [DOI] [PubMed] [Google Scholar]
  13. Hangeland J. J., Flesher J. E., Deamond S. F., Lee Y. C., Ts'O P. O., Frost J. J. Tissue distribution and metabolism of the [32P]-labeled oligodeoxynucleoside methylphosphonate-neoglycopeptide conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, in the mouse. Antisense Nucleic Acid Drug Dev. 1997 Jun;7(3):141–149. doi: 10.1089/oli.1.1997.7.141. [DOI] [PubMed] [Google Scholar]
  14. Hangeland J. J., Levis J. T., Lee Y. C., Ts'o P. O. Cell-type specific and ligand specific enhancement of cellular uptake of oligodeoxynucleoside methylphosphonates covalently linked with a neoglycopeptide, YEE(ah-GalNAc)3. Bioconjug Chem. 1995 Nov-Dec;6(6):695–701. doi: 10.1021/bc00036a006. [DOI] [PubMed] [Google Scholar]
  15. Huisman W., Bouma J. M., Gruber M. Involvement of thiol enzymes in the lysosomal breakdown of native and denatured proteins. Biochim Biophys Acta. 1973 Jan 24;297(1):98–109. doi: 10.1016/0304-4165(73)90053-6. [DOI] [PubMed] [Google Scholar]
  16. Inagaki M., Togawa K., Carr B. I., Ghosh K., Cohen J. S. Antisense oligonucleotides: inhibition of liver cell proliferation and in vivo disposition. Transplant Proc. 1992 Dec;24(6):2971–2972. [PubMed] [Google Scholar]
  17. Leventis R., Silvius J. R. Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles. Biochim Biophys Acta. 1990 Mar 30;1023(1):124–132. doi: 10.1016/0005-2736(90)90017-i. [DOI] [PubMed] [Google Scholar]
  18. Lewis J. G., Lin K. Y., Kothavale A., Flanagan W. M., Matteucci M. D., DePrince R. B., Mook R. A., Jr, Hendren R. W., Wagner R. W. A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3176–3181. doi: 10.1073/pnas.93.8.3176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lu X. M., Fischman A. J., Jyawook S. L., Hendricks K., Tompkins R. G., Yarmush M. L. Antisense DNA delivery in vivo: liver targeting by receptor-mediated uptake. J Nucl Med. 1994 Feb;35(2):269–275. [PubMed] [Google Scholar]
  20. McLean J. W., Tomlinson J. E., Kuang W. J., Eaton D. L., Chen E. Y., Fless G. M., Scanu A. M., Lawn R. M. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature. 1987 Nov 12;330(6144):132–137. doi: 10.1038/330132a0. [DOI] [PubMed] [Google Scholar]
  21. Merwin J. R., Noell G. S., Thomas W. L., Chiou H. C., DeRome M. E., McKee T. D., Spitalny G. L., Findeis M. A. Targeted delivery of DNA using YEE(GalNAcAH)3, a synthetic glycopeptide ligand for the asialoglycoprotein receptor. Bioconjug Chem. 1994 Nov-Dec;5(6):612–620. doi: 10.1021/bc00030a017. [DOI] [PubMed] [Google Scholar]
  22. Milligan J. F., Matteucci M. D., Martin J. C. Current concepts in antisense drug design. J Med Chem. 1993 Jul 9;36(14):1923–1937. doi: 10.1021/jm00066a001. [DOI] [PubMed] [Google Scholar]
  23. Mizutani T., Kato N., Hirota M., Sugiyama K., Murakami A., Shimotohno K. Inhibition of hepatitis C virus replication by antisense oligonucleotide in culture cells. Biochem Biophys Res Commun. 1995 Jul 26;212(3):906–911. doi: 10.1006/bbrc.1995.2055. [DOI] [PubMed] [Google Scholar]
  24. Morishita R., Gibbons G. H., Kaneda Y., Ogihara T., 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. 1994 Nov 4;149(1):13–19. doi: 10.1016/0378-1119(94)90406-5. [DOI] [PubMed] [Google Scholar]
  25. Nakazono K., Ito Y., Wu C. H., Wu G. Y. Inhibition of hepatitis B virus replication by targeted pretreatment of complexed antisense DNA in vitro. Hepatology. 1996 Jun;23(6):1297–1303. doi: 10.1002/hep.510230601. [DOI] [PubMed] [Google Scholar]
  26. Oberhauser B., Wagner E. Effective incorporation of 2'-O-methyl-oligoribonucleotides into liposomes and enhanced cell association through modification with thiocholesterol. Nucleic Acids Res. 1992 Feb 11;20(3):533–538. doi: 10.1093/nar/20.3.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pearson A. M., Rich A., Krieger M. Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices. J Biol Chem. 1993 Feb 15;268(5):3546–3554. [PubMed] [Google Scholar]
  28. Plank C., Zatloukal K., Cotten M., Mechtler K., Wagner E. Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. Bioconjug Chem. 1992 Nov-Dec;3(6):533–539. doi: 10.1021/bc00018a012. [DOI] [PubMed] [Google Scholar]
  29. Reinis M., Damková M., Korec E. Receptor-mediated transport of oligodeoxynucleotides into hepatic cells. J Virol Methods. 1993 Apr;42(1):99–105. doi: 10.1016/0166-0934(93)90181-p. [DOI] [PubMed] [Google Scholar]
  30. Rifai A., Brysch W., Fadden K., Clark J., Schlingensiepen K. H. Clearance kinetics, biodistribution, and organ saturability of phosphorothioate oligodeoxynucleotides in mice. Am J Pathol. 1996 Aug;149(2):717–725. [PMC free article] [PubMed] [Google Scholar]
  31. Sands H., Gorey-Feret L. J., Cocuzza A. J., Hobbs F. W., Chidester D., Trainor G. L. Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate. Mol Pharmacol. 1994 May;45(5):932–943. [PubMed] [Google Scholar]
  32. Sands H., Gorey-Feret L. J., Ho S. P., Bao Y., Cocuzza A. J., Chidester D., Hobbs F. W. Biodistribution and metabolism of internally 3H-labeled oligonucleotides. II. 3',5'-blocked oligonucleotides. Mol Pharmacol. 1995 Mar;47(3):636–646. [PubMed] [Google Scholar]
  33. Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
  34. Sugano M., Makino N. Changes in plasma lipoprotein cholesterol levels by antisense oligodeoxynucleotides against cholesteryl ester transfer protein in cholesterol-fed rabbits. J Biol Chem. 1996 Aug 9;271(32):19080–19083. doi: 10.1074/jbc.271.32.19080. [DOI] [PubMed] [Google Scholar]
  35. Sugano M., Makino N., Sawada S., Otsuka S., Watanabe M., Okamoto H., Kamada M., Mizushima A. Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits. J Biol Chem. 1998 Feb 27;273(9):5033–5036. doi: 10.1074/jbc.273.9.5033. [DOI] [PubMed] [Google Scholar]
  36. Tari A. M., Tucker S. D., Deisseroth A., Lopez-Berestein G. Liposomal delivery of methylphosphonate antisense oligodeoxynucleotides in chronic myelogenous leukemia. Blood. 1994 Jul 15;84(2):601–607. [PubMed] [Google Scholar]
  37. Van Berkel T. J., De Rijke Y. B., Kruijt J. K. Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells. J Biol Chem. 1991 Feb 5;266(4):2282–2289. [PubMed] [Google Scholar]
  38. Wagner R. W., Flanagan W. M. Antisense technology and prospects for therapy of viral infections and cancer. Mol Med Today. 1997 Jan;3(1):31–38. doi: 10.1016/S1357-4310(96)10053-8. [DOI] [PubMed] [Google Scholar]
  39. Wagner R. W. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994 Nov 24;372(6504):333–335. doi: 10.1038/372333a0. [DOI] [PubMed] [Google Scholar]
  40. Wakita T., Wands J. R. Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides. In vitro model for selection of target sequence. J Biol Chem. 1994 May 13;269(19):14205–14210. [PubMed] [Google Scholar]
  41. Wu G. Y., Wu C. H. Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides. J Biol Chem. 1992 Jun 25;267(18):12436–12439. [PubMed] [Google Scholar]
  42. Zelphati O., Wagner E., Leserman L. Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes. Antiviral Res. 1994 Sep;25(1):13–25. doi: 10.1016/0166-3542(94)90090-6. [DOI] [PubMed] [Google Scholar]

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