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Use of a hammerhead ribozyme with cationic liposomes to reduce leukocyte type 12-lipoxygenase expression in vascular smooth muscle

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Abstract

Chemically synthesized hammerhead-type ribozymes targeted against the porcine leukocyte-type 12-lipoxygenase (LO) have been developed and studied. One chimeric ribozyme consists of DNA in the non-enzymatic portions, and RNA in the enzymatic core as well as two phosphorothioate internucleotide linkages at 3′ terminus. The second ribozyme consists of ribonucleotide sequences generated by in vitro transcription. In this chapter we describe methodologies to first analyze the ribozyme catalytic activity in vitro by studying cleavage of target RNA in vitro. The subsequent sections will describe how to target the catalytic ribozyme and deliver it to porcine vascular smooth muscle cells (PVSMC) by a liposome-mediated method. Finally ways to evaluate its activity to inhibit expression of the 12-LO mRNA will be presented. These results demonstrate the feasibility of using ribozymes as novel candidates for therapeutic agents to block specific gene expression in vascular cells.

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References

  1. Natarajan R, Gonzales N, Lanting L, Nadler JL: Role of the lipoxygenase pathway in angiotensin II-induced vascular smooth muscle cell hypertrophy. Hypertension 23 (suppl. I): I142–I147, 1994

    Google Scholar 

  2. Stern N, Golub M, Nozawa K, Berger M, Knoll E, Yanagawa N, Natarajan R, Nadler JL, Tuck M: Selective inhibition of angiotensin II-mediated vasoconstriction by lipoxygenase blockade. Am J Physiol 257: H434–H443, 1989

    Google Scholar 

  3. Hamberg M, Samueisson B: Prostagladin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl Acad Sci USA 71: 3400–3404, 1974

    Google Scholar 

  4. Nugteren DH. Arachidonate lipoxygenase in blood platelets. Biochem Biophys Acta 380: 299–307, 1975

    Google Scholar 

  5. Izumi T, Hoshiko S, Radmark O, Samuelsson B: Cloning of the cDNA for human 12-lipoxygenase. Proc Natl Acad Sci USA 87: 7477–7481, 1990

    Google Scholar 

  6. Funk CD, Furci L, FitzGerald GA: Molecular cloning primary structure, and expression of the human platelet/erytholeukemia cell 12-lipoxygenase. Proc Natl Acad Sci USA 87: 5638–5642, 1990

    Google Scholar 

  7. Yoshimoto T, Yamamoto Y, Arakawa T, Suzuki H, Yamanoto S, Yokoyama C, Tanabe T, Toh H: Molecular cloning and expression of human arachidonate 12-lipoxygenase. Biochem Biophys Res Commun 172: 1230–1235, 1990

    Google Scholar 

  8. Yoshimoto T, Miyamoto Y, Ochi K, Yamamopto S: Arachidonate 12-lipxogyenase of porcine leukocyte with activity for 5-hydroxyeicosatetraenoic acid. Biochem Biophys Acta 713: 638–646, 1982

    Google Scholar 

  9. Yoshimoto T, Suzuki H. Yamamoto S, Takai T, Yokoyama C, Tanbe T: Cloning and sequence analysis of the cDNA for arachidonate lipoxygenase of porcine leukocytes. Proc Natl Acad Sci USA 87: 2142–2146, 1990

    Google Scholar 

  10. Natarajan R, Gu J, Rossi J, Gonzales N, Lanting L, Xu L, Nadler JL: Elevated glucose and angiotensin II increase 12-lipoxygenase activity and expression in porcine aortic smooth muscle cells. Proc Natl Acad Sci USA 90: 4947–4951, 1993

    Google Scholar 

  11. Natarajan R, Gonzales N, Xu L, Nadler JL. Vascular smooth muscle cells exhibit increased growth response to elevated glucose. Biochem Biophys Res Commun 187: 552–560, 1992

    Google Scholar 

  12. Gu J, Natarajan R, Ben-Zzra J, Valente G, Scott S, Yoshimoto T, Yamamoto S, Rossi J, Nadler JL: Evidence that a leukocyte type of 12-lipoxygenase is expressed and regulated by angiotensin II in human adrenal glomerulosa cells. Endocrinology 134: 70–77, 1994

    Google Scholar 

  13. Kim J, Gu J, Natarajan R, Berliner J, Nadler JL: A leukocyte type of 12-lipoxygenase is expressed in human vascular and mononuclear cells-evidence for upregulation by angiotensin II. Thromb Vasc Biol 15: 942–948, 1995

    Google Scholar 

  14. Kruger K, Grabowski PJ, Zang AJ, Sands J, Gottschling DE, Cech TR: Self-splicing RNA:autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena. Cell 31: 147–157, 1982

    Google Scholar 

  15. Guerrier-Takada C, Gardiner K, Marzch T, Pace N, Altman S: The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35: 849–857, 1983

    Google Scholar 

  16. Cech TR, Bass BL: Biological catalysis by RNA. Annu Rev Biochem 55: 599–629, 1986

    Google Scholar 

  17. Cech TR: The chemistry of self-splicing RNA and RNA enzymes. Sci 236: 1532–1539, 1987

    Google Scholar 

  18. Cech TR: Self-splicing of group 1 introns. Annu Rev Biochem 59: 543–568, 1990

    Google Scholar 

  19. Altman S: RNA enzyme-directed gene therapy. Proc Natl Acad Sci USA 90: 10898–10900, 1993

    Google Scholar 

  20. Poeschla E, Wong-Staal F: Antiviral and anticancer ribozymes. Curr Op in Onc 6: 601–606, 1994

    Google Scholar 

  21. Rossi JJ, Cantin EM, Sarver N, Chang PF: The potential use of catalytic RNAs in therapy of HIV injection and other disease. Pharmacol Ther 50: 245–254, 1991

    Google Scholar 

  22. Yu M, Ojwang J, Yamado O, Hampel A, Rappaport J, Looney D, Wong-Staal F: A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 90: 6340–6344, 1993

    Google Scholar 

  23. Yamad O, Yu M, Yee JK, Kraus G, Looney D, Wong-Staal F: Intracellular immunization of human T-cells with a hairpin ribozyme against human immunodeficiency virus type 1. Gene Therapy 1: 38–45, 1994

    Google Scholar 

  24. Sarver M, Cantin E, Chang PS, Ladne PA, Stephens DA, Zaia JA, Rossi JJ: Ribozyme as potential anti-HIV-1 therapeutic agents. Sci 247: 1222–1225, 1990

    Google Scholar 

  25. Rossi J, Elkins D, Zaia J, Sullivan S: Ribozymes as anti-HIV-A therapeutic agents: principles, applications, and problems. AIDS Res Hum Retroviruses 8: 183–189, 1992

    Google Scholar 

  26. Snyder DS, Wu Y, Wang JL, Rossi JJ, Swiderski P, Kaplan BE, Forman SJ: Ribozyme mediated inhibition of bcr-abl gene expression in a philadelphia chromosome-positive cell line. Blood 82: 600–605, 1993

    Google Scholar 

  27. Scanlon KJ, Jiao L, Funato T, Wang W, Tone T, Rossi JJ, Kasahani-Sabet M: Ribozyme-mediated cleavage of c-fosmRNA reduces gene expression of DNA synthesis enzymes and metallothionein. Proc Natl Acad Sci USA 88: 10591–10595, 1991

    Google Scholar 

  28. Kashani-Sabet M, Funato T, Florenes VA, Fodstad O, Scanlon KJ: Suppression of the neoplastic phenotype in vitroby an anti-ras ribozyme. Cancer Res 54: 900–902, 1994

    Google Scholar 

  29. Gu J, Veerapanae D, Rossi J, Natarajan R, Thomas L, Nadler JL: Ribozyme-mediated inhibitor of expression of leukocyte-type 12-lipoxygenase in porcine aortic vascular smooth muscle cells. Circ Res 77: 14–20, 1995

    Google Scholar 

  30. Uhlenbeck OC: A small catalytic deoxyribonucleotide. Nature 328: 596–600, 1987

    Google Scholar 

  31. Koizuni M, Iwai S, Ohtsuka E: Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett 239: 285–288, 1988

    Google Scholar 

  32. Jeffries AC, Symons RH: A catalytic 13-mer ribozyme. NAR 17: 1371–1377, 1989

    Google Scholar 

  33. Haseloff J, Gerlach WL: Simple RNA enzymes with new and highly specific endoribonuclease activity. Nature 334: 585–591, 1988

    Google Scholar 

  34. Weerasinghe M, Liem SE, Asad S, Read SE, Joshi S: Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retro viral vectors expressing an HIV-1 RNA-specific ribozyme. J Virol 65: 5531–5534, 1991

    Google Scholar 

  35. Steve LO KM, Biasolo MA, Dehni G, Palu G, Haseltine WA: Inhibition of replication of HIV-1 by retro viral vectors expressing tatantisense and anti-tat ribozyme RNA. Virol 190: 176–183, 1992

    Google Scholar 

  36. Sullenger BA, Cech TR: Tethering ribozymes to a retro viral packaging signal for destruction of viral RNA. Sci 262: 1566–1569, 1993

    Google Scholar 

  37. Feng M, Cabrera G, Deshane J, Scanlon KJ, Curiel DT: Neoplastic reversion accomplished by high efficiency adenoviral mediated delivery of an anti-ras ribozyme. Cancer Res 55: 2024–2028, 1995

    Google Scholar 

  38. Shimayama T, Nishikawa F, Nishikawa S, Taira K: Nuclease resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkage. NAR 21: 2605–2611, 1993

    Google Scholar 

  39. Beigelman L, McSwiggen JA, Draper KG, Gonzalez C, Jensen K, Karpeisky AM, Modak AS, Matulic-Adamic J, Direzno AB, Haeberli P, Sweedler D, Tracz D, Griman S, Wencott FE, Thackray VG, Usman N. Chemical modification of hammerhead ribozymes catalytic activity and nuclease resistance. J Biol Chem 270: 25702–25708, 1995

    Google Scholar 

  40. Taylor NR, Kaplan BE, Swiderski P, Li H, Rossi JJ. Chimeric DNARNA hammerhead ribozymes have enhanced in vitrocatalytic efficiency and increased stability in vivo. NAR 20: 4559–4565, 1992

    Google Scholar 

  41. Hency P, McCall MJ, Santiago FS, Jennings PA: A ribozyme with DNA in the hybridizing arms displays enhances cleavage ability. NAR 20: 5737–5741, 1992

    Google Scholar 

  42. Kariko K, Megyeri K, Xiao Q, Barnathan ES: Lipofectin-aided cell delivery of ribozyme targeted to human urokinase receptor mRNA. FEBS Lett 352: 41–44, 1994

    Google Scholar 

  43. Sioud M, Natvig JB, Forre Ø: Performed ribozyme destroys tumour necrosis factor mRNA in human cells. J Mol Biol 223: 831–835, 1992

    Google Scholar 

  44. Pickering JG, Jekanowski J, Weir L, Takeshita S, Losordo DW, Isner JM: Liposome-Mediated gene transfer into human vascular smooth muscle cells. Circ 89: 13–21,1994

    Google Scholar 

  45. Capaccioli S, Pasquale GD, Mini E, Mazei T, Quattrone A: Cationic lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells and in human serum. Biochem Biophys Res Commun 197: 818–825, 1993

    Google Scholar 

  46. Hertel KJ, Pardi A, Uhlenbeck OC, Koizumi M, Ohtsuka E, Uesugi S, Cedergren R, Ecketein F, Gerlach WL, Hodgson R, Symons RH: Numbering system for the hammerhead. NAR 20: 3252, 1992

    Google Scholar 

  47. Ruffner DE, Stormo GD, Uhlenbeck OC. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochem 29: 10695–10702, 1990

    Google Scholar 

  48. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current protocols in molecular biology. John Wiley & Sons, Inc. 1995: 1.0–1.13, 2.7.1, 2.10, 3.1–3.16, 8.2.2.

  49. Ogden RC, Adams DA: Electrophoresis in agarose and acrylamide gel. In: SL Berger, AR Kimmel (eds). Method in Enzymology, Vol 152, Guide to Molecular Cloning Techniques. Academic Press, 1987, pp 61–87

  50. Bertrand E, Pictet R, Grange T: Can hammerhead ribozymes be efficient tools to inactivate gene function? NAR 22: 293–300, 1994

    Google Scholar 

  51. Chang PS, Cantin E, Zaia JA, Ladne PA, Stephens DA, Sarver N, Rossi JJ: Ribozyme-mediated site specific cleavage of the HIV-1 genome. Clin Biotech 2: 23–31, 1990

    Google Scholar 

  52. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guandium thiocyante-phenol-chloroform extraction. Anal Biochem 162: 169–159, 1987

    Google Scholar 

  53. Heidenreich O, Eckstein F: Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1. J Biol Chem 267: 1904–1909, 1992

    Google Scholar 

  54. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Annal Biochem 72: 284–254, 1976

    Google Scholar 

  55. Deshler JO, Rossi JJ: Catalytic Antisense RNAs-principles and Design. Nucleic Acid Targeted Drug Design. Prospst CI, Thomas JP, Marcel Dekker Inc. New York: 557–577, 1992

    Google Scholar 

  56. Castanotto D, Bertrand E, Rossi JJ: Antisense technology and ribozymes in molecular biology: current innovation and future trends. AG Hugh (ed). Horizon Scientific Press. Norfold, England part 11, 103–113, 1995

    Google Scholar 

  57. Zuker M, Stiegler P: Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. NAR 9: 133–148, 1981

    Google Scholar 

  58. Sun L, Warrilow D, Wang L, Witherington C, Macpherson J, Symonds G: Ribozyme-mediated suppression of moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines. Proc Natl Acad Sci USA 91: 9715–9719, 1994

    Google Scholar 

  59. Pachuk CJ, Yoon K, Moelling K, Coney LR: Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences. NAR 22: 301–301, 1994

    Google Scholar 

  60. Heindenriech O, Benseler F, Fahrenholz A, Eckstein F: High activity and stability of hammerhead ribozymes containing 2?-modified pyrimidine nucleosides and phosphorothioates. J Biol Chem 269: 2131–2138, 1994

    Google Scholar 

  61. Gao X, Huang L: A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem Biophys Res Commun 179: 280–285, 1991

    Google Scholar 

  62. Caplen NJ, Alton E, Middleton PG, Dorin JK, Stevenson BJ, Gao X, Durham SR, Jefferey PK, Hodson ME, Coutelle C, Huang L, Porteous DJ, Williamson R, Geddes DM: Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nature Medicine 1: 39–46, 1995

    Google Scholar 

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Authors and Affiliations

  1. Department of Diabetes, Endocrinology and Metabolism, City of Hope Medical Center, 1500 East Duarte Road, Duarte, CA, 91010, USA

    Jia-Li Gu & Jerry Nadler

  2. Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA, 91010, USA

    John Rossi

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  1. Jia-Li Gu
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  2. Jerry Nadler
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Gu, JL., Nadler, J. & Rossi, J. Use of a hammerhead ribozyme with cationic liposomes to reduce leukocyte type 12-lipoxygenase expression in vascular smooth muscle. Mol Cell Biochem 172, 47–57 (1997). https://doi.org/10.1023/A:1006855219018

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