Skip to main content
Log in

Summary

1. Ribozymes can be designed to cleavein trans, i.e. several substrate molecules can be turned over by one molecule of the catalytic RNA. Only small molecular weight ribozymes, or small ribozymes, are discussed in this review with particular emphasis on the hammerhead ribozyme as this has been most widely used for the inhibition of gene expression by cleavage of mRNAs.

2. Cellular delivery of the ribozyme is of crucial importance for the success of inhibition of gene expression by this methodology. Two modes of delivery can be envisaged, endogenous and exogenous delivery. Of the former several variants exist, depending on the vector used. The latter is still in its infancy, even though chemical modification has rendered such ribozymes resistant against degradation by serum nucleases without impairment of catalytic efficiency.

3. Various successful applications of ribozymes for the inhibition of gene expression are discussed, with particular emphasis on HIV1 and cancer targets. These examples demonstrate the promise of this methodology.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Altman, S. (1993). RNA enzyme directed gene therapy.Proc. Natl. Acad. Sci. USA 9010898–10900.

    PubMed  Google Scholar 

  • Aurup, H., Williams, D. M., and Eckstein, F. (1992). 2′-Fluoro and 2′-amino-2′-deoxynucleoside 5′-triphosphates as substrates for T7 RNA polymerase.Biochemistry 319636–9641.

    PubMed  Google Scholar 

  • Benseler, F., Fu, D.-J., Ludwig, J., and McLaughlin, L. W. (1993). Hammerhead-like molecules containing non-nucleotidic linkers are active RNA catalysts.J. Am. Chem. Soc. 1158483–8484.

    Google Scholar 

  • Bertrand, E., Pictet, R., and Grange, T. (1994). Can hammerhead ribozymes be efficient tools to inactivate gene function?Nucleic Acids Res. 22293–300.

    PubMed  Google Scholar 

  • Berzal, H. A., Joseph, S., Chowrira, B. M., Butcher, S. E., and Burke, J. M. (1993). Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.EMBO J. 122567–2573.

    PubMed  Google Scholar 

  • Bratty, J., Chartrand, G. Ferbeyre, G., and Cedergren, R. (1993). The Hammerhead RNA domain, a model ribozyme.Biochim. Biophys. Acta 1216345–359.

    PubMed  Google Scholar 

  • Cantor, G. H., McElwain, T. F., Birkebak, T. A., and Palmer, G. H. (1993). Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.Proc. Natl. Acad. Sci. USA 9010932–10936.

    PubMed  Google Scholar 

  • Cech, T. R. (1990). Self-splicing of group I introns.Annu. Rev. Biochem. 59543–568.

    PubMed  Google Scholar 

  • Chen, C. J., Banerjea, A. C., Harmison, G. G., Haglund, K., Schubert, M. (1992). Multitargetribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits HIV-1 replication—Potential effectiveness against most presently sequenced HIV-1 isolates.Nucleic Acids Res. 204581–4589.

    PubMed  Google Scholar 

  • Cotten, M. (1990). The in vivo application of ribozymes.TIBS 8174–178.

    Google Scholar 

  • Cotten, M., and Birnstiel, M. L. (1989). Ribozyme mediated destruction of RNA in vivo.EMBO J. 83861–3866.

    PubMed  Google Scholar 

  • Cotten, M., Schaffner, G., and Birnstiel, M. L. (1989). Ribozyme, antisense RNA and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro.Mol. Cell. Biol. 94479–4487.

    PubMed  Google Scholar 

  • Cotten, M., Wagner, E., Zatloukal, K., Phillips, S., Curiel, D. T., and Birnstiel, M. L. (1992). High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles).Proc. Natl. Acad. Sci. USA 896094–6098.

    PubMed  Google Scholar 

  • Crisell, P., Thompson, S., and James, W. (1993). Inhibition of HIV-1 replication by ribozymes that show poor activityin vitro.Nucleic Acids Res. 215251–5255.

    PubMed  Google Scholar 

  • Eder, P. S., DeVine, R. J., Dagle and Walder, J. A. (1991). Substrate specificity and kinetics of degradation of antisense oligonucleotides by a 3′-exonuclease in plasma.Antisense Res. Dev. 1141–151.

    PubMed  Google Scholar 

  • Emlen, W. and Mannik, M. (1978). Kinetics and mechanisms for removal of circulating single-stranded DNA in mice.J. Exp. Med. 147684–699.

    PubMed  Google Scholar 

  • 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. 213857–3865.

    PubMed  Google Scholar 

  • Gerlach, W. L., and Young, M. J. (1991). Ribozymes for the control of gene activity in vivo. InAntisense Nucleic Acids & Proteins; Fundamentals and Applications (Mol, J. M., and Krol, A. R., Eds.), Dekker, New York, Basel, Hong Kong, pp. 157–168.

    Google Scholar 

  • Goodchild, J. (1992). Enhancement of ribozyme catalytic activity by a contiguous oligodeoxynucleotide (facilitator) and by 2′-O-methylation.Nucleic Acids Res. 204607–4612.

    PubMed  Google Scholar 

  • Hasselhoff, J., and Gerlach, W. L. (1988). Simple RNA enzymes with new and highly specific endoribonuclease activity.Nature 334585–591.

    PubMed  Google Scholar 

  • Heidenreich, O., Benseler, F., Fahrenholz, A., and Eckstein, F. (1994). High activity and stability of ribozymes containing 2′-modified pyrimidine nucleosides and phosphorothioates.J. Biol. Chem. 2692131–2138.

    PubMed  Google Scholar 

  • Heidenreich, O., and Eckstein, F. (1992). Hammerhead ribozyme mediated cleavage of the long terminal repeat of human immunodeficiency virus type 1.J. Biol. Chem. 2671904–1909.

    PubMed  Google Scholar 

  • Heinrich, J. C., Tabler, M., and Louis, C. (1993). Attenuation of white gene expression in trangenic Drosophila melanogaster: possible role of a catalytic antisense RNA.Dev. Genet. 14258–265.

    PubMed  Google Scholar 

  • Hertel, K. J., Pardi, A., Uhlenbeck, O. C., Koizumi, M., Ohtsuka, E., Uesugi, S., Cedergren, R., Eckstein, F., Gerlach, W., Hofgson, R., and Symons, R. H. (1992). Numbering system for the hammerhead.Nucleic Acids Res. 203252.

    PubMed  Google Scholar 

  • Hertel, K. J., Herschlag, D., and Uhlenbeck, O. C. (1994). A kinetic and thermodynamic framework for the hammerhead ribozyme reaction.Biochemistry 333374–3385.

    PubMed  Google Scholar 

  • Homann, M., Tzortzakaki, S., Rittner, K., Sczakiel, G., and Tabler, M. (1993). Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.Nucleic Acids Res. 212809–2814.

    PubMed  Google Scholar 

  • Kashani, S. M., Funato, T., Tone, T., Jiao, L., Wang, W., Yoshida, E., Kashfinn, B. I., Shitara, T., Wu, A. M., Moreno, J. G., Traweek, S. E., Ahlering, T. E., and Scanlon, K. J. (1992). Reversal of the malignant phenotype by an anti-ras ribozyme.Antisense Res. Dev. 23–15.

    PubMed  Google Scholar 

  • Kirsch, J., Wolters, I., Triller, A., and Betz, H. (1993). Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons.Nature 366745–748.

    PubMed  Google Scholar 

  • Koizumi, M., Kamiya, H., and Ohtsuka, E. (1992). Ribozymes designed to inhibit transformation of NIH3T3 cells by the activated c-Ha-ras gene.Gene 117179–184.

    PubMed  Google Scholar 

  • Krieg, A. M., Tonkinso, J., Matson, S., Zhao, Q., Saxon, M., Zhang, L.-M., Bhanja, U., Yakubov, L., and Stein, C. A. (1993). Modification of antisense phosphodiester oligodeoxynucleotides by a 5′ cholesteryl moiety increases association and improves efficacy.Proc. Natl. Acad. Sci. USA 901048–1052.

    PubMed  Google Scholar 

  • Krinke, L., and Wulff, D. L. (1990). The cleavage specificity of RNase III.Nucleic Acids Res. 184809–4815.

    PubMed  Google Scholar 

  • Lange, W., Daskalakis, M., Finke, J., and Dölken, G. (1994). Comparison of different ribozymes for efficient and specific cleavage of BCR/ABL related mRNAs.FEBS Lett. 338175–178.

    PubMed  Google Scholar 

  • Leonetti, J. P., and Leserman, L. D. (1993). Targeted delivery of Oligonucleotides. InAntisense Research and Applications (Cook, S. T., and Lebleu, B., Eds.), CRC Press, Boca Raton, pp. 493–504.

    Google Scholar 

  • Leonetti, J. P., Mechti, N., Degols, G., Gagnor, C., and Lebleu, B. (1991). Intracellular distribution of microinjected antisense oligonucleotides.Proc. Natl. Acad. Sci. USA 882702–2706.

    PubMed  Google Scholar 

  • L'Huillier, P. J., Davis, S. R., and Bellamy, A. R. (1992). Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells.EMBO J. 114411–4418.

    PubMed  Google Scholar 

  • Lo, K. M., Biasolo, M. A., Dehni, G., Palu, G., and Haseltine, W. A. (1992). Inhibition of replication of HIV-1 by retroviral vectors expressing tat-antisense and anti-tat ribozyme RNA.Virology 190176–183.

    PubMed  Google Scholar 

  • Long, D. M., and Uhlenbeck, O. C. (1993). Self-cleaving catalytic RNA.FASEB J. 725–30.

    PubMed  Google Scholar 

  • Mackellar, C., Graham, D., Will, D. W., Burgess, S., and Brown, T. (1992). Synthesis and physical properties of anti-HIV antisense oligonucleotides bearing terminal lipophilic groups.Nucleic Acids Res. 203411–3417.

    PubMed  Google Scholar 

  • Miller, A. D. (1992). Human gene therapy comes of age.Nature 357455–460.

    PubMed  Google Scholar 

  • Milligan, J. F., Matteuci, M. D., and Martin, J. C. (1993). Current concepts in antisense drug design.J. Med. Chem. 361923–1937.

    PubMed  Google Scholar 

  • Morgan, R. A., and Anderson, W. F. (1993). Human gene therapy.Annu. Rev. Biochem. 62191–217.

    PubMed  Google Scholar 

  • Mulligan, R. C. (1993). The basic science of gene therapy.Science 260926–932.

    PubMed  Google Scholar 

  • Nakamaye, K. L., and Eckstein, F. (1994) AUA-Cleaving hammerhead ribozymes: Attempted selection for improved cleavage.Biochemistry 331271–1277.

    PubMed  Google Scholar 

  • Nellen, W., and Lichtenstein, C. (1993). What makes an mRNA anti-sense-itive?TIBS 18419–423.

    PubMed  Google Scholar 

  • Ojwang, J. O., Hampel, A., Looney, D. J., Wong, S. F., and Rappaport, J. (1992). Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme.Proc. Natl. Acad. Sci. USA 8910802–10806.

    PubMed  Google Scholar 

  • Paolella, G., Sproat, B., and Lamond, A. I. (1992) Nuclease resistant ribozymes with high catalytic activity.EMBO J. 111913–1919.

    PubMed  Google Scholar 

  • Perriman, R., Delves, A., and Gerlach, W. L. (1992). Extended target-site specificity for a hammerhead ribozyme.Gene 13156–163.

    Google Scholar 

  • Pieken, W. W. A., Olsen, D. B., Benseler, F., Aurup, H., and Eckstein, F. (1991). Kinetic characterisation of ribonuclease resistant 2′-modified hammerhead ribozymes.Science 253314–317.

    PubMed  Google Scholar 

  • Ramalho Ortiago, J. F., Rosch, H., Selter, H., Frohlich, A., Lorenz, A., Montenarh, M., and Seliger, H. (1992). Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: A minimal modification protecting against nucleolytic degradation.Antisense Res. Dev. 2129–146.

    PubMed  Google Scholar 

  • Ratajczak, M. Z., and Gewirtz, A. M. (1994). Oligonucleotide-based therapeutics of human malignancies. InNucleic Acids & Molecular Biology (F. Eckstein and D. J. M. Lilley, Eds.), Springer-Verlag, Berlin, Heidelberg, Vol. 8, pp. 298–326.

    Google Scholar 

  • Rossi, J. J. (1992). Ribozymes.Curr. Opin. Biotechnol. 33–7.

    PubMed  Google Scholar 

  • Rossi, J. J., and Sarver, N. (1990). RNA enzymes (ribozymes) as antiviral therapeutic agents.TIBTECH 8179–183.

    Google Scholar 

  • Rossi, J. J., Elkins, D., Zaia, J. A., and Sullivan, S. (1992). Ribozymes as anti-HIV-1 therapeutic agents: Principles, applications, and problems.AIDS Res. Hum. Retroviruses 8183–189.

    PubMed  Google Scholar 

  • Ruffner, D. E., Stormo, G. D., and Uhlenbeck, O. C. (1990). Sequence requirements of the hammerhead ribozyme RNA self-cleavage reaction.Biochemistry 2910695–10702.

    PubMed  Google Scholar 

  • Ohsen-Sand, A., Catsicas, M., Staple, J. K., Jones, K. A., Yala, G., Knowles, J., Grenningloh, G., and Catsicas, S. (1993).Nature 364445–448.

    PubMed  Google Scholar 

  • Sarver, N., Cantin, E. M., Chang, P. S., Zaia, J. A., Ladne, P. A., Stephens, D. A., and Rossi, J. J. (1990). Ribozymes as potential anti-HIV-1 therapeutic agents.Science 2471222–1225.

    PubMed  Google Scholar 

  • Scanlon, K. J., Jiao, L., Funato, T., Wang, W., Tone, T., Rossi, J. J., and Kashani, S. M. (1991). Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.Proc. Natl. Acad. Sci. USA 8810591–10595.

    PubMed  Google Scholar 

  • Shimayama, T., Nishikawa, F., Nishikawa, S., and Kazunari, T. (1993). Nuclease resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.Nucleic Acids Res. 212605–2611.

    PubMed  Google Scholar 

  • Sioud, M., and Drlica, K. (1991). Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme.Proc. Natl. Acad. Sci. USA 887303–7307.

    PubMed  Google Scholar 

  • Sioud, M., Natvig, J. B., and Forre, O. (1992). Preformed ribozyme destroys tumour necrosis factor mRNA in human cells.J. Mol. Biol. 223831–835.

    PubMed  Google Scholar 

  • Snyder, D. S., Wu, Y., Wang, J. L., Rossi, J. J., Swiderski, P., Kaplan, B. E., and Forman, S. J. (1993). Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line.Blood 82600–605.

    PubMed  Google Scholar 

  • Soreq, H., Patinkin, D., Lev-Lehman, E., Grifman, M., Ginzberg, D., Eckstein, F., and Zakut, H. (1994). Antisense oligonucleotide inhibition of acetylcholinesterase gene expression induces progenitor cell expansion and suppresses hematopoietic apoptosisex vivo.Proc. Natl. Acad. Sci. USA 917907–7911.

    PubMed  Google Scholar 

  • Stein, C. A., and Cheng, Y.-C. (1993). Antisense oligonucleotides as therapeutic agents—is the bullet really magical?Science 2611004–1012.

    PubMed  Google Scholar 

  • Stein, C. A., Tonkinson, J. L., Zhang, L. M., Yakubov, L., Gervasoni, J., Taub, R., and Rotenberg, S. A. (1993). Dynamics of the internalization of phosphodiester oligodeoxynucleotides in HL60 cells.Biochemistry 324855–4861.

    PubMed  Google Scholar 

  • Sullenger, B. A., and Cech, T. R. (1993). Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA.Science 2621566–1569.

    PubMed  Google Scholar 

  • Symons, R. H. (1992). Small catalytic RNAs.Annu. Rev. Biochem. 61641–671.

    PubMed  Google Scholar 

  • Taylor, N. R., Kaplan, B. A., Swiderski, P., Li, H., and Rossi, J. J. (1992). Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.Nucleic Acids Res. 204559–4565.

    PubMed  Google Scholar 

  • Thomson, J. B., Tuschl, T., and Eckstein, F. (1993). Activity of hammerhead ribozymes containing non-nucleotidic linkers.Nucleic Acids Res. 205600–5603.

    Google Scholar 

  • Tsuchihashi, Z., Khosla, M., and Herschlag, D. (1993). Protein enhancement of hammerhead ribozyme catalysis.Science 26299–102.

    PubMed  Google Scholar 

  • Tsuji, H., Nomiyama, K., Murai, K., Akagi, K., and Fujishima, M. (1992). Comparison of the properties of ribonucleases in human liver tissue and serum.Eur. J. Clin. Chem. Biochem. 30339–341.

    Google Scholar 

  • Uhlenbeck, O. C. (1987). A small catalytic oligoribonucleotide.Nature 3284855–4861.

    Google Scholar 

  • Uhlmann, E., and Peymann, A. (1990). Antisense oligonucleotides: A new therapeutic principle.Chem. Rev. 90543–584.

    Google Scholar 

  • Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K., Curiel, D. T., and Birnstiel, M. L. (1992). Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes.Proc. Natl. Acad. Sci. USA 896099–6103.

    PubMed  Google Scholar 

  • Weerasinghe, M., Liem, S. E., Asad, S., Read, S. E., and Joshi, S. (1991). Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme.J. Virol. 655531–5534.

    PubMed  Google Scholar 

  • Yamada, O., Yu, M., Yee, J.-K., Kraus, G., Looney, D., and Wong-Staal, F. (1994). Intracellular immunization of human T-cells with a hairpin ribozyme against human immunodeficiency virus type 1.Gene Ther. 138–45.

    PubMed  Google Scholar 

  • Yang, J.-H., Usman, N., Chartrand, P., and Cedergreen, R. (1992). Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain.Biochemistry 315005–5009.

    PubMed  Google Scholar 

  • Yu, M., Ojwang, J., Yamada, O., Hampel, A., Rappaport, J., Looney, D., and Wong-Staal, F. (1993). A hairpin ribozyme inhibits expression of diverse strains of human efficiency virus type 1.Proc. Natl. Acad. Sci. USA 896099–6103.

    Google Scholar 

  • Yuan, Y., and Altman, S. (1994). Selection of guide sequences that direct efficient cleavage of mRNA by human ribonuclease P.Science 2631269–1273.

    PubMed  Google Scholar 

  • Zhao, J. J., and Pick, L. (1993). Generating loss-of-function phenotypes of the fushi tarazu gene with a targeted ribozyme in Drosophila.Nature 365448–451.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Marschall, P., Thomson, J.B. & Eckstein, F. Inhibition of gene expression with ribozymes. Cell Mol Neurobiol 14, 523–538 (1994). https://doi.org/10.1007/BF02088835

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02088835

Key words

Navigation