Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Enhanced intracellular availability and survival of hammerhead ribozymes increases target ablation in a cellular model of osteogenesis imperfecta

Gene Therapy volume 10, pages 2005–2012 (2003)Cite this article

Abstract

Antisense hammerhead ribozymes have the capability to cleave complementary RNA in a sequence-dependent manner. In osteogenesis imperfecta, a genetic disorder of connective tissue, mutant collagen type I has been shown to participate in but not sustain formation of the triple helix. Selective ablation of mutant collagen gene transcript could potentially remove the mutant gene product and reverse the dominant-negative effect exerted by the abnormal protein. In earlier studies we showed that the hammerhead ribozyme Col1A1Rz547 selectively cleaved a mutant Col1A1 gene transcript in a murine calvarial osteobleast cell line. In order to test the possible therapeutic efficacy of this approach, a dramatic downregulation of the mutant transcript must be achieved, a function directly related to high steady-state level of intracellular ribozyme. We report significantly enhanced expression of Col1A1Rz547 by vaccinia T7 polymerase following infection with an attenuated T7-pol vaccinia virus as shown both by the intracellular level of the ribozyme and the cleavage of the mutant Col1A1 gene transcript. We also describe the engineering of a multimeric ribozyme construct comprising eight subunits, which can self-cleave to monomers. These studies suggest the potential use of multimeric ribozymes expressed by a vaccinia-based system in the therapy of a variety of disorders.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Opalinska JB, Gewirtz AM . Nucleic-acid therapeutics: basic principles and recent applications. Nat Rev Drug Discov 2002; 1: 503–514.

    Article  CAS  Google Scholar 

  2. Lebedeva I, Stein CA . Antisense oligonucleotides: promise and reality. Annu Rev Pharmacol Toxicol 2001; 41: 403–419.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Uhlenbeck OC . A small catalytic oligoribonucleotide. Nature 1987; 328: 596–600.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Forster AC, Symons RH . Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell 1987; 49: 211–220.

    Article  CAS  Google Scholar 

  7. Scott WG et al. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science 1996; 274: 2065–2069.

    Article  CAS  Google Scholar 

  8. Kilpatrick MW et al. Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts. Hum Mol Genet 1996; 5: 1939–1944.

    Article  CAS  Google Scholar 

  9. Lewin AS et al. Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa. Nat Med 1998; 4: 967–971.

    Article  CAS  Google Scholar 

  10. Scherr M et al. Effective reversal of a transformed phenotype by retrovirus-mediated transfer of a ribozyme directed against mutant N-ras. Gene Therapy 1998; 5: 1227–1234.

    Article  CAS  Google Scholar 

  11. Millington-Ward S et al. A mutation-independent therapeutic strategem for osteogenesis imperfecta. Antisense Nucleic Acid Drug Dev 1999; 9: 537–542.

    Article  CAS  Google Scholar 

  12. Hammond SM, Caudy AA, Hannon GJ . Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet 2001; 2: 110–119.

    Article  CAS  Google Scholar 

  13. Elbashir SM et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411: 494–498.

    Article  CAS  Google Scholar 

  14. Hutvagner G, Zamore PD . RNAi: nature abhors a double-strand. Curr Opin Genet Dev 2002; 12: 225–232.

    Article  CAS  Google Scholar 

  15. Phylactou LA, Tsipouras P, Kilpatrick MW . Hammerhead ribozymes targeted to the FBN1 mRNA can discriminate a single base mismatch between ribozyme and target. Biochem Biophys Res Commun 1998; 249: 804–810.

    Article  CAS  Google Scholar 

  16. Byers PH, Cole WG . Osteogenesis imperfecta. In: Royce PM, Steinmann B (eds). Connective Tissue and its Heritable Disorders, 2nd edn. Wiley-Liss: New York, 2002 pp 385–430.

    Chapter  Google Scholar 

  17. Kielty CM, Grant ME . The collagen family: structure, assembly, and organization in the extracellular matrix. In: Royce PM, Steinmann B (eds). Connective Tissue and its Heritable Disorders, 2nd edn. Wiley-Liss: New York, 2002 pp 159–221.

    Chapter  Google Scholar 

  18. Barsh GS, David KE, Byers PH . Type I osteogenesis imperfecta: a non functional allele for pro α1 (I) chains of type I procollagen. Proc Natl Acad Sci USA 1982; 79: 3838–3842.

    Article  CAS  Google Scholar 

  19. Stacey A et al. Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineered mutant pro-alpha 1(I) collagen gene. Nature 1988; 332: 131–136.

    Article  CAS  Google Scholar 

  20. Bonadio J et al. Transgenic mouse model of the mild dominant form of osteogenesis imperfecta. Proc Natl Acad Sci USA 1990; 87: 7145–7149.

    Article  CAS  Google Scholar 

  21. Chipman SD et al. Defective pro alpha 2(I) collagen synthesis in a recessive mutation in mice: a model of human osteogenesis imperfecta. Proc Natl Acad Sci USA 1993; 90: 1701–1705.

    Article  CAS  Google Scholar 

  22. Forlino A et al. Use of the Cre/lox recombination system to develop a non-lethal knock-in murine model for osteogenesis imperfecta with an alpha1(I) G349C substitution. Variability in phenotype in BrtlIV mice. J Biol Chem 1999; 274: 37923–37931.

    Article  CAS  Google Scholar 

  23. Pereira R et al. Transgenic mice expressing a partially deleted gene for type I procollagen (COL1A1). A breeding line with a phenotype of spontaneous fractures and decreased bone collagen and mineral. J Clin Invest 1993; 91: 709–716.

    Article  CAS  Google Scholar 

  24. Pereira R et al. Phenotypic variability and incomplete penetrance of spontaneous fractures in an inbred strain of transgenic mice expressing a mutated collagen gene (COL1A1). J Clin Invest 1994; 93: 1765–1769.

    Article  CAS  Google Scholar 

  25. Wenstrup RJ, Fowlkes JL, Witte DP, Florer JB . Discordant expression of osteoblast markers in MC3T3-E1 cells that synthesize a high turnover matrix. J Biol Chem 1996; 271: 10271–10276.

    Article  CAS  Google Scholar 

  26. Toudjarska I et al. Delivery of a hammerhead ribozyme specifically downregulates mutant type I collagen mRNA in a murine model of osteogenesis imperfecta. Antisense Nucleic Acid Drug Dev. 2001; 11: 341–346.

    Article  CAS  Google Scholar 

  27. Ruiz J, Wu CH, Ito Y, Wu GY . Design and preparation of a multimeric self-cleaving hammerhead ribozyme. Biotechniques 1997; 22: 338–345.

    Article  CAS  Google Scholar 

  28. Carroll MW, Moss B . Poxviruses as expression vectors. Curr Opin Biotech 1997; 8: 573–577.

    Article  CAS  Google Scholar 

  29. Cheng L, Goldman E . Reversal of inhibition by the T7 concatemer junction sequence on expression from a downstream T7 promoter. Gene Expression 2001; 9: 257–264.

    Article  CAS  Google Scholar 

  30. Guhaniyogi J, Brewer G . Regulation of mRNA stability in mammalian cells. Gene 2001; 265: 11–23.

    Article  CAS  Google Scholar 

  31. Mastrangelo MJ, Eisenlohr LC, Gomella L, Lattime EC . Poxvirus vectors: orphaned and underappreciated. J Clin Invest 2000; 105: 1031–1034.

    Article  CAS  Google Scholar 

  32. Evans ME, Lesnaw JA . Infection control in gene therapy [comment]. Infect Control Hosp Epidemiol 1999; 20: 568–576.

    Article  CAS  Google Scholar 

  33. Peplinski GR, Tsung K, Norton JA . Vaccinia virus for human gene therapy. Surg Oncol Clin N Am 1998; 7: 575–588.

    Article  CAS  Google Scholar 

  34. Konetschny C, Holzer GW, Falkner FG . Retroviral vectors produced in the cytoplasmic vaccinia virus system transduce intron-containing genes. J Virol 2002; 76: 1236–1243.

    Article  CAS  Google Scholar 

  35. Carroll MW, Moss B . Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 1997; 238: 198–211.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by a grant from the Children's Brittle Bone Foundation. Modified vaccinia virus Ankara expressing bacteriophage T7 polymerase (MVA-T7) was generously provided by Dr Bernard Moss, NIAID-NIH.

Author information

Authors and Affiliations

  1. Department of Pediatrics, University of Connecticut Health Center, Farmington, CT, USA

    Y Smicun, M W Kilpatrick, I Toudjarska & P Tsipouras

  2. Cincinnati Children's Hospital Research Foundation, Cincinnati, OH, USA

    J Florer & R J Wenstrup

  3. Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA

    G Wu

Authors
  1. Y Smicun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M W Kilpatrick
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J Florer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. I Toudjarska
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R J Wenstrup
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. P Tsipouras
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Smicun, Y., Kilpatrick, M., Florer, J. et al. Enhanced intracellular availability and survival of hammerhead ribozymes increases target ablation in a cellular model of osteogenesis imperfecta. Gene Ther 10, 2005–2012 (2003). https://doi.org/10.1038/sj.gt.3302108

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302108

Keywords

This article is cited by

Search

Advanced search

Quick links