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:

Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides

Nature Biotechnology volume 17, pages 989–993 (1999)Cite this article

Abstract

A sequence-specific genomic delivery system for the correction of chromosomal mutations was designed by incorporating two different binding domains into a single-stranded oligonucleotide. A repair domain (RD) contained the native sequence of the target region. A third strand-forming domain (TFD) was designed to form a triplex by Hoogsteen interactions. The design was based upon the premise that the RD will rapidly form a heteroduplex that is anchored synergistically by the TFD. Deoxyoligonucleotides were designed to form triplexes in the human adenosine deaminase (ADA) and p53 genes adjacent to known point mutations. Transfection of ADA-deficient human lymphocytes corrected the mutant sequence in 1–2% of cells. Neither the RD or TFD individually corrected the mutation. Transfection of p53 mutant human glioblastoma cells corrected the mutation and induced apoptosis in 7.5% of cells.

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: Correction of a G→A missense mutation in codon 329 of exon 11 of the human ADA gene.
Figure 2: Molecular analysis of oligonucleotide-treated and nontreated SNB-19 tumor cells.
Figure 3: Sequence analysis of human SNB-19 tumor cells treated with 273P1 and 273P2 oligonucleotides.

Similar content being viewed by others

References

  1. Kunzelmann, K. et al. Gene targeting of CFTR DNA in CF epithelial cells. Gene Ther. 10, 859–867 (1996).

    Google Scholar 

  2. Cole-Strauss, A. et al. Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science 273, 1386–1389 (1996).

    Article  CAS  Google Scholar 

  3. Russell D.W. & Hirata, R.K. Human gene targeting by viral vectors. Nat. Genet. 18, 325–330 (1998).

    Article  CAS  Google Scholar 

  4. Chan, P.P. & Glazer, P.M. Triplex DNA: fundamentals, advances, and potential applications for gene therapy. J. Mol. Med. 75, 267–282 (1997).

    Article  CAS  Google Scholar 

  5. Hanvey, J.C., Williams, E.M. & Besterman J.M. DNA triple-helix formation at physiological pH and temperature. Antisense Res. Dev. 1, 307–317 (1991).

    Article  CAS  Google Scholar 

  6. Hsieh, P., Camerini-Otero, C.S. & Camerini-Otero, R.D. The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA. Proc. Natl Acad. Sci. USA 89, 6492–6496 (1992).

    Article  CAS  Google Scholar 

  7. Rao, B.J. & Radding, C.M. Formation of base triplets by non-Watson–Crick bonds mediates homologous recognition in RecA recombination filaments. Proc. Natl Acad. Sci. USA 91, 6161–6165 (1994).

    Article  CAS  Google Scholar 

  8. Yanez, R.J. & Porter, A.C. Therapeutic gene targeting. Gene Ther. 5, 149–159 (1998).

    Article  CAS  Google Scholar 

  9. Griffin, L.C. & Dervan, P.B. Recognition of thymine-adenine base pairs by guanine in a pyrimidine triple helix motif. Science 245, 967–971 (1989).

    Article  CAS  Google Scholar 

  10. Fossella, J.A., Kim, Y.J., Shih, H., Richards, E.G. & Fresco, J.R. Relative specificities in binding of Watson–Crick base pairs by third strand residues in a DNA pyrimidine triplex motif. Nucleic Acids Res. 21, 4511–4515 (1993).

    Article  CAS  Google Scholar 

  11. Vlieghe, D. et al. Parallel and antiparallel (G•GC)2 triple helix fragments in a crystal structure. Science 273, 1702–1706 (1996).

    Article  CAS  Google Scholar 

  12. Majumdar, A. et al. Targeted gene knockout mediated by triple helix forming oligonucleotides. Nat. Genet. 20, 2112–2214 (1998).

    Article  Google Scholar 

  13. Svinarchuk, F. et al. Recruitment of transcription factors to the target site by triplex-forming oligonucleotides. Nucleic Acids Res. 25, 3459–3464 (1997).

    Article  CAS  Google Scholar 

  14. Panyutin, I.G. & Neumann, R.D. Sequence-specific DNA double-strand breaks induced by triplex forming 125I labeled oligonucleotides. Nucleic Acids Res. 22, 4979–4982 (1994).

    Article  CAS  Google Scholar 

  15. Grigoriev, M. et al. A triple helix-forming oligonucleotide–intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence. J. Biol. Chem. 267, 3389–3395 (1992).

    CAS  PubMed  Google Scholar 

  16. Escude, C. et al. Rational design of a triple helix-specific intercalating ligand. Proc. Natl Acad. Sci. USA 95, 3591–3596 (1998).

    Article  CAS  Google Scholar 

  17. Wang, G., Levy, D.D., Seidman, M.M. & Glazer, P.M. Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation. Mol. Cell. Biol. 15, 1759–1768 (1995).

    Article  CAS  Google Scholar 

  18. Volkmann, S., Jendis, J., Frauendorf, A. & Moelling, K. Inhibition of HIV-1 reverse transcription by triple-helix forming oligonucleotides with viral RNA. Nucleic Acids Res. 23, 1204–1212 (1995).

    Article  CAS  Google Scholar 

  19. Sun, J.S., Garestier, T. & Helene, C. Oligonucleotide directed triple helix formation. Curr. Opin. Struct. Biol. 6, 327–333 (1996).

    Article  CAS  Google Scholar 

  20. Gamper Jr.,H.B., Hou, Y.-M., Stamm, M.R., Podyminogin, M.A. & Meyer, R.B. Strand invasion of supercoiled DNA by oligonucleotides with a triplex guide sequence. J. Am. Chem. Soc. 120, 2182–2183 (1998).

    Article  CAS  Google Scholar 

  21. Chan et al. Targeted correction of an episomal gene in mammalian cells by a short DNA fragment tethered to a triplex-forming oligonucleotide. J. Biol. Chem. 274, 11541–11548 (1999).

    Article  CAS  Google Scholar 

  22. Hirschhorn, R. Adenosine deaminase deficiency: molecular basis and recent developments. Clin. Immunol. Pathol. 76, 219–227 (1995).

    Google Scholar 

  23. Blaese, R.M., Culver, K.W. & Anderson, W.F. The ADA human gene therapy clinical protocol. Hum. Gene Ther. 1, 331–362 (1990).

    Article  Google Scholar 

  24. Markert, M.L., Norby-Slycord, C. & Ward, F.E. A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution. Am. J. Hum. Genet. 45, 354–361 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Hirschhorn, R., Martiniuk, F. & Rosen, F.S. Adenosine deaminase activity in normal tissues and tissues from a child with severe combined immunodeficiency and adenosine deaminase deficiency. Clin. Immunol. Immunopathol. 9, 287–292 (1978).

    Article  CAS  Google Scholar 

  26. Hussain, S.P. & Harris, C.C. Molecular epidemiology of human cancer: Contribution of mutation spectra studies of tumor suppressor genes. Cancer Res. 58, 4023–4037 (1998).

    CAS  PubMed  Google Scholar 

  27. Fujiwara, T. et al. Retroviral-mediated transduction of p53 gene increases TGF-beta expression in a human glioblastoma cell line. Int. J. Cancer 56, 834–839 (1994).

    Article  CAS  Google Scholar 

  28. Wang, G., Seidman, M.M. & Glazer, P.M. Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. Science 27, 802–805 (1996).

    Article  Google Scholar 

  29. Sturzbecher, H.W., Donzelmann, B., Henning, W., Knippschild, U. & Buchhop, S. p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. EMBO J. 15, 1992–2002 (1996).

    Article  CAS  Google Scholar 

  30. Seto, S., Carrera, C.J., Wasson, D.B. & Carson, D.A. Inhibition of DNA repair by deoxyadenosine in resting human lymphocytes. J. Immunol. 136, 2839–2843 (1986).

    CAS  PubMed  Google Scholar 

  31. Goncz, K.K., Kunzelmann, K., Xu, Z., & Gruenert, D.C. Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments. Hum. Mol. Genet. 7, 1913–1919 (1998).

    Article  CAS  Google Scholar 

  32. Cole-Strauss, A. et al. Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science 273, 1386–1389 (1996).

    Article  CAS  Google Scholar 

  33. Kren, B.T., Cole-Strauss, A., Kmiec, E.B. & Steer, C.J. Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide. Hepatology 25,1462–1468 (1997).

    Article  CAS  Google Scholar 

  34. Strauss, M. The site-specific correction of genetic defects. Nat. Med. 4, 274–275 (1998).

    Article  CAS  Google Scholar 

  35. Blaese, R. M. et al. T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science 270, 475–480 (1995).

    Article  CAS  Google Scholar 

  36. Maher, L.J., Dervan, P.B & Wold, B. Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA. J. Biochem. 29, 8820–8826 (1990).

    Article  CAS  Google Scholar 

  37. Agrawal, S. et al. Mixed-backbone oligonucleotides as second generation antisense oligonucleotides: in vitro and in vivo studies. Proc. Natl. Acad. Sci USA 94, 2620–2625 (1997).

    Article  CAS  Google Scholar 

  38. Tang, M.X., Redemann, C.T. & Szoka, Jr., F.C. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem. 7, 703–714 (1996).

    Article  CAS  Google Scholar 

  39. Culver, K.W. et al. Lymphocytes as cellular vehicles for gene therapy in mouse and man. Proc. Natl Acad. Sci. USA 88, 3155–3159 (1991).

    Article  CAS  Google Scholar 

  40. Li, X. et al. Application of biotin, digoxigenin or fluorescein conjugated deoxynucleotides to label DNA strand breaks for analysis of cell proliferation and apoptosis using flow cytometry. Biotech. Histochem. 70, 234–242 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. English and B. Joshi for assistance with biological assays; L. Markert for ADA -deficient cells; and C. Harris and D. Brown for helpful discussions and critical review of this manuscript

Author information

Author notes

  1. Kenneth W. Culver

    Present address: Novartis Pharmaceuticals Corp., East Hanover, NJ, 07936

  2. Yuri Khripine

    Present address: Intergen Co., Gaithersburg, MD, 20877

  3. Alexander Khorlin

    Present address: Genelabs Technologies, Inc., Redwood City, CA, 94063

Authors and Affiliations

  1. Codon Pharmaceuticals, Inc., Gaithersburg, 20877, MD

    Wang-Ting Hsieh, Yentram Huyen, Vivian Chen & Jilan Liu

Authors
  1. Kenneth W. Culver
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wang-Ting Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yentram Huyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vivian Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jilan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuri Khripine
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alexander Khorlin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kenneth W. Culver.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Culver, K., Hsieh, WT., Huyen, Y. et al. Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides. Nat Biotechnol 17, 989–993 (1999). https://doi.org/10.1038/13684

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/13684

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing