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:

Session III – Homologous Replacement/Gene Targeting

Modification of hepatic genomic DNA using RNA/DNA oligonucleotides

Gene Therapy volume 9, pages 686–690 (2002)Cite this article

Abstract

The ideal gene therapy is one that repairs the precise genetic defect without additional modification of the genome. Such a strategy has been developed for correcting single nucleotide mutations by using RNA/DNA oligonucleotides, or chimeraplasts. This approach for in situ repair is based on the delivery of exogenous DNA designed to mediate genomic base conversion, insertion, or deletion, thereby, correcting the genetic mutation. Using in vivo delivery systems to hepatocytes via the asialoglycoprotein receptor, we targeted rat liver DNA and successfully modified the genomic sequence by chimeraplasty. The changes in both the hepatic genes, and their associated phenotypes remained stable for 2 years. In addition, we also examined the potential to alter sequence defects in mitochondrial DNA. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeraplast-mediated DNA changes. Using an in vitro DNA repair assay of mutagenized plasmids and an Escherichia coli readout system, we showed that extracts from highly purified rat liver mitochondria have the essential enzymatic activity to mediate precise single-nucleotide changes at a frequency similar to liver nuclear extracts. Moreover, single-stranded oligonucleotides carrying a single nucleotide mismatch with the target sequence were capable of promoting gene conversion using either mitochondrial or nuclear extracts. Several approaches now exist for the precise repair of genetic mutations using either single-stranded or RNA/DNA chimeric oligonucleotides.

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

Similar content being viewed by others

References

  1. Ferber D . Gene therapy: Safer and virus-free Science 2001 294: 1638–1642

    Article  CAS  Google Scholar 

  2. Nishikawa M, Huang L . Nonviral vectors in the new millennium: delivery barriers in gene transfer Hum Gene Ther 2001 12: 861–870

    Article  CAS  Google Scholar 

  3. Wu GY, Wu CH . Receptor-mediated gene delivery and expression in vivo J Biol Chem 1988 263: 14621–14624

    CAS  PubMed  Google Scholar 

  4. Roy Chowdhury N et al. Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis in vivo. Prolonged persistence in cytoplasmic vesicles after partial hepatectomy J Biol Chem 1993 268: 1265–11271

    Google Scholar 

  5. Findeis MA, Wu CH, Wu GY . Ligand-based carrier systems for delivery of DNA to hepatocytes Meths Enzymol 1994 247: 341–351

    Article  CAS  Google Scholar 

  6. Kren BT et al. Oligonucleotide-mediated site-directed gene repair Meth Enzymol 2002 346: 14–31

    Article  CAS  Google Scholar 

  7. Kotani H, Kmiec EB . Transcription activates RecA-promoted homologous pairing of nucleosomal DNA Mol Cell Biol 1994 14: 1949–1955

    Article  CAS  Google Scholar 

  8. Kotani H, Kmiec EB . A role for RNA synthesis in homologous pairing events Mol Cell Biol 1994 14: 6097–6106

    Article  CAS  Google Scholar 

  9. Kmiec EB . Targeted gene repair Gene Therapy 1999 6: 1–3

    Article  CAS  Google Scholar 

  10. Gamper HB Jr. et al. A plausible mechanism for gene correction by chimeric oligonucleotides Biochemistry 2000 39: 5808–5816

    Article  CAS  Google Scholar 

  11. Yoon K, Cole-Strauss A, Kmiec EB . Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide Proc Natl Acad Sci USA 1996 93: 2071–2076

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Kren BT et al. Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide Hepatology 1997 25: 1462–1468

    Article  CAS  Google Scholar 

  14. Kren BT, Bandyopadhyay P, Steer CJ . In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides Nature Med 1998 4: 285–290

    Article  CAS  Google Scholar 

  15. Bandyopadhyay P et al. Enhanced gene transfer into HuH-7 cells and primary rat hepatocytes using targeted liposomes and polyethylenimine BioTechniques 1998 25: 282–292

    Article  CAS  Google Scholar 

  16. Bandyopadhyay P et al. Site-directed nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomal systems and lactosylated polyethylenimine to the asialoglycoprotein receptor J Biol Chem 1999 274: 10163–10172

    Article  CAS  Google Scholar 

  17. Higgins GM, Anderson RM . Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal Arch Pathol 1931 12: 186–202

    Google Scholar 

  18. Higgins GM, Anderson RM . Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal Steer CJ et al. Liver-directed gene therapy. In: Arias IM et al (eds). The Liver: Biology and Pathobiology. 4th edn. Lippincott Williams and Wilkins: New York, 2001, pp 941–962

    Google Scholar 

  19. Kren BT et al. Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler-Najjar syndrome type I with a chimeric oligonucleotide Proc Natl Acad Sci USA 1999 96: 10349–10354

    Article  CAS  Google Scholar 

  20. Iyanagi T, Watanabe T, Uchiyama Y . The 3-methylcholanthrene-inducible UDP-glucuronosyltransferase deficiency in the hyperbilirubinemic rat (Gunn rat) is caused by a -1 frameshift mutation J Biol Chem 1989 264: 21302–21307

    CAS  PubMed  Google Scholar 

  21. Roy-Chowdhury J et al. Molecular basis for the lack of bilirubin-specific and 3-methylcholanthrene-inducible UDP-glucuronosyltransferase activities in Gunn rats. The two isoforms are encoded by distinct mRNA species that share an identical single base deletion J Biol Chem 1991 266: 18294–18298

    CAS  PubMed  Google Scholar 

  22. Brown MD et al. Phylogenetic analysis of Leber's hereditary optic neuropathy mitochondrial DNA's indicates multiple independent occurrences of the common mutations Hum Mutat 1995 6: 311–325

    Article  CAS  Google Scholar 

  23. Hanna MG, Nelson IP . Genetics and molecular pathogenesis of mitochondrial respiratory chain diseases Cell Mol Life Sci 1999 55: 691–706

    Article  CAS  Google Scholar 

  24. Friedberg EC, Walker GC, Siede W . DNA Repair and Mutagenesis ASM Press 1995

    Google Scholar 

  25. Bogenhagen DF . Repair of mtDNA in vertebrates Am J Hum Genet 1999 64: 1276–1281

    Article  CAS  Google Scholar 

  26. Kren BT et al. Gene Repair using RNA/DNA oligonucleotides Semin Liver Dis 1999 19: 93–104

    Article  CAS  Google Scholar 

  27. Cole-Strauss A et al. Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract Nucleic Acids Res 1999 27: 1323–1330

    Article  CAS  Google Scholar 

  28. Rodrigues CM et al. Ursodeoxycholic acid may inhibit deoxycholic acid-induced apoptosis by modulating mitochondrial transmembrane potential and reactive oxygen species production Mol Med 1998 4: 165–178

    Article  CAS  Google Scholar 

  29. Thyagarajan B, Padua RA, Campbell C . Mammalian mitochondria possess homologous DNA recombination activity J Biol Chem 1996 271: 27536–27543

    Article  CAS  Google Scholar 

  30. Chen Z et al. Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair Biochem Biophys Res Commun 2001 285: 188–194

    Article  CAS  Google Scholar 

  31. Thyagaraan B, Cruise JL, Campbell C . Elevated levels of homologous recombination activity in the regenerating rat liver Somatic Cell Mol Genet 1996 22: 31–39

    Article  Google Scholar 

  32. Gamper HB et al. The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts Nucleic Acids Res 2000 28: 4332–4339

    Article  CAS  Google Scholar 

  33. Igoucheva O, Alexeev V, Yoon K . Targeted gene correction by small single-stranded oligonucleotides in mammalian cells Gene Therapy 2001 8: 391–399

    Article  CAS  Google Scholar 

  34. Igoucheva O et al. A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system Gene Therapy 1999 6: 1960–1971

    Article  CAS  Google Scholar 

  35. Tagalakis AD et al. Gene correction of the apolipoprotein (Apo) E2 phenotype to wild-type ApoE3 by in situ chimeraplasty J Biol Chem 2001 276: 13226–13230

    Article  CAS  Google Scholar 

  36. Liu L, Rice MC, Kmiec EB . In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides Nucleic Acids Res 2001 29: 4238–4250

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded in part by National Institutes of Health Grants P01 HD32652 to BTK, RO1 DK39137 to NRC, RO1 DK46057 to JRC, P01 HL65578 and P01 HL55552 to CJS, and ValiGen, Inc. to CJS.

Author information

Author notes

  1. R Felsheim

    Present address: Biological Process Technology Institute, University of Minnesota, Saint Paul, MN 55108, USA

Authors and Affiliations

  1. Department of Medicine, University of Minnesota, Minneapolis, MN, USA

    Z Chen, R Felsheim & C J Steer

  2. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA

    C J Steer

  3. Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, USA

    N Roy Chowdhury & J Roy Chowdhury

  4. Department of Medicine, Mayo Mail Code 36, 420 Delaware Street SE, Minneapolis, 55455, Minnesota, USA

    B T Kren

Authors
  1. B T Kren
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Z Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R Felsheim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N Roy Chowdhury
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J Roy Chowdhury
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C J Steer
    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

Kren, B., Chen, Z., Felsheim, R. et al. Modification of hepatic genomic DNA using RNA/DNA oligonucleotides. Gene Ther 9, 686–690 (2002). https://doi.org/10.1038/sj.gt.3301762

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301762

Keywords

This article is cited by

Search

Advanced search

Quick links