Skip to main content

Thank you for visiting 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.

Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs


Expanded trinucleotide repeats1 cause many neurological diseases. These include Machado-Joseph disease (MJD)2 and Huntington's disease (HD)3, which are caused by expanded CAG repeats within an allele of the ataxin-3 (ATXN3) and huntingtin (HTT) genes, respectively. Silencing expression of these genes is a promising therapeutic strategy, but indiscriminate inhibition of both the mutant and wild-type alleles may lead to toxicity, and allele-specific approaches have required polymorphisms that differ among individuals. We report that peptide nucleic acid and locked nucleic acid antisense oligomers that target CAG repeats can preferentially inhibit mutant ataxin-3 and HTT protein expression in cultured cells. Duplex RNAs were less selective than single-stranded oligomers. The activity of the peptide nucleic acids does not involve inhibition of transcription, and differences in mRNA secondary structure or the number of oligomer binding sites may be important. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length may offer new options for developing treatments for MJD, HD and related hereditary diseases.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: PNAs, LNAs and inhibition of HTT expression by PNA-peptide conjugates.
Figure 2: Effect of PNA modification, LNAs and siRNAs on selectivity.
Figure 3: Selectivity is affected by the number of repeats in mutant HTT.
Figure 4: Potent and selective inhibition of mutant ataxin-3 in GM06151 fibroblasts.

Accession codes




  1. 1

    Orr, H.T. & Zoghbi, H.Y. Trinucleotide repeat disorders. Annu. Rev. Neurosci. 30, 575–621 (2007).

    CAS  Article  Google Scholar 

  2. 2

    Paulson, H.L. Dominantly inherited ataxias: Lessons learned from Machado-Joseph disease/spinocerbeller ataxia type 3. Semin. Neurol. 27, 133–142 (2007).

    Article  Google Scholar 

  3. 3

    Walker, F.O. Huntington's disease. Lancet 369, 218–228 (2007).

    CAS  Google Scholar 

  4. 4

    Gusella, J.F. & MacDonald, M.E. Huntington's disease: seeing the pathogenic process through a genetic lens. Trends Biochem. Sci. 31, 533–540 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Kremer, B. et al. A worldwide study of the Huntington's disease mutation: The sensitivity and specificity of measuring CAG repeats. N. Engl. J. Med. 330, 1401–1406 (1994).

    CAS  Article  Google Scholar 

  6. 6

    Boado, R.J., Kazantsev, A., Apostol, B.L., Thompson, L.M. & Pardridge, W.M. Antisense-mediated down-regulation of the mutant human huntingtin gene. J. Pharmacol. Exp. Ther. 295, 239–243 (2000).

    CAS  PubMed  Google Scholar 

  7. 7

    Harper, S.Q. et al. RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc. Natl. Acad. Sci. USA 102, 5820–5825 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Denovan-Wright, E.M. & Davidson, B.L. RNAi: a potential therapy for the dominantly inherited nucleotide repeat diseases. Gene Ther. 13, 525–531 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Wang, Y.-L. et al. Clinico-pathological rescue of a model mouse of Huntington's disease by siRNA. Neurosci. Res. 53, 241–249 (2005).

    CAS  Article  Google Scholar 

  10. 10

    DiFiglia, M. et al. Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proc. Natl. Acad. Sci. USA 104, 17204–17209 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Miller, V.M. et al. Allele-specific silencing of dominant disease genes. Proc. Natl. Acad. Sci. USA 100, 7195–7200 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Schwarz, D.S. et al. Designing siRNA that distinguish between genes that differ by a single nucleotide. PLoS Genet. 2, e140 (2006).

    Article  Google Scholar 

  13. 13

    Rodriguez-Lebron, E. & Paulson, H.L. Allele-specific RNA interference for neurological disease. Gene Ther. 13, 576–581 (2006).

    CAS  Article  Google Scholar 

  14. 14

    van Bilsen, P.H.J. et al. Identification and allele-specific silencing of the mutant huntingtin allele in Huntington's disease patient-derived fibroblasts. Hum. Gene Ther. 19, 710–718 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Alves, S. et al. Allele-specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model of Machado-Joseph disease. PLoS One 3, e3341 (2008).

    Article  Google Scholar 

  16. 16

    Zhang, Y., Engelman, J. & Friedlander, R.M.J. Allele-specific silencing of mutant Huntington's disease gene. J. Neurochem. 108, 82–90 (2009).

    CAS  Article  Google Scholar 

  17. 17

    De Souza, E.B., Cload, S.T., Pendergrast, P.S. & Sah, D.W. Novel therapeutic modalities to address nondrugable protein interaction targets. Neuropsychopharmacology 34, 142–158 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Nasir, J. et al. Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell 81, 811–823 (1995).

    CAS  Article  Google Scholar 

  19. 19

    White, J.K. et al. Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease CAG expansion. Nat. Genet. 17, 404–410 (1997).

    CAS  Article  Google Scholar 

  20. 20

    Sobczak, K., de Mezer, M., Michlewski, G., Krol, J. & Krzyzosiak, W.J. RNA structure of trinucleotide repeats associated with human neurological diseases. Nucleic Acids Res. 31, 5469–5482 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Marin, V.L. & Armitage, B.A. RNA guanine quadruplex invasion by complementary and homologous PNA probes. J. Am. Chem. Soc. 127, 8032–8033 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Hu, J. & Corey, D.R. Inhibiting gene expression with peptide nucleic acid (PNA)-peptide conjugates that target chromosomal DNA. Biochemistry 46, 7581–7589 (2007).

    CAS  Article  Google Scholar 

  23. 23

    Slow, E.J. et al. Selective striatal neuronal loss in a YAC128 mouse model for Huntington disease. Hum. Mol. Genet. 12, 1555–1567 (2003).

    CAS  Article  Google Scholar 

  24. 24

    Tang, T.-S. Disturbed Ca2+ signaling and apoptosis of medium spiny neurons in Huntington's disease. Proc. Natl. Acad. Sci. USA 102, 2602–2607 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Knudsen, H. & Nielsen, P.E. Antisense properties of duplex- and triplex-forming PNAs. Nucleic Acids Res. 24, 494–500 (1996).

    CAS  Article  Google Scholar 

  26. 26

    Janowski, B.A. et al. Inhibiting transcription of chromosomal DNA with antigene peptide nucleic acids. Nat. Chem. Biol. 1, 210–215 (2005).

    CAS  Article  Google Scholar 

  27. 27

    Corey, D.R. 48,000-fold acceleration of hybridization of chemically modified oligonucleotides to duplex DNA. J. Am. Chem. Soc. 117, 9373–9374 (1995).

    CAS  Article  Google Scholar 

  28. 28

    Koch, T. et al. Locked nucleic acid: Properties and therapeutic aspects. in Therapeutic Oligonucleotides (ed. Kurreck, J.) 103–141, (RSC Biomolecular Sciences, Royal Society of Chemistry, Cambridge, UK, 2008).

    Chapter  Google Scholar 

  29. 29

    Kurreck, J., Wyszko, E., Gillen, C. & Erdmann, V.A. Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res. 30, 1911–1918 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Bumcrot, D., Manoharan, M., Koteliansky, V. & Sah, D.W. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2, 711–719 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Maruyama, H. et al. Molecular features of the CAG repeats and clinical manifestation of Machado-Joseph disease. Hum. Mol. Genet. 4, 807–812 (1995).

    CAS  Article  Google Scholar 

  32. 32

    Smith, R.A. et al. Antisense oligonucleotide therapy for neurodegenerative disease. J. Clin. Invest. 116, 2290–2296 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Wahlestedt, C. et al. Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc. Natl. Acad. Sci. USA 97, 5633–5638 (2000).

    CAS  Article  Google Scholar 

  34. 34

    Corsten, M.F. et al. MicroRNA-21 knockdown disrupts glioma growth in vivo and displays synergistic cytotoxicity with neural precursor cell delivered S-TRAIL in human gliomas. Cancer Res. 67, 8994–9000 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Corey, D.R. RNAi learns from antisense. Nat. Chem. Biol. 3, 8–11 (2007).

    CAS  Article  Google Scholar 

Download references


This work was supported by the High-Q Foundation, the US National Institutes of Health (NIGMS 60642 and 73042 to D.R.C.; NINDS RO1NS056224 to I.B.; NIBIB EB 05556 to J.C.S.), and the Robert A. Welch Foundation (I-1244 and I-1336) and Ataxia MJD research project. We thank B. Janowski for helpful comments and Y. Li for help maintaining the YAC128 mouse colony.

Author information




J.H. and M.M. designed and performed experiments in patient-derived fibroblast cells. J.W. and J.H. designed and performed experiments in MSN cells. K.T.G. and J.C.S assisted with experiments. K.A. and S.G. supplied LNAs. D.R.C. and I.B. supervised experiments.

Corresponding author

Correspondence to David R Corey.

Ethics declarations

Competing interests

D.R.C and J.H. have filed a patent application related to this research. S.G. and K.A. are employed by Sigma-Aldrich.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 Supplementary Tables 1,2 and Supplementary Methods (PDF 6266 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hu, J., Matsui, M., Gagnon, K. et al. Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs. Nat Biotechnol 27, 478–484 (2009).

Download citation

Further reading


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