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.

  • Original Article
  • Published:

Meganucleases can restore the reading frame of a mutated dystrophin

Abstract

Mutations in Duchenne muscular dystrophy (DMD) are either inducing a nonsense codon or a frameshift. Meganucleases (MGNs) can be engineered to induce double-strand breaks (DSBs) at specific DNA sequences. These breaks are repaired by homologous recombination or by non-homologous end joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. To verify whether MGNs could be used to restore the normal reading frame of a dystrophin gene with a frameshift mutation, we inserted in a plasmid coding for the dog μ-dystrophin sequences containing a MGN target. The number of base pairs in these inserted sequences changed the reading frame. One of these modified target μ-dystrophin plasmids and an appropriate MGN were then transfected in 293FT cells. The MGN induced micro-deletion or micro-insertion in the μ-dystrophin that restored dystrophin expression. MGNs also restored μ-dystrophin expression in myoblasts in vitro and in muscle fibers in vivo. The mutation of the targeted μ-dystrophin was confirmed by PCR amplification followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons. These experiments are thus a proof of principle that MGNs that are adequately engineered to target appropriate sequences in the human dystrophin gene should be able to restore the normal reading frame of that gene in DMD patients with an out-of-frame deletion. New MGNs engineered to target a sequence including or near nonsense mutation could also be used to delete it.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Hoffman EP, Brown Jr RH, Kunkel LM . Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987; 51: 919–928.

    Article  CAS  Google Scholar 

  2. Trimarco A, Torella A, Piluso G, Maria Ventriglia V, Politano L, Nigro V . Log-PCR: a new tool for immediate and cost-effective diagnosis of up to 85% of dystrophin gene mutations. Clin Chem 2008; 54: 973–981.

    Article  CAS  Google Scholar 

  3. England SB, Nicholson LV, Johnson MA, Forrest SM, Love DR, Zubrzycka-Gaarn EE et al. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature 1990; 343: 180–182.

    Article  CAS  Google Scholar 

  4. Ohshima S, Shin J-H, Yuasa K, Nishiyama A, Kira J, Okada T et al. Transduction efficiency and immune response associated with the administration of AAV8 vector into dog skeletal muscle. Mol Ther 2009; 17: 73–80.

    Article  CAS  Google Scholar 

  5. Liu M, Yue Y, Harper SQ, Grange RW, Chamberlain JS, Duan D . Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury. Mol Ther 2005; 11: 245–256.

    Article  CAS  Google Scholar 

  6. Lai Y, Yue Y, Liu M, Duan D . Synthetic intron improves transduction efficiency of trans-splicing adeno-associated viral vectors. Hum Gene Ther 2006; 17: 1036–1042.

    Article  CAS  Google Scholar 

  7. Wang Z, Kuhr CS, Allen JM, Blankinship M, Gregorevic P, Chamberlain JS et al. Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression. Mol Ther 2007; 15: 1160–1166.

    Article  CAS  Google Scholar 

  8. Odom GL, Gregorevic P, Allen JM, Finn E, Chamberlain JS . Microutrophin delivery through rAAV6 increases lifespan and improves muscle function in dystrophic dystrophin/utrophin-deficient mice. Mol Ther 2008; 16: 1539–1545.

    Article  CAS  Google Scholar 

  9. Harper SQ, Hauser MA, DelloRusso C, Duan D, Crawford RW, Phelps SF et al. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat Med 2002; 8: 253–261.

    Article  CAS  Google Scholar 

  10. Peault B, Rudnicki M, Torrente Y, Cossu G, Tremblay JP, Partridge T et al. Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Mol Ther 2007; 15: 867–877.

    Article  CAS  Google Scholar 

  11. Deasy BM, Jankowski RJ, Huard J . Muscle-derived stem cells: characterization and potential for cell- mediated therapy. Blood Cells Mol Dis 2001; 27: 924–933.

    Article  CAS  Google Scholar 

  12. Ikemoto M, Fukada S-I, Uezumi A, Masuda S, Miyoshi H, Yamamoto H et al. Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice. Mol Ther 2007; 15: 2178–2185.

    Article  CAS  Google Scholar 

  13. Sampaolesi M, Blot S, D’Antona G, Granger N, Tonlorenzi R, Innocenzi A et al. Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 2006; 444: 574–579.

    Article  CAS  Google Scholar 

  14. Skuk D, Goulet M, Roy B, Chapdelaine P, Bouchard J-P, Roy R et al. Dystrophin expression in muscles of Duchenne muscular dystrophy patients after high-density injections of normal myogenic cells. J Neuropathol Exp Neurol 2006; 65: 371–386.

    Article  CAS  Google Scholar 

  15. Skuk D, Goulet M, Roy B, Piette V, Cote CH, Chapdelaine P et al. First test of a ‘high-density injection’ protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen months follow-up. Neuromuscul Disord 2007; 17: 38–46.

    Article  Google Scholar 

  16. Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007; 447: 87–91.

    Article  CAS  Google Scholar 

  17. Wilton S . PTC124, nonsense mutations and Duchenne muscular dystrophy. Neuromuscul Disord 2007; 17: 719–720.

    Article  Google Scholar 

  18. Williams JH, Schray RC, Sirsi SR, Lutz GJ . Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice. BMC Biotechnol 2008; 8: 35.

    Article  Google Scholar 

  19. Jearawiriyapaisarn N, Moulton HM, Buckley B, Roberts J, Sazani P, Fucharoen S et al. Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. Mol Ther 2008; 16: 1624–1629.

    Article  CAS  Google Scholar 

  20. Yokota T, Duddy W, Partridge T . Optimizing exon skipping therapies for DMD. Acta Myol 2007; 26: 179–184.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N et al. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res 2009; 37: 5405–5419.

    Article  CAS  Google Scholar 

  22. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee Y-L, Kim KA et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 2007; 25: 1298–1306.

    Article  CAS  Google Scholar 

  23. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005; 435: 646–651.

    Article  CAS  Google Scholar 

  24. Stoddard BL . Homing endonuclease structure and function. Q Rev Biophys 2005; 38: 49–95.

    Article  CAS  Google Scholar 

  25. Choulika A, Perrin A, Dujon B, Nicolas JF . Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol 1995; 15: 1968–1973.

    Article  CAS  Google Scholar 

  26. Rouet P, Smih F, Jasin M . Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol 1994; 14: 8096–8106.

    Article  CAS  Google Scholar 

  27. Paques F, Duchateau P . Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr Gene Ther 2007; 7: 49–66.

    Article  CAS  Google Scholar 

  28. Boothroyd CE, Dreesen O, Leonova T, Ly KI, Figueiredo LM, Cross GA et al. A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei. Nature 2009; 459: 278–281.

    Article  CAS  Google Scholar 

  29. Paques F, Haber JE . Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 1999; 63: 349–404.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Doyon JB, Pattanayak V, Meyer CB, Liu DR . Directed evolution and substrate specificity profile of homing endonuclease I-SceI. J Am Chem Soc 2006; 128: 2477–2484.

    Article  CAS  Google Scholar 

  31. Ashworth J, Havranek JJ, Duarte CM, Sussman D, Monnat Jr RJ, Stoddard BL et al. Computational redesign of endonuclease DNA binding and cleavage specificity. Nature 2006; 441: 656–659.

    Article  CAS  Google Scholar 

  32. Arnould S, Chames P, Perez C, Lacroix E, Duclert A, Epinat J-C et al. Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. J Mol Biol 2006; 355: 443–458.

    Article  CAS  Google Scholar 

  33. Smith J, Grizot S, Arnould S, Duclert A, Epinat JC, Chames P et al. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res 2006; 34: e149.

    Article  Google Scholar 

  34. Redondo P, Prieto J, Munoz IG, Alibes A, Stricher F, Serrano L et al. Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 2008; 456: 107–111.

    Article  CAS  Google Scholar 

  35. Arnould S, Perez C, Cabaniols J-P, Smith J, Gouble A, Grizot S et al. Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol 2007; 371: 49–65.

    Article  CAS  Google Scholar 

  36. Kinoshita I, Vilquin JT, Asselin I, Chamberlain J, Tremblay JP . Transplantation of myoblasts from a transgenic mouse overexpressing dystrophin produced only a relatively small increase of dystrophin-positive membrane. Muscle Nerve 1998; 21: 91–103.

    Article  CAS  Google Scholar 

  37. Brunet E, Simsek D, Tomishima M, DeKelver R, Choi VM, Gregory P et al. Chromosomal translocations induced at specified loci in human stem cells. Proc Natl Acad Sci USA 2009; 106: 10620–10625.

    Article  CAS  Google Scholar 

  38. Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM . Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J Biol Chem 2000; 275: 9390–9395.

    Article  CAS  Google Scholar 

  39. Galetto R, Duchateau P, Paques F . Targeted approaches for gene therapy and the emergence of engineered meganucleases. Expert Opin Biol Ther 2009; 9: 1289–1303.

    Article  CAS  Google Scholar 

  40. Wang B, Li J, Qiao C, Chen C, Hu P, Zhu X et al. A canine minidystrophin is functional and therapeutic in mdx mice. Gene Therapy 2008; 15: 1099–1106.

    Article  CAS  Google Scholar 

  41. Chapdelaine P, Vignola K, Fortier MA . Protein estimation directly from SDS-PAGE loading buffer for standardization of samples from cell lysates or tissue homogenates before Western blot analysis. Biotechniques 2001; 31: 478–480, 482.

    Article  CAS  Google Scholar 

  42. Chapdelaine P, Delahaye S, Gauthier E, Tremblay RR, Dube JY . A one-hour procedure for the preparation of genomic DNA from frozen tissues. Biotechniques 1993; 14: 163–164.

    CAS  PubMed  Google Scholar 

  43. Quenneville SP, Chapdelaine P, Rousseau J, Beaulieu J, Caron NJ, Skuk D et al. Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts. Mol Ther 2004; 10: 679–687.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to J P Tremblay.

Ethics declarations

Competing interests

Dr Pâques is the scientific director of Cellectis Inc. Thus, his salary is obtained from that company and he has shares in that company. The project in the Tremblay laboratory was supported by a grant from the Canadian Institute of Health Research. A patent based on the results described in this paper was applied for by Cellectis Inc.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chapdelaine, P., Pichavant, C., Rousseau, J. et al. Meganucleases can restore the reading frame of a mutated dystrophin. Gene Ther 17, 846–858 (2010). https://doi.org/10.1038/gt.2010.26

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2010.26

Keywords

This article is cited by

Search

Quick links