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.

  • Letter
  • Published:

MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation

An Author Correction to this article was published on 05 October 2018

This article has been updated


Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system1. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules1. Using a mouse model with a heteroplasmic mtDNA mutation2, we tested whether mitochondrial-targeted TALENs (mitoTALENs)3,4 could reduce the mutant mtDNA load in muscle and heart. AAV9-mitoTALEN was administered via intramuscular, intravenous, and intraperitoneal injections. Muscle and heart were efficiently transduced and showed a robust reduction in mutant mtDNA, which was stable over time. The molecular defect, namely a decrease in transfer RNAAla levels, was restored by the treatment. These results showed that mitoTALENs, when expressed in affected tissues, could revert disease-related phenotypes in mice.

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

Access options

Buy this article

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

Fig. 1: Development of a mitoTALEN for the mouse mutant m.5024C>T mtDNA.
Fig. 2: AAV9-MitoTALEN is expressed in skeletal muscle and shifts mtDNA heteroplasmy in a predicted manner.
Fig. 3: AAV9-mitoTALENs induce a significant and persistent shift in mtDNA heteroplasmy in heart and skeletal muscle after systemic injection.

Similar content being viewed by others

Data availability

All the data contained in this manuscript is available to investigators.

Change history

  • 05 October 2018

    In the version of this article originally published, there was an error in Fig. 1a. The m.5024C>T mutation, shown as a green T, was displaced by one base. The error has been corrected in the print, HTML and PDF versions of this article.


  1. Craven, L., Alston, C. L., Taylor, R. W. & Turnbull, D. M. Recent advances in mitochondrial disease. Annu. Rev. Genomics Hum. Genet. 18, 257–275 (2017).

    Article  CAS  Google Scholar 

  2. Kauppila, J. H. et al. A phenotype-driven approach to generate mouse models with pathogenic mtDNA mutations causing mitochondrial disease. Cell Rep. 16, 2980–2990 (2016).

    Article  CAS  Google Scholar 

  3. Bacman, S. R., Williams, S. L., Pinto, M., Peralta, S. & Moraes, C. T. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nat. Med. 19, 1111–1113 (2013).

    Article  CAS  Google Scholar 

  4. Hashimoto, M. et al. MitoTALEN: a general approach to reduce mutant mtDNA Loads and restore oxidative phosphorylation function in mitochondrial diseases. Mol. Ther. 23, 1592–1599 (2015).

    Article  CAS  Google Scholar 

  5. Gammage, P. A. et al. Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs. Nucleic Acids Res. 44, 7804–7816 (2016).

    Article  CAS  Google Scholar 

  6. Lehmann, D. et al. Pathogenic mitochondrial mt-tRNA(Ala) variants are uniquely associated with isolated myopathy. Eur. J. Hum. Genet. 23, 1735–1738 (2015).

    Article  CAS  Google Scholar 

  7. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    Article  CAS  Google Scholar 

  8. Miller, J. C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  Google Scholar 

  9. Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39, 359–372 (2011).

    Article  Google Scholar 

  10. Szymczak, A. L. et al. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat. Biotechnol. 22, 589–594 (2004).

    Article  CAS  Google Scholar 

  11. Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M. & Hoogstraten-Miller, S. Retro-orbital injections in mice. Lab. Anim. (NY) 40, 155–160 (2011).

    Article  Google Scholar 

  12. Srivastava, S. & Moraes, C. T. Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans. Hum. Mol. Genet. 14, 893–902 (2005).

    Article  CAS  Google Scholar 

  13. Fukui, H. & Moraes, C. T. Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons. Hum. Mol. Genet. 18, 1028–1036 (2009).

    Article  CAS  Google Scholar 

  14. Bacman, S. R., Williams, S. L. & Moraes, C. T. Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks. Nucleic Acids Res. 37, 4218–4226 (2009).

    Article  CAS  Google Scholar 

  15. El-Hattab, A. W. & Scaglia, F. Mitochondrial cardiomyopathies. Front. Cardiovasc. Med. 3, 25 (2016).

    Article  Google Scholar 

  16. Kelly, R. D., Mahmud, A., McKenzie, M., Trounce, I. A. & St John, J. C. Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A. Nucleic Acids Res. 40, 10124–10138 (2012).

    Article  CAS  Google Scholar 

  17. Nissanka, N., Bacman, S. R., Plastini, M. J. & Moraes, C. T. The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions. Nat. Commun. 9, 2491 (2018).

    Article  Google Scholar 

  18. Peeva, V. et al. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat. Commun. 9, 1727 (2018).

    Article  Google Scholar 

  19. Gammage, P. A. et al. Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo. Nat. Med. (2018).

    Article  CAS  Google Scholar 

  20. Petruzzella, V. et al. Extremely high levels of mutant mtDNAs co-localize with cytochrome c oxidase-negative ragged-red fibers in patients harboring a point mutation at nt 3243. Hum. Mol. Genet. 3, 449–454 (1994).

    Article  CAS  Google Scholar 

  21. Doyon, Y. et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat. Methods 8, 74–79 (2011).

    Article  CAS  Google Scholar 

  22. Lochmuller, H., Johns, T. & Shoubridge, E. A. Expression of the E6 and E7 genes of human papillomavirus (HPV16) extends the life span of human myoblasts. Exp. Cell Res. 248, 186–193 (1999).

    Article  CAS  Google Scholar 

  23. Lamb, B. M., Mercer, A. C. & Barbas, C. F. 3rd Directed evolution of the TALE N-terminal domain for recognition of all 5’ bases. Nucleic Acids Res. 41, 9779–9785 (2013).

    Article  CAS  Google Scholar 

  24. Bacman, S. R., Williams, S. L., Garcia, S. & Moraes, C. T. Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease. Gene Ther. 17, 713–720 (2010).

    Article  CAS  Google Scholar 

  25. Moraes, C. T., Ricci, E., Bonilla, E., DiMauro, S. & Schon, E. A. The mitochondrial tRNA(Leu(UUR)) mutation in mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS): genetic, biochemical, and morphological correlations in skeletal muscle. Am. J. Hum. Genet. 50, 934–949 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Pinto, M., Nissanka, N. & Moraes, C. T. Lack of parkin anticipates the phenotype and affects mitochondrial morphology and mtDNA levels in a mouse model of Parkinson’s disease. J. Neurosci. 38, 1042–1053 (2018).

    Article  CAS  Google Scholar 

  27. Davies, S. M. et al. MRPS27 is a pentatricopeptide repeat domain protein required for the translation of mitochondrially encoded proteins. FEBS Lett. 586, 3555–3561 (2012).

    Article  CAS  Google Scholar 

Download references


This work was supported primarily by the National Institutes of Health Grant 5R01EY010804, with additional support from 1R01AG036871 and 1R01NS079965 (NIH) and the Muscular Dystrophy Association (CTM). We also acknowledge support from The JDM Fund for Mitochondrial Research and the Biscardi family. N.N. is supported by an American Heart Association predoctoral fellowship (16PRE30480009). We are grateful to the University of Miami Flow Cytometry core facility (SCCC) for expert assistance.

Author information

Authors and Affiliations



S.R.B. was involved with the concept, design and execution of the cell and mouse experiments, and wrote and revised the manuscript. J.H.K.K. characterized the mouse model and the tRNA alanine northern experiments. C.V.P. produced and characterized the cell lines with high levels of the m.5024C>T mtDNA mutation. N.N. performed the experiments with mouse and the detection of mtDNA deletions. M.M. performed the tRNA alanine northern experiments. M.P. prepared the positive controls for mtDNA deletions. S.L.W. was involved with the concept, troubleshooting and recombinant virus administration. N.-G.L. characterized the mouse model. J.B.S. characterized the mouse model and the tRNA alanine northern experiments. C.T.M. was involved with the concept and design, and also wrote and revised the manuscript. All authors read and edited the manuscript.

Corresponding authors

Correspondence to Sandra R. Bacman or Carlos T. Moraes.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bacman, S.R., Kauppila, J.H.K., Pereira, C.V. et al. MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation. Nat Med 24, 1696–1700 (2018).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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