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.

  • Article
  • Published:

The deletion of mutant SOD1 via CRISPR/Cas9/sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model

Gene Therapy volume 27, pages 157–169 (2020)Cite this article

Subjects

Abstract

The superoxide dismutase 1 (SOD1) mutation is one of the most notable causes of amyotrophic lateral sclerosis (ALS), and modifying the mutant SOD1 gene is the best approach for the treatment of patients with ALS linked to the mutations in this gene. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9)/sgRNA delivered by the adeno-associated virus (AAV) system is a powerful tool for genome editing in the central nervous system (CNS). Here, we tested the capacity of the AAV-SaCas9-sgRNA system to modify mutant SOD1 in SOD1G93A transgenic mice and found that AAV9-SaCas9-sgRNA5 deleted the SOD1 gene, improved the lifespan of SOD1G93A mice by 54.6%, and notably ameliorated the performance of ALS transgenic mice. An immunochemical analysis showed that the expression of mutant SOD1 was very weak in motor neurons expressing SaCas9-sgRNA5. Consequently, the area showing muscle atrophy was more notably restored in the group treated with SaCas9-sgRNA5 compared with the group treated with SaCas9-sgLacZ. In addition, deep sequencing did not show the indel mutation in the gene highly matched to sgRNA5. Hence, AAV9-SaCas9-sgRNA-based gene editing is a feasible potential treatment for patients with ALS linked to SOD1 mutations.

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

Fig. 1: Activity of SOD1 gene editing in vitro and in vivo.
Fig. 2: The hind-limb stretching evaluation for SOD1G93A mice treated with AAV9-SaCas9-sgRNAs (sg1, sg5, or a combination) and control AAV9-SaCas9-sgLacZ (LacZ).
Fig. 3: Performance analysis of SOD1G93A mice treated with AAV9-SaCas9-sgRNAs (sg1, sg5, or a combination) and control AAV9-SaCas9-sgLacZ (LacZ).
Fig. 4: Motor neuron protection conferred by the administration of SOD1 sgRNAs at a titer of 3 × 1013 vg/mL.
Fig. 5: Therapeutic effect of AAV9-SaCas9-sgRNA5 on SOD1G93A mice.
Fig. 6: Motor neuron protection conferred by AAV9-SaCas9-sgRNA5 (sg5) at the higher titer (1 × 1014 vg/mL).
Fig. 7

Similar content being viewed by others

Data availability

The genome sequence was obtained using an open source collaborative initiative and is available in Ensemble (http://asia.ensembl.org/Homo_sapiens/Info/Index). The CRISPR guide for off-target activities is the result of an open source collaborative initiative and is available in Cas-OFFinder (http://www.rgenome.net/cas-offinder/).

References

  1. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993;362:59–62.

    Article  CAS  Google Scholar 

  2. Taylor JP, Brown RH Jr, Cleveland DW. Decoding ALS: from genes to mechanism. Nature. 2016;539:197–206.

    Article  Google Scholar 

  3. Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science. 1994;264:1772–5.

    Article  CAS  Google Scholar 

  4. Ripps ME, Huntley GW, Hof PR, Morrison JH, Gordon JW. Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis. Proc Natl Acad Sci USA. 1993;92:689–93.

    Article  Google Scholar 

  5. Bruijn LI, Becher MW, Lee MK, Anderson KL, Jenkins NA, Copeland NG, et al. ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron. 1997;18:327–38.

    Article  CAS  Google Scholar 

  6. McCampbell A, Cole T, Wegener AJ, Tomassy GS, Setnicka A, Farley BJ, et al. Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models. J Clin Investig. 2018;128:3558–67.

    Article  Google Scholar 

  7. Miller TM, Pestronk A, David W, Rothstein J, Simpson E, Appel SH, et al. An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomised, first-in-man study. Lancet Neurol. 2013;12:435–42.

    Article  CAS  Google Scholar 

  8. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21.

    Article  CAS  Google Scholar 

  9. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.

    Article  CAS  Google Scholar 

  10. Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33:102–6.

    Article  CAS  Google Scholar 

  11. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520:186–91.

    Article  CAS  Google Scholar 

  12. Gaj T, Ojala DS, Ekman FK, Byrne LC, Limsirichai P, Schaffer DV. In vivo genome editing improves motor function and extends survival in a mouse model of ALS. Sci Adv. 2017;3:eaar3952. https://doi.org/10.1126/sciadv.aar3952.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Alexander GM, Erwin KL, Byers N, Deitch JS, Augelli BJ, Blankenhorn EP, et al. Effect of transgene copy number on survival in the G93A SOD1 transgenic mouse model of ALS. Brain Res Mol Brain Res. 2004;130:7–15.

    Article  CAS  Google Scholar 

  14. Kim JY, Grunke SD, Levites Y, Golde TE, Jankowsky JL. Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. J Vis Exp. 2014;15:51863.

    Google Scholar 

  15. Ralph GS, Radcliffe PA, Day DM, Carthy JM, Leroux MA, Lee DC, et al. Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat Med. 2005;11:429–33.

    Article  CAS  Google Scholar 

  16. Raoul C, Abbas-Terki T, Bensadoun JC, Guillot S, Haase G, Szulc J, et al. Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat Med. 2005;11:423–8.

    Article  CAS  Google Scholar 

  17. Rizvanov AA, Mukhamedyarov MA, Palotás A, Islamov RR. Retrogradely transported siRNA silences human mutant SOD1 in spinal cord motor neurons. Exp Brain Res. 2009;195:1–4.

    Article  CAS  Google Scholar 

  18. Winer L, Srinivasan D, Chun S, Lacomis D, Jaffa M, Fagan A, et al. SOD1 in cerebral spinal fluid as a pharmacodynamic marker for antisense oligonucleotide therapy. JAMA Neurol. 2013;70:201–7.

    Article  Google Scholar 

  19. Foust KD, Nurre E, Montgomery CL, Hernandez A, Chan CM, Kaspar BK. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol. 2009;27:59–65.

    Article  CAS  Google Scholar 

  20. Saunders NR, Joakim EkC, Dziegielewska KM. The neonatal blood-brain barrier is functionally effective, and immaturity does not explain differential targeting of AAV9. Nat Biotechnol. 2009;9:804–5.

    Article  Google Scholar 

  21. Armbruster N, Lattanzi A, Jeavons M, Van Wittenberghe L, Gjata B, Marais T, et al. Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy. Mol Ther Methods Clin Dev. 2016;3:16060.

    Article  Google Scholar 

  22. Tabebordbar M, Zhu K, Cheng JK, Chew WL, Widrick JJ, Yan WX, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016;351:407–11.

    Article  CAS  Google Scholar 

  23. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351:403–7.

    Article  CAS  Google Scholar 

  24. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016;351:400–3.

    Article  CAS  Google Scholar 

  25. Rothstein JD, Martin LJ, Kuncl RW. Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N Engl J Med. 1992;326:1464–8.

    Article  CAS  Google Scholar 

  26. Lin CL, Bristol LA, Jin L, Dykes-Hoberg M, Crawford T, Clawson L, et al. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron. 1998;20:589–602.

    Article  CAS  Google Scholar 

  27. Howland DS, Liu J, She Y, Goad B, Maragakis NJ, Kim B, et al. Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS). Proc Natl Acad Sci USA. 2002;99:1604–9.

    Article  CAS  Google Scholar 

  28. Tong J, Huang C, Bi F, Wu Q, Huang B, Liu X, et al. Expression of ALS-linked TDP-43 mutant in astrocytes causes non-cell-autonomous motor neuron death in rats. EMBO J. 2013;32:1917–26.

    Article  CAS  Google Scholar 

  29. Frakes AE, Ferraiuolo L, Haidet-Phillips AM, Schmelzer L, Braun L, Miranda CJ, et al. Microglia induce motor neuron death via the classical NF-κB pathway in amyotrophic lateral sclerosis. Neuron. 2014;8:1009–23.

    Article  Google Scholar 

  30. Fine SM, Angel RA, Perry SW, Epstein LG, Rothstein JD, Dewhurst S, et al. Tumor necrosis factor alpha inhibits glutamate uptake by primary human astrocytes. Implications for pathogenesis of HIV-1 dementia. J Biol Chem. 1996;271:15303–6.

    Article  CAS  Google Scholar 

  31. Ouali Alami N, Schurr C, Olde Heuvel F, Tang L, Li Q, Tasdogan A, et al. NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS. EMBO J. 2018;37:1–23.

    Article  Google Scholar 

  32. de Belleroche J, Orrell R, King A. Familial amyotrophic lateral sclerosis/motor neurone disease (FALS): a review of current developments. J Med Genet. 1995;32:841–7.

    Article  Google Scholar 

  33. Rogers ML, Smith KS, Matusica D, Fenech M, Hoffman L, Rush RA, et al. Non-viral gene therapy that targets motor neurons in vivo. Front Mol Neurosci. 2014;7:1–12.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Professor Shao-Cong Sun (The University of Texas MD Anderson Cancer Center) for the critical reading and editing of this manuscript.

Funding

This work was supported by grants from the Natural Science Foundation of China (31371089 and 81171210).

Author information

Authors and Affiliations

  1. Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei, PR China

    Weisong Duan, Moran Guo, Le Yi, Yakun Liu, Zhongyao Li, Yaling Liu, Hui Bu, Xueqin Song & Chunyan Li

  2. Neurological Laboratory of Hebei Province, Shijiazhuang, 050000, Hebei, PR China

    Weisong Duan, Moran Guo, Le Yi, Yakun Liu, Zhongyao Li, Yaling Liu, Hui Bu, Xueqin Song & Chunyan Li

  3. Institute of Cardiocerebrovascular Disease, Shijiazhuang, 050000, Hebei, PR China

    Weisong Duan, Zhongyao Li, Yaling Liu, Hui Bu, Xueqin Song & Chunyan Li

  4. Jiangsu Nhwa Pharm. Co., Ltd, Nantong, Jiangsu, PR China

    Yanqin Ma & Guisen Zhang

Authors
  1. Weisong Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Moran Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Le Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yakun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhongyao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yanqin Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Guisen Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yaling Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hui Bu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xueqin Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Chunyan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Xueqin Song or Chunyan Li.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

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

Duan, W., Guo, M., Yi, L. et al. The deletion of mutant SOD1 via CRISPR/Cas9/sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model. Gene Ther 27, 157–169 (2020). https://doi.org/10.1038/s41434-019-0116-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41434-019-0116-1

This article is cited by

Search

Advanced search

Quick links