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:

A novel gene editing system to treat both Tay–Sachs and Sandhoff diseases

Gene Therapy volume 27, pages 226–236 (2020)Cite this article

Subjects

Abstract

The GM2-gangliosidoses are neurological diseases causing premature death, thus developing effective treatment protocols is urgent. GM2-gangliosidoses result from deficiency of a lysosomal enzyme β-hexosaminidase (Hex) and subsequent accumulation of GM2 gangliosides. Genetic changes in HEXA, encoding the Hex α subunit, or HEXB, encoding the Hex β subunit, causes Tay–Sachs disease and Sandhoff disease, respectively. Previous studies have showed that a modified human Hex µ subunit (HEXM) can treat both Tay–Sachs and Sandhoff diseases by forming a homodimer to degrade GM2 gangliosides. To this end, we applied this HEXM subunit in our PS813 gene editing system to treat neonatal Sandhoff mice. Through AAV delivery of the CRISPR system, a promoterless HEXM cDNA will be integrated into the albumin safe harbor locus, and lysosomal enzyme will be expressed and secreted from edited hepatocytes. 4 months after the i.v. of AAV vectors, plasma MUGS and MUG activities reached up to 144- and 17-fold of wild-type levels (n = 10, p < 0.0001), respectively. More importantly, MUGS and MUG activities in the brain also increased significantly compared with untreated Sandhoff mice (p < 0.001). Further, HPLC-MS/MS analysis showed that GM2 gangliosides in multiple tissues, except the brain, of treated mice were reduced to normal levels. Rotarod analysis showed that coordination and motor memory of treated mice were improved (p < 0.05). Histological analysis of H&E stained tissues showed reduced cellular vacuolation in the brain and liver of treated Sandhoff mice. These results demonstrate the potential of developing a treatment of in vivo genome editing for Tay–Sachs and Sandhoff patients.

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: Construct design and gRNA validation by Surveyor assay.
Fig. 2
Fig. 3
Fig. 4
Fig. 5: Histological analysis showed that cellular vacuolation was reduced in the brain and liver of treated Sandhoff mice.
Fig. 6

Similar content being viewed by others

References

  1. Ou L, Przybilla MJ, Whitley CB. SAAMP 2.0: an algorithm to predict genotype-phenotype correlation of lysosomal storage diseases. Clin Genet. 2018;93:1008–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Ou L, Przybilla MJ, Whitley CB. Phenotype prediction for mucopolysaccharidosis type I by in silico analysis. Orphan J Rare Dis. 2017;12:125.

    Google Scholar 

  3. Ou L, Kim S, Whitley CB, Jarnes-Utz JR. Genotype-phenotype correlation of gangliosidosis mutations using in silico tools and homology modeling. Mol Genet Metab Rep. 2019;20:100495.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Jarnes Utz JR, Kim S, King K, Ziegler R, Schema L, Redtree ES, et al. Infantile gangliosidoses: Mapping a timeline of clinical changes. Mol Genet Metab. 2017;121:170–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Whitley CB, Anderson RA, McIvor RS. Heterozygosity for the DN allele (G533>A) of the β-hexosaminidase α subunit gene identified by direct DNA sequencing in a family with the B1 variant of GM2-gangliosidosis. Neuropediatrics. 1992;23:96–101.

    CAS  PubMed  Google Scholar 

  6. Karumuthil-Melethil S, Nagabhushan Kalburgi S, Thompson P, Tropak M, Kaytor MD, Keimel JG, et al. Novel vector design and hexosaminidase variant enabling self-complementary adeno-associated virus for the treatment of Tay–Sachs disease. Hum Gene Ther. 2016;27:509–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Osmon KJ, Woodley E, Thompson P, Ong K, Karumuthil-Melethil S, Keimel JG, et al. Systemic gene transfer of a hexosaminidase variant using an scAAV9.47 vector corrects GM2 gangliosidosis in Sandhoff mice. Hum Gene Ther. 2016;27:497–508.

    CAS  PubMed  Google Scholar 

  8. Bradbury AM, Cochran JN, McCurdy VJ, Johnson AK, Brunson BL, Gray-Edwards H, et al. Therapeutic response in feline Sandhoff disease despite immunity to intracranial gene therapy. Mol Ther. 2013;21:1306–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Ou L, DeKelver R, Rhode M, Tom S, Radeke R, St Martin SJ, et al. ZFN-mediated in vivo genome editing corrects Murine Hurler Syndrome. Mol Ther. 2019;27:178–87.

    CAS  PubMed  Google Scholar 

  10. Harmatz P, Lau HA, Helderman C, Leslie N, Foo CWP, Vaidya SA, et al. EMPOWERS: a phase 1/2 clinical trial of SB-318 ZFN-mediated in vivo human genome editing for treatment of MPS I (Hurler syndrome). Mol Genet Metab. 2019;126:S68. abstract 147.

    Google Scholar 

  11. Tropak MB, Yonekawa S, Karumuthil-Melethil S, Thompson P, Wakarchuk W, Gray SJ, et al. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo. Mol Ther Methods Clin Dev. 2016;3:15057.

    PubMed  PubMed Central  Google Scholar 

  12. Sango K, Yamanaka S, Hoffmann A, Okuda Y, Grinberg A, Westphal H, et al. Mouse models of Tay–Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism. Nat Genet. 1995;11:170–6.

    CAS  PubMed  Google Scholar 

  13. Aronovich EL, Hall BC, Bell JB, McIvor RS, Hackett PB. Quantitative analysis of α-L-iduronidase expression in immunocompetent mice treated with the Sleeping Beauty transposon system. PLoS ONE. 2013;8:e78161.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang D, El-Amouri SS, Dai M, Kuan CY, Hui DY, Brady RO, et al. Engineering a lysosomal enzyme with a derivative of receptor-binding domain of apoE enables delivery across the blood–brain barrier. Proc Natl Acad Sci USA. 2013;110:2999–3004.

    CAS  PubMed  Google Scholar 

  15. Ou L, Przybilla MJ, Koniar BL, Whitley CB. Elements of lentiviral vector design study toward gene therapy for treating mucopolysaccharidosis I. Mol Genet Metab Rep. 2016;8:93–7.

    Google Scholar 

  16. Przybilla MJ, Ou L, Tăbăran A, Jiang X, Sidhu R, Kell PJ, et al. Comprehensive behavioral and biochemical outcomes of novel murine models of GM1-gangliosidosis and Morquio syndrome type B. Mol Genet Metab. 2019;126:139–50.

    CAS  PubMed  Google Scholar 

  17. Ogawa N, Hirose Y, Ohara S, Ono T, Watanabe Y. A simple quantitative bradykinesia test in MPTP-treated mice. Res Commun Chem Pathol Pharmacol. 1985;50:435–41.

    CAS  PubMed  Google Scholar 

  18. Hockly E, Woodman B, Mahal A, Lewis CM, Bates G. Standardization and statistical approaches to therapeutic trials in the R6/2 mouse. Brain Res Bull. 2003;61:469–79.

    PubMed  Google Scholar 

  19. Martin-Fernandez M, Jamison S, Robin LM, Zhao Z, Martin ED, Aguilar J, et al. Synapse-specific astrocyte gating of amygdala-related behavior. Nat Neurosci. 2017;20:1540–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Sands MS, Davidson BL. Gene therapy for lysosomal storage diseases. Mol Ther. 2006;13:839–49.

    CAS  PubMed  Google Scholar 

  21. Ou L, Herzog T, Koniar BL, Gunther R, Whitley CB. High-dose enzyme replacement therapy in murine Hurler syndrome. Mol Genet Metab. 2014;111:116–22.

    CAS  PubMed  Google Scholar 

  22. Cho SY, Lee J, Ko AR, Kwak MJ, Kim S, Sohn YB, et al. Effect of systemic high dose enzyme replacement therapy on the improvement of CNS defects in a mouse model of mucopolysaccharidosis type II. Orphanet J Rare Dis. 2015;10:141.

    PubMed  PubMed Central  Google Scholar 

  23. Rozaklis T, Beard H, Hassiotis S, Garcia AR, Tonini M, Luck A, et al. Impact of high-dose, chemically modified sulfamidase on pathology in a murine model of MPS IIIA. Exp Neurol. 2011;230:123–30.

    CAS  PubMed  Google Scholar 

  24. Vogler C, Levy B, Grubb JH, Galvin N, Tan Y, Kakkis E, et al. Overcoming the blood-brain barrier with high-dose enzyme replacement therapy in murine mucopolysaccharidosis VII. Proc Natl Acad Sci USA. 2005;102:14777–82.

    CAS  PubMed  Google Scholar 

  25. Lee WC, Courtenay A, Troendle FJ, Stallings-Mann ML, Dickey CA, DeLucia MW, et al. Enzyme replacement therapy results in substantial improvements in early clinical phenotype in a mouse model of globoid cell leukodystrophy. FASEB J. 2005;19:1549–51.

    CAS  PubMed  Google Scholar 

  26. Matzner U, Herbst E, Hedayati KK, Lüllmann-Rauch R, Wessig C, Schröder S, et al. Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy. Hum Mol Genet. 2005;14:1139–52.

    CAS  PubMed  Google Scholar 

  27. Blanz J, Stroobants S, Lüllmann-Rauch R, Morelle W, Lüdemann M, D'Hooge R, et al. Reversal of peripheral and central neural storage and ataxia after recombinant enzyme replacement therapy in alpha-mannosidosis mice. Hum Mol Genet. 2008;17:3437–45.

    CAS  PubMed  Google Scholar 

  28. Dunder U, Kaartinen V, Valtonen P, Väänänen E, Kosma VM, Heisterkamp N, et al. Enzyme replacement therapy in a mouse model of aspartylglycosaminuria. FASEB J. 2000;14:361–7.

    CAS  PubMed  Google Scholar 

  29. Laoharawee K, DeKelver RC, Podetz-Pedersen KM, Rohde M, Sproul S, Nguyen HO, et al. Dose-dependent prevention of metabolic and neurologic disease in murine MPS II by ZFN-mediated in vivo genome editing. Mol Ther. 2018;26:1127–36.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Hu L, Sun Y, Villasana LE, Paylor R, Klann E, Pautler RG. Early changes in the apparent diffusion coefficient (ADC) in a mouse model of Sandhoff's disease occur prior to disease symptoms and behavioral deficits. Magn Reson Med. 2009;62:1175–84.

    PubMed  PubMed Central  Google Scholar 

  31. Aschauer DF, Kreuz S, Rumpel S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS ONE. 2013;8:e76310.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Itakura T, Kuroki A, Ishibashi Y, Tsuji D, Kawashita E, Higashine Y, et al. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice. Biol Pharm Bull. 2006;29:1564–9.

    CAS  PubMed  Google Scholar 

  33. Cachón-González MB, Wang SZ, Lynch A, Ziegler R, Cheng SH, Cox TM. Effective gene therapy in an authentic model of Tay-Sachs-related diseases. Proc Natl Acad Sci USA. 2006;103:10373–8.

    PubMed  Google Scholar 

  34. Kytzia HJ, Sandhoff K. Evidence for two different active sites on human beta-hexosaminidase A. Interaction of GM2 activator protein with beta-hexosaminidase A. J Biol Chem. 1985;260:7568–72.

    CAS  PubMed  Google Scholar 

  35. Tsuji D, Akeboshi H, Matsuoka K, Yasuoka H, Miyasaki E, Kasahara Y, et al. Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis. Ann Neurol. 2011;69:691–701.

    CAS  PubMed  Google Scholar 

  36. Maegawa GH, Banwell BL, Blaser S, Sorge G, Toplak M, Ackerley C, et al. Substrate reduction therapy in juvenile GM2 gangliosidosis. Mol Genet Metab. 2009;98:215–24.

    CAS  PubMed  Google Scholar 

  37. Osher E, Fattal-Valevski A, Sagie L, Urshanski N, Amir-Levi Y, Katzburg S, et al. Pyrimethamine increases β-hexosaminidase A activity in patients with late onset Tay Sachs. Mol Genet Metab. 2011;102:356–63.

    CAS  PubMed  Google Scholar 

  38. Jacobs J, Willemsen M, Groot-Loonen J, Wevers RA, Hoogerbrugge PM. Allogeneic BMT followed by substrate reduction therapy in a child with subacute Tay–Sachs disease. Bone Marrow Transplant. 2005;36:925–6.

    CAS  PubMed  Google Scholar 

  39. Kyrkanides S, Miller JH, Brouxhon SM, Olschowka JA, Federoff HJ. beta-hexosaminidase lentiviral vectors: transfer into the CNS via systemic administration. Brain Res Mol Brain Res. 2005;133:286–98.

    CAS  PubMed  Google Scholar 

  40. Cachón-González MB, Wang SZ, McNair R, Bradley J, Lunn D, Ziegler R, et al. Gene transfer corrects acute GM2 gangliosidosis-potential therapeutic contribution of perivascular enzyme flow. Mol Ther. 2012;20:1489–500.

    PubMed  PubMed Central  Google Scholar 

  41. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415–9.

    CAS  PubMed  Google Scholar 

  42. Nakai H, Yant SR, Storm TA, Fuess S, Meuse L, Kay MA. Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol. 2001;75:6969–76.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Calcedo R, Wilson JM. Humoral Immune Response to AAV. Front Immunol. 2013;4:341.

    PubMed  PubMed Central  Google Scholar 

  44. Walia JS, Altaleb N, Bello A, Kruck C, LaFave MC, Varshney GK, et al. Long-term correction of Sandhoff disease following intravenous delivery of rAAV9 to mouse neonates. Mol Ther. 2015;23:414–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Maeder ML, Stefanidakis M, Wilson CJ, Baral R, Barrera LA, Bounoutas GS, et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med. 2019;25:229–33.

    CAS  PubMed  Google Scholar 

  46. Zuo E, Sun Y, Wei W, Yuan T, Ying W, Sun H, et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science. 2019;364:289–92.

    CAS  PubMed  Google Scholar 

  47. Ou L, Przybilla MJ, Whitley CB. Metabolomics profiling reveals profound metabolic impairments in mice and patients with Sandhoff disease. Mol Genet Metab. 2019;126:151–6.

    CAS  PubMed  Google Scholar 

  48. Passini MA, Lee EB, Heuer GG, Wolfe JH. Distribution of a lysosomal enzyme in the adult brain by axonal transport and by cells of the rostral migratory stream. J Neurosci. 2002;22:6437–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Golebiowski D, van der Bom IM, Kwon CS, Miller AD, Petrosky K, Bradbury AM, et al. Direct intracranial injection of AAVrh8 encoding monkey β-N-acetylhexosaminidase causes neurotoxicity in primate brain. Hum Gene Ther. 2017;28:510–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Dogbevia G, Grasshoff H, Othman A, Penno A, Schwaninger M. Brain endothelial specific gene therapy improves experimental Sandhoff disease. J Cereb Blood Flow Metab. 2019:271678X19865917. https://doi.org/10.1177/0271678X19865917.

    Google Scholar 

  51. Ou L, Przybilla MJ, Koniar B, Whitley CB. RTB lectin-mediated delivery of lysosomal α-l-iduronidase mitigates disease manifestations systemically including the central nervous system. Mol Genet Metab. 2018;123:105–11.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Dr Michael Benneyworth, Mouse Behavior Core, University of Minnesota for technical assistance in behavior tests.

Funding

LO is a fellow of the Lysosomal Disease Network (U54NS065768). The Lysosomal Disease Network is a part of the Rare Diseases Clinical Research Network (RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), and NCATS. This consortium is funded through a collaboration between NCATS, the National Institute of Neurological Disorders and Stroke (NINDS), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

Author information

Authors and Affiliations

  1. Gene Therapy Center, Department of Pediatrics, University of Minnesota, Minneapolis, MN, 55455, USA

    Li Ou & Chester B. Whitley

  2. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA

    Michael J. Przybilla & Chester B. Whitley

  3. Comparative Pathology Shared Resource, University of Minnesota Masonic Cancer Center, Saint Paul, MN, 55108, USA

    Alexandru-Flaviu Tăbăran, Paula Overn & M. Gerard O’Sullivan

  4. Department of Medicine, Washington University School of Medicine, Saint Louis, MO, USA

    Xuntian Jiang, Rohini Sidhu, Pamela J. Kell & Daniel S. Ory

Authors
  1. Li Ou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael J. Przybilla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexandru-Flaviu Tăbăran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paula Overn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Gerard O’Sullivan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xuntian Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rohini Sidhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pamela J. Kell
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Daniel S. Ory
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Chester B. Whitley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Li Ou.

Ethics declarations

Conflict of interest

LO and CBW are inventors of a pending patent (PCT/US2018/065747) based on the PS gene editing system. All other authors declare no conflict of interest.

Additional information

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ou, L., Przybilla, M.J., Tăbăran, AF. et al. A novel gene editing system to treat both Tay–Sachs and Sandhoff diseases. Gene Ther 27, 226–236 (2020). https://doi.org/10.1038/s41434-019-0120-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41434-019-0120-5

This article is cited by

Search

Advanced search

Quick links