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.

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system

Nature Biotechnology volume 37, pages 884–894 (2019)Cite this article

Subjects

Abstract

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington’s disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Fig. 1: A divalent siRNA chemical configuration enables gene silencing in the mouse brain.
Fig. 2: Di-siRNA efficacy is sustained in mice 6 months after a single bilateral ICV injection.
Fig. 3: Gene silencing in the NHP CNS with di-siRNAs.
Fig. 4: Di-siRNA enables silencing in the spinal cord of NHPs.
Fig. 5: Assessment of brain toxicity 1 month after a single ICV injection of di-siRNA in NHPs.
Fig. 6: RNA-seq shows limited off-target effects of di-siRNA.

Data availability

The RNA-seq data from nonhuman primate samples have been deposited in GEO under accession code GSE130132, including processed transcriptome read counts.

References

  1. Khvorova, A. & Watts, J. K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 35, 238–248 (2017).

    Article  CAS  Google Scholar 

  2. Ostergaard, M. E. et al. Efficient synthesis and biological evaluation of 5’-galnac conjugated antisense oligonucleotides. Bioconjug. Chem. 26, 1451–1455 (2015).

    Article  Google Scholar 

  3. Rajeev, K. G. et al. Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo. Chembiochem. 16, 903–908 (2015).

    Article  CAS  Google Scholar 

  4. Kordasiewicz, H. B. et al. Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis. Neuron. 74, 1031–1044 (2012).

    Article  CAS  Google Scholar 

  5. Finkel, R. S. et al. Nusinersen versus sham control in infantile-onset spinal muscular atrophy. N. Engl. J. Med. 377, 1723–1732 (2017).

    Article  CAS  Google Scholar 

  6. Coelho, T. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 369, 819–829 (2013).

    Article  CAS  Google Scholar 

  7. Alterman, J. F. et al. Hydrophobically modified siRNAs silence huntingtin mRNA in primary neurons and mouse brain. Mol. Ther. Nucleic Acids 4, e266 (2015).

    Article  CAS  Google Scholar 

  8. Nikan, M. et al. Docosahexaenoic acid conjugation enhances distribution and safety of siRNA upon local administration in mouse brain. Mol. Ther. Nucleic Acids 5, e344 (2016).

    Article  CAS  Google Scholar 

  9. Osborn, M. F. & Khvorova, A. Improving small interfering RNA delivery in vivo through lipid conjugation. Nucleic Acid Ther. 28, 128–136 (2018).

    Article  CAS  Google Scholar 

  10. Nikan, M. et al. Synthesis and evaluation of parenchymal retention and efficacy of a metabolically stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA conjugate in mouse brain. Bioconjug. Chem. 28, 1758–1766 (2017).

    Article  CAS  Google Scholar 

  11. Ly, S. et al. Visualization of self-delivering hydrophobically modified siRNA cellular internalization. Nucleic Acids Res. 45, 15–25 (2017).

    Article  CAS  Google Scholar 

  12. Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 24, 374–387 (2014).

    Article  CAS  Google Scholar 

  13. Flierl, U. et al. Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators. J. Exp. Med. 212, 129–137 (2015).

    Article  CAS  Google Scholar 

  14. Sewing, S. et al. Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia. PLoS ONE 12, e0187574 (2017).

    Article  Google Scholar 

  15. Crooke, S. T., Wang, S., Vickers, T. A., Shen, W. & Liang, X. H. Cellular uptake and trafficking of antisense oligonucleotides. Nat. Biotechnol. 35, 230–237 (2017).

    Article  CAS  Google Scholar 

  16. Hassler, M. R. et al. Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo. Nucleic Acids Res. 46, 2185–2196 (2018).

    Article  CAS  Google Scholar 

  17. Behlke, M. A. Progress towards in vivo use of siRNAs. Mol. Ther. 13, 644–670 (2006).

    Article  CAS  Google Scholar 

  18. Winkler, J., Stessl, M., Amartey, J. & Noe, C. R. Off-target effects related to the phosphorothioate modification of nucleic acids. Chem. Med. Chem. 5, 1344–1352 (2010).

    Article  CAS  Google Scholar 

  19. Amarzguioui, M., Holen, T., Babaie, E. & Prydz, H. Tolerance for mutations and chemical modifications in a siRNA. Nucl. Acids Res. 31, 589–595 (2003).

    Article  CAS  Google Scholar 

  20. Harborth, J. et al. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucl. Acid Drug Devel. 13, 83–105 (2003).

    Article  CAS  Google Scholar 

  21. Nair, J. K. et al. Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates. Nucleic Acids Res. 45, 10969–10977 (2017).

    Article  CAS  Google Scholar 

  22. Fitzgerald, K. et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 376, 41–51 (2017).

    Article  CAS  Google Scholar 

  23. Ray, K. K. et al. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 376, 1430–1440 (2017).

    Article  CAS  Google Scholar 

  24. Lee, Y. C. et al. Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features. J. Biol. Chem. 258, 199–202 (1983).

    CAS  PubMed  Google Scholar 

  25. Roehl, I., Schuster, M. & Seiffert, S. US Patent 20110201006 A1 (2011).

  26. Liu, C. C., Liu, C. C., Kanekiyo, T., Xu, H. & Bu, G. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat. Rev. Neurol. 9, 106–118 (2013).

    Article  CAS  Google Scholar 

  27. Mahley, R. W., Weisgraber, K. H. & Huang, Y. Apolipoprotein E4: a causative factor and therapeutic target in neuropathology, including Alzheimer’s disease. Proc. Natl Acad. Sci. USA 103, 5644–5651 (2006).

    Article  CAS  Google Scholar 

  28. Zetterberg, H., Jacobsson, J., Rosengren, L., Blennow, K. & Andersen, P. M. Association of APOE with age at onset of sporadic amyotrophic lateral sclerosis. J. Neurol. Sci. 273, 67–69 (2008).

    Article  CAS  Google Scholar 

  29. Didiot, M. C. et al. Nuclear localization of huntingtin mRNA Is specific to cells of neuronal origin. Cell. Rep. 24, 2553–2560 e2555 (2018).

    Article  CAS  Google Scholar 

  30. Achuta, V. S. et al. Tissue plasminogen activator contributes to alterations of neuronal migration and activity-dependent responses in fragile X mice. J. Neurosci. 34, 1916–1923 (2014).

    Article  CAS  Google Scholar 

  31. Gu, X. et al. N17 Modifies mutant huntingtin nuclear pathogenesis and severity of disease in HD BAC transgenic mice. Neuron 85, 726–741 (2015).

    Article  CAS  Google Scholar 

  32. DiFiglia, M. et al. Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14, 1075–1081 (1995).

    Article  CAS  Google Scholar 

  33. Herndon, E. S. et al. Neuroanatomic profile of polyglutamine immunoreactivity in Huntington disease brains. J. Neuropathol. Exp. Neurol. 68, 250–261 (2009).

    Article  CAS  Google Scholar 

  34. Weyer, A. & Schilling, K. Developmental and cell type-specific expression of the neuronal marker NeuN in the murine cerebellum. J. Neurosci. Res. 73, 400–409 (2003).

    Article  CAS  Google Scholar 

  35. Takala, R. S. et al. Glial fibrillary acidic protein and ubiquitin C-Terminal hydrolase-L1 as outcome predictors in traumatic brain injury. World Neurosurg. 87, 8–20 (2016).

    Article  Google Scholar 

  36. Lind, D., Franken, S., Kappler, J., Jankowski, J. & Schilling, K. Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization. J. Neurosci. Res. 79, 295–302 (2005).

    Article  CAS  Google Scholar 

  37. Dou, C. L., Li, S. & Lai, E. Dual role of brain factor-1 in regulating growth and patterning of the cerebral hemispheres. Cereb. Cortex 9, 543–550 (1999).

    Article  CAS  Google Scholar 

  38. Janas, M. M. et al. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat. Commun. 9, 723 (2018).

    Article  Google Scholar 

  39. Birmingham, A. et al. 3’ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).

    Article  CAS  Google Scholar 

  40. Zuccato, C., Valenza, M. & Cattaneo, E. Molecular mechanisms and potential therapeutical targets in Huntington’s disease. Physiol. Rev. 90, 905–981 (2010).

    Article  CAS  Google Scholar 

  41. Evers, M. M. et al. AAV5-miHTT gene therapy demonstrates broad distribution and strong human mutant huntingtin lowering in a huntington’s disease minipig model. Mol. Ther. 26, 2163–2177 (2018).

    Article  CAS  Google Scholar 

  42. Southwell, A. L. et al. Huntingtin suppression restores cognitive function in a mouse model of Huntington’s disease. Sci. Transl. Med. 10, eaar3959 (2018).

    Article  Google Scholar 

  43. Pfister, E. L. et al. Artificial miRNAs reduce human mutant huntingtin throughout the striatum in a transgenic sheep model of Huntington’s disease. Hum. Gene. Ther. 29, 663–673 (2018).

    Article  CAS  Google Scholar 

  44. Grondin, R. et al. Six-month partial suppression of huntingtin is well tolerated in the adult rhesus striatum. Brain 135, 1197–1209 (2012).

    Article  Google Scholar 

  45. Tabrizi, S. et al. Effects of IONIS-HTTRx in patients with early Huntington’s disease, results of the first HTT-lowering drug trial (CT.002). Neurology 90, CT.002 (2018).

    Google Scholar 

  46. Bhagat, L. et al. Novel oligonucleotides containing two 3’-ends complementary to target mRNA show optimal gene-silencing activity. J. Med. Chem. 54, 3027–3036 (2011).

    Article  CAS  Google Scholar 

  47. Malcolm, D. W., Sorrells, J. E., Van Twisk, D., Thakar, J. & Benoit, D. S. Evaluating side effects of nanoparticle-mediated siRNA delivery to mesenchymal stem cells using next generation sequencing and enrichment analysis. Bioeng. Transl. Med. 1, 193–206 (2016).

    Article  CAS  Google Scholar 

  48. Wang, G., Liu, X., Gaertig, M. A., Li, S. & Li, X. J. Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis. Proc. Natl Acad. Sci. USA 113, 3359–3364 (2016).

    Article  CAS  Google Scholar 

  49. Pfister, E. L. et al. Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington’s disease patients. Curr. Biol. 19, 774–778 (2009).

    Article  CAS  Google Scholar 

  50. Godinho, B. et al. Transvascular delivery of hydrophobically modified siRNAs: Gene silencing in the rat brain upon disruption of the blood-brain barrier. Mol. Ther. 26, 2580–2591 (2018).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This project was funded by the NIH/NINDS (grant no. R01 NS104022; for A.K.), NIH/OD (grant no. S10 OD020012; for A.K.), CHDI (Research Agreement no. A-6119; for N.A.), Alzheimer’s Drug Discovery Foundation (grant no. 20170101; for A.K.), Milton-Safenowitz Fellowship (no. 17-PDF-363; for B.M.D.C.G.) and The Berman–Topper Fund (for A.K. and N.A.). We would like to thank the University of Massachusetts Medical School Animal Medicine Department and veterinary technicians for their contributions to the large-animal studies. We would like to thank M.-C. Didiot for her mouse brain cartoon, E. Mohn and S. Hildebrand for editing the manuscript and Charles River Laboratories for help with neuropathology.

Author information

Author notes

  1. These authors contributed equally: Julia F. Alterman, Bruno M.D.C. Godinho, Matthew R. Hassler, Chantal M. Ferguson.

Authors and Affiliations

  1. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA

    Julia F. Alterman, Bruno M. D. C. Godinho, Matthew R. Hassler, Chantal M. Ferguson, Dimas Echeverria, Reka A. Haraszti, Andrew H. Coles, Faith Conroy, Rachael Miller, Loic Roux, Paul Yan, Emily G. Knox, Anton A. Turanov, Athma A. Pai, Neil Aronin & Anastasia Khvorova

  2. Department of Neurology, Massachusetts General Institute for Neurodegenerative Disease, Boston, MA, USA

    Ellen Sapp & Marian DiFiglia

  3. Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA

    Faith Conroy, Rachael Miller & Neil Aronin

  4. Department of Radiology, New England Center for Stroke Research, University of Massachusetts Medical School, Worcester, MA, USA

    Robert M. King, Heather L. Gray-Edwards & Matthew J. Gounis

  5. Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA

    Robert M. King

  6. Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA

    Gwladys Gernoux, Christian Mueller & Miguel Sena-Esteves

  7. Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA, USA

    Christian Mueller

  8. Department of Neurosurgery, University of Massachusetts Medical School, Worcester, MA, USA

    Richard P. Moser

  9. Department of Animal Medicine, University of Massachusetts Medical School, Worcester, MA, USA

    Nina C. Bishop & Samer M. Jaber

  10. Department of Pathology, University of Massachusetts Medical School, Worcester, MA, USA

    Samer M. Jaber

  11. Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA

    Miguel Sena-Esteves

  12. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA

    Anastasia Khvorova

Authors
  1. Julia F. Alterman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bruno M. D. C. Godinho
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthew R. Hassler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chantal M. Ferguson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dimas Echeverria
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ellen Sapp
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Reka A. Haraszti
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Andrew H. Coles
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Faith Conroy
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Rachael Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Loic Roux
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Paul Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Emily G. Knox
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Anton A. Turanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Robert M. King
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Gwladys Gernoux
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Christian Mueller
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Heather L. Gray-Edwards
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Richard P. Moser
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Nina C. Bishop
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Samer M. Jaber
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Matthew J. Gounis
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Miguel Sena-Esteves
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Athma A. Pai
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Marian DiFiglia
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Neil Aronin
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Anastasia Khvorova
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.F.A., B.M.D.C.G., M.R.H. and A.K. conceived the project. J.F.A., B.M.D.C.G., M.R.H., C.M.F., M.D, N.A. and A.K. contributed to the experimental design. J.F.A., B.M.D.C.G., C.M.F, E.S., C.M.F., R.A.H., A.H.C., F.C., R.M., L.R., P.Y., A.A.T., E.G.K. and A.A.P. contributed experimentally. M.R.H. and D.E. synthesized the compounds. J.F.A., B.M.D.C.G., C.M.F., A.H.C., R.M.K., H.L.G.E., R.P.M., N.C.B., S.M.J., M.J.G. and M.S.E. carried out large-animal studies. G.G. and C.M. provided naive nonhuman primate samples. J.F.A., B.M.D.C.G., M.R.H., C.M.F. and A.K. wrote the manuscript.

Corresponding author

Correspondence to Anastasia Khvorova.

Ethics declarations

Competing interests

A.K., J.F.A., M.R.H. and B.M.D.C.G. have filed a patent application for branched oligonucleotides.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figures 1–21, Supplementary Tables 1 and 2 and Supplementary Notes 1 and 2

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alterman, J.F., Godinho, B.M.D.C., Hassler, M.R. et al. A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system. Nat Biotechnol 37, 884–894 (2019). https://doi.org/10.1038/s41587-019-0205-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-019-0205-0

This article is cited by

Search

Advanced search

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