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

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.

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OTHERS CHEMICAL ENGINEERING OF THERAPEUTIC SIRNAS FOR ALLELE-SPECIFIC GENE SILENCING IN HUNTINGTON’S DISEASE MODELS Article Open access 03 October 2022 DEVELOPMENT OF AN AAV-RNAI STRATEGY TO

SILENCE THE DOMINANT VARIANT _GNAO1_ C.607G>A LINKED TO ENCEPHALOPATHY Article 14 April 2025 THE THERAPEUTIC IMPLICATIONS OF _ALL-IN-ONE_ AAV-DELIVERED EPIGENOME-EDITING PLATFORM IN

NEURODEGENERATIVE DISORDERS Article Open access 23 August 2024 DATA AVAILABILITY The RNA-seq data from nonhuman primate samples have been deposited in GEO under accession code GSE130132,

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

AFFILIATIONS * 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 *

Department of Neurology, Massachusetts General Institute for Neurodegenerative Disease, Boston, MA, USA Ellen Sapp & Marian DiFiglia * Department of Medicine, University of Massachusetts

Medical School, Worcester, MA, USA Faith Conroy, Rachael Miller & Neil Aronin * 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 * Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA Robert

M. King * Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA Gwladys Gernoux, Christian Mueller & Miguel Sena-Esteves * Department of Pediatrics,

University of Massachusetts Medical School, Worcester, MA, USA Christian Mueller * Department of Neurosurgery, University of Massachusetts Medical School, Worcester, MA, USA Richard P. Moser

* Department of Animal Medicine, University of Massachusetts Medical School, Worcester, MA, USA Nina C. Bishop & Samer M. Jaber * Department of Pathology, University of Massachusetts

Medical School, Worcester, MA, USA Samer M. Jaber * Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA Miguel Sena-Esteves * Program in Molecular

Medicine, University of Massachusetts Medical School, Worcester, MA, USA Anastasia Khvorova Authors * Julia F. Alterman View author publications You can also search for this author inPubMed 

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publications You can also search for this author inPubMed 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

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Tables 1 and 2 and Supplementary Notes 1 and 2 REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE 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: 25 October 2018 * Accepted: 27 June 2019 * Published: 02 August 2019 * Issue Date: August 2019 * DOI:

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