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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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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.

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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.

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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.

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Correspondence to Anastasia Khvorova.

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

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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

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