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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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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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.
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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