Abstract
Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2′-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer’s disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.
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All datasets generated and analyzed in the study are provided within the manuscript files. Source data are provided with this paper.
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Acknowledgements
The authors thank Alnylam’s Medicinal Chemistry Team, Medium Scale Synthesis Team, High Throughput Synthesis Team and Duplex Annealing Team for siRNA synthesis. They also thank A. Bisbe, P. Miller and R. Malone for siRNA purification; C. Wong, S. Maczko, M. Mendez, I. Duarte and D. Rooney for in vivo study support; A. Guan for tissue processing; J. Varao for sample management; B. Fitzsimmons for tissue drug concentration analysis; B. Carito, P. Gedman, C.-E. Stephany and K. Bittner for histology support; Alnylam’s Research Analytical Team for metabolic stability studies; and the research team at CRL Finland, especially J. Puolivali and F. Khan.
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K.M.B., J.K.N., M.M.J., S.M., M.K., C.B, D.F., K.F., M.A.M. and V.J. conceived the projects. K.M.B., J.K.N., M.M.J., S.M., Y.I.A.-R., L.T.H.D., H.P., C.S.T., E.C.-R., C.B., D.F., J.K., J.A., A.C., M.K.S., I.Z., M.K., M.A.M. and V.J. contributed to experimental design. L.T.H.D., H.P., C.S.T., E.C.-R., C.B., D.F., J.K., J.A., R.M., J.L., S.M., M.S., T.C., M.J., K.W., J.R., L.W., A.K., D.G., M.W.M., J.P., R.M., T.R., J.B., D.C., S.A., L.J., Y.J., S.L., J.G., T.N., S.C., S.L.B, U.P., A.K., A.B.R. and S.C. contributed experimentally. R.M., J.E.S. and W.D. provided non-human primate evaluations. C.S.T., T.C., K.W., J.R. and L.W. synthesized the duplexes. K.M.B., J.K.N., M.M.J., Y.I.A.-R., L.T.H.D., M.M., K.F., J.-T.W., M.A.M. and V.J. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Histopathological evaluation of the nonhuman primate (NHP) spinal cord at three months following a single IT dose of 60 mg C16-siRNA.
Representative H&E (a), LFB (b) and IBA-1 (c) staining of NHP thoracic spinal cord. Microscopic findings in the spinal cord were limited to procedure-related changes (that is, IT bolus administration in lumbar region). These consisted of minimal to mild degeneration of nerve fibers with dilated axonal sheaths and few digestion chambers containing gitter cells (arrowheads) that were localized to the white matter of spinal cord (cervical, thoracic, and lumbar segments). Limited to procedure-affected areas, there were minimal decreases in LFB staining intensity (arrows) and a minimal increase in the number of IBA-1 (brown chromogen) positive microglia (open arrows). Three animals were analyzed per group with similar results. GM = grey matter; WM = white matter.
Extended Data Fig. 2 Histopathological evaluation of the nonhuman primate (NHP) brain at three months following a single IT dose of 60 mg C16-siRNA.
Representative H&E (a), LFB (b) and IBA-1 (c) staining of NHP brainstem (medulla oblongata region adjacent to aqueduct and underlying pons). Microscopic findings in the brain were limited to procedure-related changes (that is, CSF collection from cisterna magna). These consisted of minimal to mild degeneration of nerve fibers with dilated axonal sheaths and few digestion chambers containing gitter cells (arrowheads) that were localized to the peripheral white matter areas. Limited to procedure-affected areas, there were minimal decreases in LFB staining intensity (arrows) and a minimal increase in the number of IBA-1 (purple chromogen) positive microglia (open arrows). Three animals were analyzed per group with similar results.
Extended Data Fig. 3 Histopathological evaluation of the nonhuman primate (NHP) liver at three months following a single IT dose of 60 mg C16-siRNA.
Representative H&E staining of NHP liver (H = hepatocyte; S = sinusoid; KC = Kupffer cell). There were no remarkable microscopic findings. Three animals were analyzed per group with similar results.
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Brown, K.M., Nair, J.K., Janas, M.M. et al. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates. Nat Biotechnol 40, 1500–1508 (2022). https://doi.org/10.1038/s41587-022-01334-x
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DOI: https://doi.org/10.1038/s41587-022-01334-x
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