Abstract
RNA interference (RNAi) has great potential to treat human disease1,2,3. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle4,5,6,7,8,9. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates9. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.
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Acknowledgements
We thank Y. Tor (UCSD) for critical input, Y. Su (UCSD) for help with mass spectrometry, and Z. Rudolph and L.D.F. Vasconcelos for technical assistance. B.R.M., A.S.H. and A.D.S. were supported by a T32 Cancer Biology Training grant (NCI); A.S.H. was also supported by a Blasker Award from The San Diego Foundation; A.P. was supported by a C.I.R.M. Fellowship; C.P.-A. was supported by fellowships from Knut and Alice Wallenberg Foundation and Sweden-America Foundation; J.C.H. was supported by the CT2 training grant (N.C.I.); P.L. was supported by a Swedish Research Council grant; A.D.K. was supported by a IRACDA Training grant (N.I.H.). This work was supported by the W.M. Keck Foundation (S.F.D.), the Department of Defense (S.F.D.), SCOR grant from the Leukemia & Lymphoma Society (S.F.D.), the Pardee Foundation (S.F.D.), a grant from an anonymous donor (S.F.D.) and the Howard Hughes Medical Institute (S.F.D.).
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B.R.M. designed and performed experiments, analyzed and interpreted data, synthesized RNN oligonucleotides; K.G. designed and synthesized phosphotriesters, phosphoramidites, conjugating approaches, and analyzed and interpreted data; A.S.H. performed in vivo experiments, analyzed and interpreted data, and synthesized RNN oligonucleotides; C.P.-A. and A.D.S. performed experiments, analyzed and interpreted data, and synthesized RNN oligonucleotides; A.v.d.B., J.C.H., A.E., A.D.K. A.P., P.L. and M.K. performed experiments and analyzed data; C.F.D. synthesized RNN oligonucleotides; N.Y. and E.G. helped with data analysis; X.-S.C. synthesized peptides; S.F.D. conceived of siRNN concept, designed experiments, and analyzed and interpreted data. B.R.M., K.G. and S.F.D. wrote the manuscript, and all authors refined the manuscript.
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The authors declare competing financial interests. S.F.D., B.R.M. and K.G. (UCSD) have filed patents on this work that were licensed by Solstice Biologics, Inc. (San Diego). S.F.D. and B.R.M. are cofounders of Solstice Biologics. S.F.D. is a board director of Solstice Biologics.
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Meade, B., Gogoi, K., Hamil, A. et al. Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. Nat Biotechnol 32, 1256–1261 (2014). https://doi.org/10.1038/nbt.3078
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DOI: https://doi.org/10.1038/nbt.3078
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