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.
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Castanotto, D. & Rossi, J.J. The promises and pitfalls of RNA-interference-based therapeutics. Nature 457, 426–433 (2009).
Pecot, C.V., Calin, G.A., Coleman, R.L., Lopez-Berestein, G. & Sood, A.K. RNA interference in the clinic: challenges and future directions. Nat. Rev. Cancer 11, 59–67 (2011).
Davidson, B.L. & McCray, P.B. Jr. Current prospects for RNA interference-based therapies. Nat. Rev. Genet. 12, 329–340 (2011).
Behlke, M.A. Chemical modification of siRNAs for in vivo use. Oligonucleotides 18, 305–320 (2008).
Rettig, G.R. & Behlke, M.A. Progress toward in vivo use of siRNAs-II. Mol. Ther. 20, 483–512 (2012).
Bramsen, J.B. & Kjems, J. Chemical modification of small interfering RNA. Methods Mol. Biol. 721, 77–103 (2011).
Meade, B.R. & Dowdy, S.F. The road to therapeutic RNA interference (RNAi): tackling the 800 pound siRNA delivery gorilla. Discov. Med. 8, 253–256 (2009).
Zhou, J., Shum, K.-T., Burnett, J.C. & Rossi, J.J. Nanoparticle-based delivery of RNAi therapeutics: progress and challenges. Pharmaceuticals 6, 85–107 (2013).
Kanasty, R., Dorkin, J.R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977 (2013).
Lipinski, C.A., Lombardo, F., Dominy, B.W. & Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26 (2001).
Joshua-Tor, L. & Hannon, G.J. Ancestral roles of small RNAs: an Ago-centric perspective. Cold Spring Harb. Perspect. Biol. 3, a003772 (2011).
Carthew, R.W. & Sontheimer, E.J. Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655 (2009).
Reese, C.B. Oligo- and poly-nucleotides: 50 years of chemical synthesis. Org. Biomol. Chem. 3, 3851–3868 (2005).
Gates, K.S. An overview of chemical processes that damage the cellular DNA: spontaneous hydrolysis, alkylation, and reactions with radicals. Chem. Res. Toxicol. 22, 1747–1760 (2009).
Grajkowski, A., Wilk, A., Chmielewski, M.K., Phillips, L.R. & Beaucage, S.L. The 2-(N-formyl-N-methyl) aminoethyl group as a potential phosphate/thiophosphate protecting group in solid-phase oligodeoxyribonucleotide synthesis. Org. Lett. 3, 1287–1290 (2001).
Dellinger, D.J., Sheehan, D.M., Christensen, N., Lindberg, J.G. & Caruthers, M.H. Solid phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides. J. Am. Chem. Soc. 125, 940–950 (2003).
Tanabe, K., Ando, Y. & Nishimoto, S. Reversible modification of oligodeoxynucleotides: click reaction at phosphate group and alkali treatment. Tetrahedron Lett. 52, 7135–7137 (2011).
Krishna, H. & Caruthers, M.H. Alkynyl phosphonate DNA: a versatile “click”able backbone for DNA-based biological applications. J. Am. Chem. Soc. 134, 11618–11631 (2012).
Tosquellas, G. et al. The pro-oligonucleotide approach: solid phase synthesis and preliminary evaluation of model pro-dodecathymidylates. Nucleic Acids Res. 26, 2069–2074 (1998).
Périgaud, C. et al. Rational design for cytosolic delivery of nucleoside monophosphates: “SATE” and “DTE” as enzyme-labile transient phosphate protecting groups. Bioorg. Med. Chem. Lett. 3, 2521–2526 (1993).
Lefebvre, I. et al. Mononucleoside phosphotriester derivatives with S-acyl-2-thioethyl bioreversible phosphate-protecting groups: intracellular delivery of 3′-azido-2',3′-dideoxythymidine 5′-monophosphate. J. Med. Chem. 38, 3941–3950 (1995).
Faraj, A. et al. Intracellular metabolism of beta-L-ddAMP-bis(tbutylSATE), a potent inhibitor of hepatitis B virus replication. Nucleosides Nucleotides 18, 987–988 (1999).
Bologna, J.C., Morvan, F. & Imbach, J.L. The prooligonucleotide approach: synthesis of mixed phosphodiester and SATE phosphotriester prooligonucleotides using H-Phosphonate and phosphoramidite chemistries. Eur. J. Org. Chem. 9, 2353–2358 (1999).
Guzaev, A.P., Balow, G. & Manoharan, M. Synthesis of chimerical oligonucleotides containing internucleosidic phosphodiester and S-pivaloyl mercaptoethyl phosphotriester linkages. Nucleosides Nucleotides 18, 1391–1392 (1999).
Peyrottes, S. et al. SATE pronucleotide approaches: an overview. Mini Rev. Med. Chem. 4, 395–408 (2004).
Breslow, R. & Xu, R. Recognition and catalysis in nucleic acid chemistry. Proc. Natl. Acad. Sci. USA 90, 1201–1207 (1993).
Beaucage, S.L. Solid-phase synthesis of siRNA oligonucleotides. Curr. Opin. Drug Discov. Devel. 11, 203–216 (2008).
Wadia, J.S., Stan, R.V. & Dowdy, S.F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315 (2004).
van den Berg, A. & Dowdy, S.F. Protein transduction domain delivery of therapeutic macromolecules. Curr. Opin. Biotechnol. 22, 888–893 (2011).
Robbins, M., Judge, A. & MacLachlan, I. siRNA and innate immunity. Oligonucleotides 19, 89–102 (2009).
Whitehead, K.A., Dahlman, J.E., Langer, R.S. & Anderson, D.G. Silencing or stimulation? siRNA delivery and the immune system. Annu. Rev. Chem. Biomol. Eng. 2, 77–96 (2011).
Judge, A.D. et al. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol. Ther. 13, 494–505 (2006).
Petersen, S. Self-delivering bio-labile phosphate protected pro-oligos for oligonucleotide based therapeutics and mediating RNA interference. USPTO 8,691,971 (2014).
Sliedregt, L.J.A.M. et al. Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor. J. Med. Chem. 42, 609–618 (1999).
Gao, S. et al. The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice. Mol. Ther. 17, 1225–1233 (2009).
Pon, R.T. & Yu, S. Hydroquinone-O,O'-diacetic acid ('Q-linker') as a replacement for succinyl and oxalyl linker arms in solid phase oligonucleotide synthesis. Nucleic Acids Res. 25, 3629–3635 (1997).
Kuijpers, W.H., Huskens, J., Koole, L.H. & van Boeckel, C.A. Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent. Nucleic Acids Res. 18, 5197–5205 (1990).
Eguchi, A. et al. Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nat. Biotechnol. 27, 567–571 (2009).
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.).
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.
About this article
Cite this article
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
Pharmacodynamic and Pharmacokinetic Properties of Full Phosphorothioate Small Interfering RNAs for Gene Silencing In Vivo
Nucleic Acid Therapeutics (2020)
Signal Transduction and Targeted Therapy (2020)
Double Click-Functionalized siRNA Polyplexes for Gene Silencing in Epidermal Growth Factor Receptor-Positive Tumor Cells
ACS Biomaterials Science & Engineering (2020)
Reduction of interstrand charge repulsion of DNA duplexes by salts and by neutral phosphotriesters – Contrary effects for harnessing duplex formation
Journal of the Taiwan Institute of Chemical Engineers (2020)
Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages
Chemistry – A European Journal (2020)