5′‐(E)‐Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA–GalNAc Conjugates

Abstract

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.

Preclinical and Clinical Advances of GalNAc-Decorated Nucleic Acid Therapeutics

“…Parmar et al. (Alnylam team) 61 also demonstrated that incorporation of (E) -5′-VP results in up to 20-fold-improved in vitro potency and up to a 3-fold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.…”

“…This nucleotide can be selected from DNA, RNA, locked nucleic acid (LNA), 2′-F, or 2′-F-5′-methyl 60 . (3) It may comprise a 5′-vinyl phosphonate (5′-VP) because this phosphate mimic can enhance the potency of synthetic siRNA 61, 62. (4) Strategies regarding the sequence length and structure of siRNA, blocks of phosphorothioate internucleotide linkages, and AU pairing at the 5′ end of the antisense strand are similar to the abovementioned principles.…”

Oligonucleotides targeting coagulation factor mRNAs: use in thrombosis and hemophilia research and therapy

“…Thus far, the delivery of oligonucleotides to the liver in mammals is an area where significant progress has been made: siRNAs can be packaged in liposomes with a high tropism for the liver, and they can be conjugated to specific ligands (the so called- N-acetylgalactosamine (GalNAC) ligands, which allows the uptake via hepatocyte-specific asialoglycoprotein receptors) which mediate receptor-mediated take-up by hepatocytes [14–17]. A liposome-based formulation and hepatic delivery has also been described for other oligonucleotides [18].…”

“…Currently, a human-specific antithrombin siRNA named Fitusiran is being tested in phase 1 clinical trials. This siRNA, amongst others candidate siRNAs which are tested in clinical trials, is chemically modified to improve its systemic stability and is conjugated to a GalNAc-ligand, which allows hepatocyte-specific uptake [17]. The phase 1 clinical trial is done in a diverse group of individuals, including healthy volunteers, patients with hemophilia A or B, and with or without inhibitors against supplemented recombinant proteins [74].…”

Gene Manipulation with Micro RNAs at Single-Human Cancer Cell

“…Consistent with the biochemistry of the slicer function, the PIWI domain of Argonaut has been shown to resemble a RNase H enzyme. A binding site in the Mid domain anchors the phosphate backbone of the guide strand at the 5′ end [4], whereas the 3′ end attaches to the PAZ domain [5]. A highly conserved DEDH catalytic tetramer in the PIWI active site is the critical slicing component in Argonaut [6, 7].…”