5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates.
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.
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Rubina
R
Parmar
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Jennifer L S
JL
Willoughby
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Jingxuan
J
Liu
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Donald J
DJ
Foster
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Benjamin
B
Brigham
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Christopher S
CS
Theile
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Klaus
K
Charisse
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Akin
A
Akinc
Merck and Co., Inc, 770 Sumneytown Pike, West Point, PA, 19486, USA.
Erin
E
Guidry
Merck and Co., Inc, 770 Sumneytown Pike, West Point, PA, 19486, USA.
Yi
Y
Pei
Merck and Co., Inc, 770 Sumneytown Pike, West Point, PA, 19486, USA.
Walter
W
Strapps
Merck and Co., Inc, 770 Sumneytown Pike, West Point, PA, 19486, USA.
Mark
M
Cancilla
Merck and Co., Inc, 770 Sumneytown Pike, West Point, PA, 19486, USA.
Matthew G
MG
Stanton
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Kallanthottathil G
KG
Rajeev
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Laura
L
Sepp-Lorenzino
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Muthiah
M
Manoharan
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Rachel
R
Meyers
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA.
Martin A
MA
Maier
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA, 02142, USA. vjadhav@alnylam.com.
Vasant
V
Jadhav
D000116:Acetylgalactosamine / Q000737:chemistry*; D000818:Animals; D001055:Apolipoproteins B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D060565:Argonaute Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D005164:Factor IX / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000097:blood; D051379:Mice; D008810:Mice, Inbred C57BL; D063065:Organophosphonates / Q000737:chemistry* / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014753:Vinyl Compounds / Q000737:chemistry* / Q000494:pharmacology
D001055:Apolipoproteins B; D060565:Argonaute Proteins; C118642:Hexim1 protein, mouse; D008077:Lipoproteins, LDL; D063065:Organophosphonates; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D014157:Transcription Factors; D014753:Vinyl Compounds; C433467:vinylphosphonic acid; D005164:Factor IX; D000116:Acetylgalactosamine
Clp1 kinase; RISC loading; oligonucleotides; phosphate mimic; siRNA