title: Identification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activity.
abstract: The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog. In this report we used crystal structure of the 5'-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5'-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA tprakash@isisph.com.
authors.forename: Thazha P
authors.initials: TP
authors.lastname: Prakash
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Walt F
authors.initials: WF
authors.lastname: Lima
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Murray
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Wenyu
authors.initials: W
authors.lastname: Li
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Garth A
authors.initials: GA
authors.lastname: Kinberger
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Alfred E
authors.initials: AE
authors.lastname: Chappell
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Hans
authors.initials: H
authors.lastname: Gaus
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Punit P
authors.initials: PP
authors.lastname: Seth
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Balkrishen
authors.initials: B
authors.lastname: Bhat
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
authors.affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Eric E
authors.initials: EE
authors.lastname: Swayze
mesh_terms: D000818:Animals; D053305:Apolipoprotein C-III / Q000235:genetics / Q000378:metabolism; D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D008078:Cholesterol, LDL / Q000097:blood; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry; D054730:Protein Interaction Domains and Motifs; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014280:Triglycerides / Q000097:blood
chemical_list: C466422:AGO2 protein, human; D053305:Apolipoprotein C-III; D060565:Argonaute Proteins; D008078:Cholesterol, LDL; D010710:Phosphates; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D014280:Triglycerides