title: Chemical modification study of antisense gapmers.
abstract: A series of insertion patterns for chemically modified nucleotides [2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), methoxyethyl (MOE), locked nucleic acid (LNA), and G-Clamp] within antisense gapmers is studied in vitro and in vivo in the context of the glucocorticoid receptor. Correlation between lipid transfection and unassisted (gymnotic--using no transfection agent) in vitro assays is seen to be dependent on the chemical modification, with the in vivo results corresponding to the unassisted assay in vitro. While in vitro mRNA knockdown assays are typically reasonable predictors of in vivo results, G-Clamp modified antisense oligonucleotides have poor in vivo mRNA knockdown as compared to transfected cell based assays. For LNA gapmers, knockdown is seen to be highly sensitive to the length of the antisense and number of LNA insertions, with longer 5LNA-10DNA-5LNA compounds giving less activity than 3LNA-10DNA-3LNA derivatives. Additionally, the degree of hepatoxicity for antisense gapmers with identical sequences was seen to vary widely with only subtle changes in the chemical modification pattern. While the optimization of knockdown and hepatic effects remains a sequence specific exercise, general trends emerge around preferred physical properties and modification patterns.
authors.affiliation: Oligonucleotide Therapeutic Unit, Pfizer, Cambridge, MA, USA. robert.stanton@pfizer.com
authors.forename: Robert
authors.initials: R
authors.lastname: Stanton
authors.forename: Simone
authors.initials: S
authors.lastname: Sciabola
authors.forename: Christopher
authors.initials: C
authors.lastname: Salatto
authors.forename: Yan
authors.initials: Y
authors.lastname: Weng
authors.forename: Debra
authors.initials: D
authors.lastname: Moshinsky
authors.forename: Jeremy
authors.initials: J
authors.lastname: Little
authors.forename: Evan
authors.initials: E
authors.lastname: Walters
authors.forename: John
authors.initials: J
authors.lastname: Kreeger
authors.forename: Debra
authors.initials: D
authors.lastname: DiMattia
authors.forename: Tracy
authors.initials: T
authors.lastname: Chen
authors.forename: Tracey
authors.initials: T
authors.lastname: Clark
authors.forename: Mei
authors.initials: M
authors.lastname: Liu
authors.forename: Jessie
authors.initials: J
authors.lastname: Qian
authors.forename: Marc
authors.initials: M
authors.lastname: Roy
authors.forename: Robert
authors.initials: R
authors.lastname: Dullea
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D003562:Cytidine / Q000031:analogs & derivatives / Q000737:chemistry; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009841:Oligonucleotides / Q000008:administration & dosage / Q000009:adverse effects / Q000737:chemistry / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000009:adverse effects / Q000737:chemistry / Q000235:genetics*; D010078:Oxazines / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry; D014162:Transfection; D044366:Transition Temperature
chemical_list: C470812:9-(2-aminoethoxy)phenoxazine; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D010078:Oxazines; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; C477371:locked nucleic acid; D006151:Guanosine; D003562:Cytidine; D000241:Adenosine; D013936:Thymidine