title: Evaluation of C-5 propynyl pyrimidine-containing oligonucleotides in vitro and in vivo.
abstract: Inclusion of C-5 propynyl pyrimidines in phosphorothioate antisense oligonucleotides (ASOs) has been shown to significantly increase their potency for inhibiting gene expression in vitro. This increased potency is believed to be the result of enhanced binding affinity to target RNA. Our results show that C-5 propynyl pyrimidine-modified oligonucleotides caused an increase in the melting temperature (T(m)) of both oligodeoxynucleotides (ODNs) and 2'-O-(2-methoxy)ethyl (2'-MOE)-modified oligonucleotides. The in vitro data show a moderate increase in potency for an antisense oligodeoxynucleotide containing C-5 propynyl pyrimidines targeting the murine PTEN (MMAC1) transcript. Second-generation 2'-MOE chimeric ASOs containing C-5 propynyl pyrimidines showed no improvement in potency in PTEN target reduction in vitro or in vivo compared to their nonpropyne-modified parent. These results suggest that increasing affinity for target RNA beyond that achieved with the 2'-MOE modification does not further increase potency in cell-based assays. To evaluate whether this observation held true for in vivo applications, we evaluated both compounds in mice. We were unable to establish a dose-response relationship with C-5 propynyl pyrimidine-modified ODNs because of severe toxicity. The toxicity was characterized by mortality in animals receiving 50 mg/kg and an increase in infiltrating cells and apoptotic cells in livers of mice receiving 20 mg/kg. C-5 propynyl pyrimidine-modified chimeric oligonucleotides exhibited decreased hepatotoxicity compared with C-5 propynyl-modified ODNs but did not exhibit an increase in potency compared with unmodified chimeric oligonucleotides. The hepatotoxicity could be further limited if incorporation of propynyl pyrimidines was restricted to 2'-MOE nucleosides.
authors.affiliation: Isis Pharmaceuticals, Carlsbad, CA 92008, USA.
authors.forename: Lijiang
authors.initials: L
authors.lastname: Shen
authors.forename: Andrew
authors.initials: A
authors.lastname: Siwkowski
authors.forename: Edward V
authors.initials: EV
authors.lastname: Wancewicz
authors.forename: Elena
authors.initials: E
authors.lastname: Lesnik
authors.forename: Madeline
authors.initials: M
authors.lastname: Butler
authors.forename: Donna
authors.initials: D
authors.lastname: Witchell
authors.forename: Guillermo
authors.initials: G
authors.lastname: Vasquez
authors.forename: Bruce
authors.initials: B
authors.lastname: Ross
authors.forename: Oscar
authors.initials: O
authors.lastname: Acevedo
authors.forename: Gopal
authors.initials: G
authors.lastname: Inamati
authors.forename: Henri
authors.initials: H
authors.lastname: Sasmor
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
authors.forename: Brett P
authors.initials: BP
authors.lastname: Monia
mesh_terms: D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001219:Aspartate Aminotransferases / Q000097:blood; D001483:Base Sequence; D001921:Brain / Q000098:blood supply; D002460:Cell Line; D004334:Drug Administration Schedule; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D007274:Injections, Intraperitoneal; D008099:Liver / Q000187:drug effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D015394:Molecular Structure; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D009929:Organ Size / Q000187:drug effects; D011742:Pyrimidine Nucleotides / Q000737:chemistry*; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D016376:Oligonucleotides, Antisense; D011742:Pyrimidine Nucleotides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012313:RNA; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase