title: On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide.
abstract: Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.
authors.affiliation: Department of Neurogenetics, AMC, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. k.fluiter@amc.uva.nl
authors.forename: Kees
authors.initials: K
authors.lastname: Fluiter
authors.forename: Miriam
authors.initials: M
authors.lastname: Frieden
authors.forename: Jeroen
authors.initials: J
authors.lastname: Vreijling
authors.forename: Christoph
authors.initials: C
authors.lastname: Rosenbohm
authors.forename: Marit B
authors.initials: MB
authors.lastname: De Wissel
authors.forename: Signe M
authors.initials: SM
authors.lastname: Christensen
authors.forename: Troels
authors.initials: T
authors.lastname: Koch
authors.forename: Henrik
authors.initials: H
authors.lastname: Ørum
authors.forename: Frank
authors.initials: F
authors.lastname: Baas
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D011905:Genes, ras / Q000187:drug effects*; D008297:Male; D051379:Mice; D009374:Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry*; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013237:Stereoisomerism; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D009711:Nucleotides; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C477371:locked nucleic acid; D012313:RNA; D004247:DNA