On the in vitro and in vivo Properties of Four Locked Nucleic Acid Nucleotides Incorporated into an Anti-H-Ras Antisense Oligonucleotide

• Published in: Chembiochem : a European journal of chemical biology 2005-06, Vol.6 (6), p.1104-1109
• Main Authors: Fluiter, Kees, Frieden, Miriam, Vreijling, Jeroen, Rosenbohm, Christoph, De Wissel, Marit B, Christensen, Signe M, Koch, Troels, Ørum, Henrik, Baas, Frank
• Format: Article
• Language: English
• Subjects: Animals; antisense agents; Cell Division - drug effects; DNA - chemistry; DNA - metabolism; Dose-Response Relationship, Drug; Genes, ras - drug effects; Male; Mice; Neoplasms, Experimental - drug therapy; Neoplasms, Experimental - metabolism; nucleic acids; Nucleotides - chemistry; Oligonucleotides; Oligonucleotides, Antisense - chemistry; Oligonucleotides, Antisense - metabolism; Oligonucleotides, Antisense - pharmacology; RNA - chemistry; RNA - metabolism; RNA, Messenger - metabolism; Stereoisomerism; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
• ISSN: 1439-4227; 1439-7633; 1439-4227
• DOI: 10.1002/cbic.200400419

Locked nucleic acid (β-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, β-D-amino LNA (amino-LNA), β-D-thio LNA (thio-LNA), β-D-LNA (LNA), and its stereoisomer α-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 α-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 α-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 α-L-LNA is a very promising member of the family of LNA analogues in antisense applications.