Signe M. Christensen

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

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.

Synthesis of 2′-Amino-LNA Purine Nucleosides

The first reported synthesis of 2′-amino-LNA purine nucleosides via a transnucleosidation is accomplished enabling the preparation of oligonucleotides incorporating 2′-amino-LNA with all four natural bases.

Improved Synthesis of 2′-Amino-LNA

Abstract 2′-Amino-LNA phosphoramidite (10) was synthesised by means of a new strategy, which is convergent with the synthesis of 2′-oxy-LNA up until a late stage intermediate (1).