On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide
On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide
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DOI：
10.1002/cbic.200400419
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发表时间：
2005-06-01
期刊：
影响因子：
3.2
通讯作者：
Baas, F
中科院分区：
文献类型：
--
作者：
Fluiter, K;Frieden, M;Baas, F
锁核酸（β-D-LNA）单体是具有亚甲基2 '-O，4'-C键的构象限制核苷酸，其对匹配DNA或RNA具有前所未有的高亲和力。在这项研究中，我们比较了四种不同LNA的体外和体内性质，即β-D-氨基LNA（amino-LNA）、β-D-硫代LNA（thio-LNA）、β-D-LNA（LNA）及其立体异构体α-L-LNA的反义寡核苷酸（ODN）。用不同的LNA类似物在5 ′-和3 ′-末端修饰一种众所周知的针对H-Ras的反义ODN设计（LNA-DNA-LNA gapmer设计）。在癌细胞培养物和携带前列腺肿瘤异种移植物的裸小鼠模型中测试了所得的缺口聚体。测定靶点敲除的功效、生物分布和抑制肿瘤生长的能力。所有抗H-Ras ODNs在体外都能非常有效地敲低H-Ras mRNA，在低于5 nm的浓度下达到最大效果。此外，含有α-L-LNA的抗H-Ras ODN在H-Ras敲低中明显具有最高功效。所有LNA类型在血清中显示出很大的稳定性。与其他LNA类型相比，含有氨基LNA的ODN显示增加的心脏、肝脏和肺的摄取。α-L-LNA和LNA gapmer ODNs都具有高的肿瘤生长抑制功效，并且在测试剂量下是无毒的。值得注意的是，体内肿瘤生长抑制可以在低至0.5 mg/kg/天的剂量下观察到。这些结果表明，在反义应用中，α-L-LNA是LNA类似物家族中非常有前途的成员。
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 on increased uptake by heart, liver, and lungs as compared to the other LNA types. Both a-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.