Antisense oligonucleotides.

Among the approaches which have been followed to convert a natural antisense phosphodiester oligonucleotide into a potential therapeutic agent, conjugation chemistry seems to be one of the most attractive. Indeed, natural phosphodiester oligonucleotide have the ideal properties (sequence specific hybridization, RNaseH activation, low or no toxicity, water solubility, easy and relative inexpensive synthesis in bulk quantities) to function as antisense oligomers. Their disadvantages are situated in their nuclease lability so that they are rapidly degraded in a biological medium, and to their low cellular uptake due to their polyanionic character. We investigated the minimum molecular modifications necessary to transform natural, partial self-complementary, phosphodiester oligonucleotides into a nuclease stable construct which is taken up in sufficient amounts in tumor cells to exert a selective antiproliferative effect. This study revealed that small aliphatic diols connected at the 3'-end gives oligonucleotides which are stable against nuclease degradation and which demonstrate potent and selective biological activity. Because of the low toxicity of both phosphodiester oligonucleotides and most aliphatic diols no cytotoxicity and no side effects are expected for these constructs. Moreover, these oligonucleotides may be synthesized easily in large amounts for an affordable price. As a first potential application we demonstrate that a 1,3-propanediol modified 12-mer directed at the point-mutation in codon 12 of the Ha-ras mRNA demonstrates a selective antiproliferative effect at a concentration which is 500 times lower than the one observed with unmodified antisense oligonucleotides. The EC50 value of +/-nM warrants further development of these constructs as antitumoral drugs for these cancers showing a high frequency of Ha-ras oncogene expression with point mutation at codon 12.