BIOLOGICAL AVAILABILITY AND NUCLEASE RESISTANCE EXTEND THE IN-VITRO ACTIVITY OF A PHOSPHOROTHIOATE-3'HYDROXYPROPYLAMINE OLIGONUCLEOTIDE

Augmented biological activity in vitro has been demonstrated in oligon ucleotides (oligos) modified to provide nuclease resistance, to enhanc e cellular uptake or to increase target affinity. How chemical modific ation affects the duration of effect of an oligo with potent activity has not been investigated directly. We postulated that modification wi th internucleotide phosphorothioates and 3' alkylamine provided additi onal nuclease protection which could significantly extend the biologic al activity of a 26 mer, (T2). We showed this analog, sT2a, could maxi mally inhibit interferon gamma-induced HLA-DR mRNA synthesis and surfa ce expression in both HeLa and retinal pigmented epithelial cells and could continue to be effective, in the absence of oligo, 15 days follo wing initial oligo treatment; an effect not observed with its 3'amine counterpart, T2a. In vitro stability studies confirmed that sT2a confe rred the greatest stability to nucleases and that cellular accumulatio n of P-32-sT2a in both cell types was also greater than other T2 oligo s. Using confocal microscopy, we revealed that the intracellular distr ibution of sT2a favored greater nuclear accumulation and release of ol igo from cytoplasmic vesicles; a pattern not observed with T2a. These results suggest that phosphorothioate-3'amine modification could incre ase the duration of effect of T2 oligo by altering nuclease resistance as well as intracellular accumulation and distribution; factors known to affect biological availability.