Stability of antisense oligonucleotides during incubation with a mixture of isolated lysosomal enzymes

Abstract

Cellular uptake of antisense oligonucleotides occurs predominantly via the endocytic route but the subsequent intracellular trafficking and metabolic fate is still poorly understood. We have examined the stability of 20-mer phosphodiester (D-oligo) and phosphorothioate (S-oligo) oligonucleotides ([ 32P]-radiolabelled either internally and at the 3′- or 5′- end) during exposure to a mixture of isolated rat liver lysosomal enzymes (Tritosomes). Whereas the oligonucleotides remained stable in aqueous buffer at pH 5.4 for at least 48 h, the D-oligos (at 10 μM) were degraded relatively rapidly ( t 50% = 30–40 min) by Tritosomes and at a rate similar to S-oligos ( t 50% = 50 min). Degradation of both was verified using reverse-phase HPLC and in this case for D- and S-oligos (2 μM) the t 50% was 90 min and 150 min, respectively. Enzymatic digestion by lysosomal nucleases, and not simple acid-catalysed hydrolysis, accounts for the rapid degradation of oligonucleotides.