Covalently Bound Polyamines Enhance the Antisense Effect of bcl-2 Targeted Phosphorothioates

Antisense and siRNA oligonucleotides are indispensable scientific tools for transient gene knockdown and the study of gene function. However, their huge promises as therapeutics have not yet been fulfilled. To date, only one product has reached the market and several promising antisense compounds have recently failed in their respective phase III clinical trials. The main problems to be solved are poor cellular uptake and insufficient pharmacokinetic properties. Tethering cationic molecules to oligonucleotides result in zwitterionic molecules with better membrane permeation ability and excellent target affinity. Polyamines are involved in gene function regulation and are counterions of nucleic acids in vivo. Due to their polycationic character and the ideal distance between amino groups, they are perfect ligands for creating zwitterionic oligonucleotides. Using an easy and versatile procedure for attaching ligands to the 2¿-position, the natural polyamines putrescine, spermidine and spermine as well as a chemically prepared pentaamine have been conjugated to phosphorothioate oligonucleotides targeted at bcl-2. CD spectra revealed that polyamine conjugation caused no changes in secondary structure. The thermal duplex stability of all conjugates is slightly lower than that of unconjugated phosporothioate, but increases progressively with increasing polyamine length. In a human melanoma cell culture assay, these conjugates show progressively higher target downregulation ability with increasing polyamine chain length. Conjugates with spermine and the pentaamine have a higher efficiency than the control phosphorothioate oblimersen, a drug currently being used in clinical trials. The higher in vitro efficiency of the pentaamine conjugated oligonucleotide is presumably due to the higher cellular uptake or higher nuclease stability.