Inhibition of transthyretin-met30 expression using Inosine15.1-Hammerhead ribozymes in cell culture

Hereditary amyloidosis is primarily caused by mutations within the transthyretin gene. More than 75 mutations within transthyretin have been reported in causing amyloidosis. The most common mutation is the val30met mutation in the transthyretin protein (TTR-met30) caused by a mononucleic substitution from G to A (GUC to AUG) in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The aim of thi work is the development of a specific cleavage of TTR met30 mRNA in the cell culture system using hammer-head ribozymes. We showed previously that chemically modified nuclease stable Inosine (15.1)-Hammerhead ribozymes are able to target the TTR-met30 mRNA with high specificity on the RNA level (Biochem. Biophys. Res. Commun. 260, 313-317, 1999). Now we present data confirming our observations on the cellular level. We used the wild-type human normal (hn) TTR expressing cell line HepG2 and the stable transfected cell line 293-TTR-met30 for TTR-met30 experiments. We cleaved the TTR-met30 and hnTTR mRNA with specific nuclease stable chemically modified Inosine(15.1)-Hammerhead ribozymes and analyzed the protein after immunoprecipitation and subsequent Western blotting. We were able to down-regulate the TTR concentration by 54.5% (100% = 1.5 mg/l TTR) and also specifically to target the TTR-met30 expression in the cell culture system. The therapeutic effect was improved using cationic liposomes resulting in a total downregulation by 92.1 and 62.7% targeting hnTTR mRNA and TTR-met30 mRNA respectively. The successful employment of Inosine(15.1)-Hammerhead ribozymes in cell culture is therefore a promising tool for the development of a gene therapeutic strategy for hereditary amyloidosis. (C) 2000 Academic Press.