Inhibition of transthyretin-met30 expression using Inosine(15.1)-Hammerhead ribozymes in cell culture

Hereditary amyloidosis is primarily caused by mutations within the transthy retin gene. More than 75 mutations within transthyretin have been reported in causing amyloidosis. The most common mutation is the val30met mutation i n the transthyretin protein (TTR-met30) caused by a mononucleic substitutio n from G to A (GUC to AUG) in the transthyretin gene resulting in the excha nge for the amino acids valine to methionine in the corresponding protein s equence. The aim of thi work is the development of a specific cleavage of T TR met30 mRNA in the cell culture system using hammer-head ribozymes. We sh owed previously that chemically modified nuclease stable Inosine (15.1)-Ham merhead ribozymes are able to target the TTR-met30 mRNA with high specifici ty on the RNA level (Biochem. Biophys. Res. Commun. 260, 313-317, 1999). No w we present data confirming our observations on the cellular level. We use d the wild-type human normal (hn) TTR expressing cell line HepG2 and the st able transfected cell line 293-TTR-met30 for TTR-met30 experiments. We clea ved the TTR-met30 and hnTTR mRNA with specific nuclease stable chemically m odified Inosine(15.1)-Hammerhead ribozymes and analyzed the protein after i mmunoprecipitation and subsequent Western blotting. We were able to down-re gulate the TTR concentration by 54.5% (100% = 1.5 mg/l TTR) and also specif ically to target the TTR-met30 expression in the cell culture system. The t herapeutic effect was improved using cationic liposomes resulting in a tota l downregulation by 92.1 and 62.7% targeting hnTTR mRNA and TTR-met30 mRNA respectively. The successful employment of Inosine(15.1)-Hammerhead ribozym es in cell culture is therefore a promising tool for the development of a g ene therapeutic strategy for hereditary amyloidosis. (C) 2000 Academic Pres s.