NR AOSR
AU Franz,B.S.; Pietrella,M.; Löwer,J.; Montrasio,F.
TI Inhibition of PrP expression with small interfering RNA oligonucleotides in Neuro 2a cells
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - IV-21
PT Konferenz-Poster
AB
Prion protein expression is necessary although not sufficient to enable prion replication. Furthermore, in vivo models showed that both the incubation time and the progression to terminal disease are inversely related to PrPc expression levels. As possible "therapeutical" approach for prion diseases it is therefore conceivable to aim at the reduction of the total levels of expressed PrPc. Small interfering RNAs (siRNAs) have been successfully used in eukaryotic systems to achieve post-transcriptional gene silencing. The aim of our study was to first assess whether siRNAs could be used to downregulate PrPc expression levels in a cell culture system and, in a second step, to asses whether post-transcriptional PrP silencing can interfere with prion replication and accumulation.
Neuro 2a cells were either transiently transfected with four synthetic siRNAs molecules targeted to different sequences within the murine PrP coding region or stably transfected with a siRNA-expression vector harboring either a PrP-specific siRNA sequence or a scrambled control sequence. PrPc-expression levels were then tested by FACS, Western-blot and immunofluorescence analyses.
Transient transfection of Neuro 2a with anti-PrP siRNAs determined a significant reduction of the total amount of PrPc as compared to the controls. The efficacy in gene-silencing of the four tested siRNAs differed and was not enhanced by higher concentrations or combinations of them as shown by FACS analyses. Stably transfected Neuro-2a cells showed an evident reduction of PrPc levels for more than four months. Scrapie-infected Bos-2 cells were transiently transfected. PrPsc levels showed a clear reduction, increasing 96 hours past transfection as shown by Western-blot.
Having shown that siRNA technology can be successfully used to decrease PrPc expression levels, it remains to be determined whether it is possible to interfere with prion replication and accumulation on a long-term basis by the gene silencing approach.
AD Bettina S. Franz, Marco Pietrella, Johannes Löwer, Fabio Montrasio, Department Pr1 - TSE-Research, Paul-Ehrlich-Institute, Langen, Germany
SP englisch
PO Deutschland