Efficient and specific down-regulation of prion protein expression by RNAi G.Tilly, a,1 J.Chapuis, b,1 D.Vilette, b H.Laude, b andJ.L.Vilotte a, * a Laboratoire de G en etique Biochimique et de Cytog en etique, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France b Unit e de Virologie et Immunologie Mol eculaires, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France Received24March2003 Abstract Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challengebytheinfectiousagent.Theseresultsindicatedthatadown-regulationofthe Prnp geneexpressionisapotentialthera- peutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specificallyreducethelevelofPrPcintransfectedcells.It,thus,indicatesthatRNAiisanattractivetherapeuticapproachtofight againstpriondiseases. Ó 2003ElsevierScience(USA).Allrightsreserved. Keywords: Prions;RNAi;Transfectedcells;U6-vectors Prion diseases are fatal neurodegenerative disorders associatedwithanabnormalisoformofthePrPchost- encoded protein ([1–5] for recent reviews). The central roleofthe Prnp geneinpriondiseasewasclearlydem- onstrated with the creation by several independent groups of Prnp-knockout mice [6–8]. These transgenic linesremainhealthyfollowingscrapieinfectionanddid not accumulate or replicate the infectious agent. The lackofPrPcdidnotdramaticallyaffectthedevelopment and behaviour of the mice. Furthermore, post-natal knockoutofprionproteinwasrecentlyshownnottobe associated with detrimental sequelae and to confer to the mice the resistance to scrapie [9]. Several lines of evidence also correlate a reduction of the level of ex- pressionofPrPcwithanincreaseoftheincubationtime in scrapie-inoculated mice (see above mentioned re- views).Altogether,theseresultssuggestthatareduction of the amount of PrPc is a potential therapeutic ap- proach to at least delay the development of the fatal disordersassociatedwithprions. Severalstrategiesexisttoreducetheexpressionofa gene.Classicaltransgenesiscanbeappliedtoknockout a gene either in all cells of an animal or in restricted tissuesbytheuseoftheCre/LoxPsystem[10].However, such strategies are not practically applicable for thera- peutic issues. Inversely, specific degradation and/or translationinhibitionofmRNAcansignificantlyreduce the level of the encoded protein. Antisense and ribo- zymes have successfully been used [11–13], but unless thetargetedgeneisnaturallyexpressedatlowlevels,the achievedlevelofinhibitionisoftenlimited.Theuseof small double-stranded RNA, also called RNAi, was recently shown to result in high and specific inhibition oftargetedgeneexpression([14–20]forrecentreviews). However, the delivery of RNAi to the cells was often restricted to the transfection of in vitro synthesised double-stranded oligonucleotides due to the lack of adapted expression vectors. Recently, the use of RNA polymerase III promoters, such as U6 [21–24] or H1 [25],wasshowntoallowtheexpressionofshortdouble- stranded RNA mimicking the structure of RNase III endonuclease Dicer RNAi that was effective in down- regulating gene expression. In the present report, we assessed whether a U6 promoter-driven expression BiochemicalandBiophysicalResearchCommunications305(2003)548–551 www.elsevier.com/locate/ybbrc BBRC * Correspondingauthor.Fax:+33-1-34-65-24-78. E-mail address: vilotte@jouy.inra.fr (J.L.Vilotte). 1 Thesetwoauthorscontributedequallytothiswork. 0006-291X/03/$-seefrontmatter Ó 2003ElsevierScience(USA).Allrightsreserved. doi:10.1016/S0006-291X(03)00805-2