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RNAi Montserrat Enjuto

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RNAi Montserrat Enjuto

  1. 1. RNA de interferència RNAi Sample & Assay Technologies -1-
  2. 2. Seminari RNAi Definició RNAi: RNA interferència Mol·lècules de RNA de doble cadena són capaces de inhibir la expressió gènica, de forma específica. Dogma: DNA – RNA – Prot Quin paper té a les cèl·lules? Implicacions, noves teràpies Sample & Assay Technologies -2-
  3. 3. Definició de RNA interferència Mol·lècules de RNA de doble cadena són capaces de inhibir la expressió gènica, de forma específica mRNA Sample & Assay Technologies -3-
  4. 4. Dogma DNA-RNA-Proteïnes Sample & Assay Technologies -4-
  5. 5. Dogma DNA-RNA-Proteïnes Regulació de la expressió: Pre-transcripcional Post-transcripcional Pre-trancripcional: Enrrollament de la cromatina Controladors en les zones promotores dels gens Post-trancripcional: Capping Splicing Poliadenilació Sample & Assay Technologies -5-
  6. 6. Nobelprice 2006 Fire&Mello 2004 2001 Genome siRNA in 1998 wide screens 1990 mammalian ds RNA in Observed in plant 1998 Cells (Thuscl) C. Elegans 2005 and fungi RNAi used 2002 Fire&Mello First clinical (cosuppression) in in vivo trials Drosophila gene silencing Sample & Assay Technologies -6-
  7. 7. Reconeixement 2 Octubre de 2006 Concedeixen el Premi Nobel de Fisiologia i Medicina a Andrew Z. Fire i Craig C. Mello Pel seu descubriment: quot;RNA interference: gene silencing by double-stranded RNA“ “Interferència del RNA: silenciament gènic per RNA de doble cadena” Sample & Assay Technologies -7-
  8. 8. Mecanisme del RNAi ds RNA 1. RNAdc és introduit a dins de la cèl·lula DICER siRNA 2. Dicer digereix dsRNA en 3’OH 5`P Sense strand dsRNA de 21 pb (siRNA) Antisense strand 3`OH 5’P 3. siRNA s’integra en Helicase el complexe RISC 4. Separació de les hebres siRNA, RISC la hebra antisentit s’uneix Complexe inductor del a la diana complementària: mRNA silenciament del RNA 3`OH 5’P 5. Degradació del mRNA diana AAAAA 3`OH 5`P Silenciament AAAAA Post-transcripcional Sample & Assay Technologies -8-
  9. 9. Mecanisme absent en mamifers ds RNA AT Dicer ds RNA cleavage P ADP+Pi 5`P 3`OH siRNA- short interfering RNA 21-23 nt double stranded RNA 3`OH 5`P Sample & Assay Technologies -9-
  10. 10. Mecanisme del RNAi cel. mamífer ds RNA 1. RNAdc és introduit a dins de la cèl·lula DICER siRNA 2. Dicer digereix dsRNA en 3’OH 5`P Sense strand Silenciament dsRNA de 21 pb (siRNA) Antisense strand gènic en cèl·lules 3`OH 5’P de mamifers 3. siRNA s’integra en Helicase el complexe RISC 4. Separació de les hebres siRNA, RISC la hebra antisentit s’uneix Complexe inductor del a la diana complementària: mRNA silenciament del RNA 3`OH 5’P 5. Degradació del mRNA diana AAAAA 3`OH 5`P Silenciament AAAAA Post-transcripcional Sample & Assay Technologies - 10 -
  11. 11. The 2nd Mechanisms of RNAi pri-miRNA or MMD degradation*∗ X ∗ THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, protein No. 45, Issue of November 7, pp. 44312–44319, 2003 Sample & Assay Technologies - 11 -
  12. 12. Definició de RNAi RNAi: RNA interferència Mol·lècules de RNA de doble cadena son capaces de inhibir la expressió gènica, de forma específica. Dogma: DNA – RNA – Prot Quin paper té en les cèl·lules?: Defensa contra virus, Protecció contra gens saltarins (transposons) Mecanisme de regulació de la expressió Implicacions, noves teràpies Sample & Assay Technologies - 12 -
  13. 13. Efectes Off-target relacionats amb el miRNA Ruta siRNA ruta miRNA siRNAs poden entrar en la ruta miRNA AAAAA AAAAA CDS 3`UTR CDS 3`UTR lugar complementari Degradació del mRNA Repressió traduccional del mRNA & siRNA off-targeting en gens NO diana Sample & Assay Technologies - 13 -
  14. 14. miRNA miRNAs bloqueja la traducció del mRNA diana 5`P 3`OH RISC like complex mRNA 3` AAAAA 3´ 5` 5`P 3`OH RIBOSOME 7 2 3`UTR Seed Region Sample & Assay Technologies - 14 -
  15. 15. RNAi Definició RNAi: RNA interferència Mol·lècules de RNA de doble cadena són capaces de inhibir la expressió gènica, de forma específica. Dogma: DNA – RNA – Prot Quin paper té a les cèl·lules? Implicacions, noves teràpies Sample & Assay Technologies - 15 -
  16. 16. RNAi – The Cure? Degeneración macular relacionada con la edad: Mayor causa de ceguera Exceso de proteína VEGF protein lleva a un crecimiento excesivo de los vasos sanguíneos Terapia: silenciamiento de VEGF por inyección de siRNA Primer ensayo clínico en 2005 Gene Therapy (2006) 13, 225–234 Cancer: Terapias RNAi como soporte a la quimioterapiaRNAi Resistencia a fármacos mediada por P-glicoproteína Terapia: Silenciamiento P-glicoproteína en células leucémicas resistentes a fármacos, esto conduce a la recuperación de la sensibilidad a los fármacos. Sample & Assay Technologies - 16 -
  17. 17. Preguntes? Sample & Assay Technologies - 17 -
  18. 18. Kinetics of RNAi in cell culture Transient silencing [%] Transfection of siRNA re-transfect siRNA target mRNA protein threshold time Theoretical model (Current Medicinal Chemistry, 2003, 10(3) 245-256) Sample & Assay Technologies - 18 -

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