RNA Interference (RNAi) Ryan Duval

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RNA Interference (RNAi) Ryan Duval

  1. 1. RNA Interference (RNAi) Ryan Duval Endodontics
  2. 2. Discovery of RNAi <ul><li>First observed in petunias </li></ul><ul><li>Napoli et al 1990 </li></ul><ul><li>Observed “cosuppression” </li></ul><ul><li>Occurring at post-transcriptional level (plants and fungi) </li></ul><ul><li>Fire et al 1998 silencing of genes in nematodes (nice Prize ‘06) </li></ul><ul><li>Mammalian cells – chemically synth or expressed from plasmid or viral vector </li></ul>
  3. 3. Goals of RNA interference <ul><li>Defending cells against parasitic genes </li></ul><ul><li>Defense from viruses </li></ul><ul><li>Defense from transposons </li></ul><ul><li>Directing development and gene expression in general </li></ul>
  4. 4. RNAi <ul><li>Gene silencing mediated by double-stranded RNA </li></ul><ul><li>Silencing of gene expression </li></ul><ul><ul><li>Results from cleavage and degreadation of a target gene’s mRNA </li></ul></ul><ul><ul><li>Also results from blocking translocation of intact mRNA </li></ul></ul><ul><li>Usually about 20 – 25 base pairs long </li></ul>RNA Interference Howard Hughes Medical Institiute
  5. 5. Workings of RNAi <ul><li>Dicer recognizes and cuts the double-stranded RNA (dsRNA not common) </li></ul><ul><li>Results: short 21 to 25 base-pair molecules called “small interfering RNAs” (siRNAs) </li></ul><ul><li>siRNAs bind to several proteins (3’ overhangs) </li></ul><ul><li>Forming RISC (RNA-induced silencing complex) </li></ul>DICER
  6. 6. RNAi contd. <ul><li>RISC becomes activated when the siRNA its carying is unzipped (utilizing ATP) </li></ul><ul><li>Activated RISC bind to target mRNA </li></ul><ul><li>RISC subunits then cleave mRNA </li></ul><ul><li>Other proteins degrade mRNA & prevent protein production </li></ul>
  7. 7. RISC <ul><li>The two dsRNA pathways </li></ul><ul><ul><li>Exogenous </li></ul></ul><ul><ul><ul><li>Coming from infection by VIRUS w/RNA genome </li></ul></ul></ul><ul><ul><ul><li>By lab manipulation </li></ul></ul></ul><ul><ul><li>Endogenous </li></ul></ul><ul><ul><ul><li>Pre-microRNA expressed from RNA-coding genes in the genome </li></ul></ul></ul><ul><li>Both pathways converge at the RISC complex </li></ul>
  8. 8. RISC <ul><li>How the activated RISC complex locates complementary mRNAs within the cell is UNKNOWN? </li></ul><ul><li>Located in P-bodies (cytoplasmic bodies) </li></ul><ul><li>The active components of RISC are ARGONAUTE proteins </li></ul><ul><ul><li>Endonuclease </li></ul></ul><ul><ul><li>Cleaves the the target mRNA strand complementary to bound siRNA </li></ul></ul>
  9. 9. Overview <ul><li>20-25nt length (siRNA) </li></ul><ul><li>siRNA separated into single strands </li></ul><ul><li>Single strands integrated into RISC </li></ul><ul><li>siRNA induce cleavage of the mRNA </li></ul><ul><li>Preventing it from being used as a translation template </li></ul>
  10. 10. Recognizing the dsRNA <ul><li>Detected and bound by effector protein </li></ul><ul><li>RDE-4 in nematodes (C. elegans) </li></ul><ul><li>R2D2 in Drosophila </li></ul><ul><li>Both stimulate DICER </li></ul>
  11. 11. Gene knockdown <ul><li>A drastic decrease in the expression of a targeted gene </li></ul><ul><li>Studying the effects of the decrease can show the physiologic role of the gene product </li></ul><ul><li>RNAi may not totally abolish expression of the gene = knockdown vs knockout </li></ul>
  12. 12. RNAi applications <ul><li>Silencing CD44 (hyaluronan receptor) gene in nasopharyngeal carcinoma =  malignant potential of the cells. [Jod et al 2007, Shi Oncol Rep 2007] </li></ul><ul><li>Expression of p27 (common protein in oral squamous cell carcinoma), siRNA inhibited the cell proliferation in vitro and in vivo of the p27. [Kudo et al Oral Oncol 2005] </li></ul><ul><li>siRNA used for the treatment of ankylosis and periodontal disease. [Yamada et al J Biol Chem 2001, 2007] </li></ul>
  13. 13. Various RNAi uses <ul><li>Topical microbicide treatments of HSV II [Jiang, Milner; Oncogene 2002] </li></ul><ul><li>Knockdown host receptors for HIV [Crowe; AIDS Supp 2003] </li></ul><ul><li>Silencing Hep A and Hep B genes [Kusov et al; J Virol 2006] </li></ul><ul><li>Silencing Influenza gene expression [Jia, Zhang, Liu; Biotechnol Lett 2006] </li></ul><ul><li>Inhibition of LPS induced osteoclast formation and cytokine stimulation [Fahid et al; JOE 2008] </li></ul>
  14. 14. Application of Small Interfering RNA for Inhibition of LPS-Induced Osteoclast Formation and Cytokine Stimulation <ul><li>Purpose: Suppression of NFATc1 (transcription factor) expression in monocytes and osteoclast cells using RNAi technique </li></ul>
  15. 15. Background <ul><li>Bone homeostasis </li></ul><ul><li>RANKL (TNF family) </li></ul><ul><li>Induces osteoclastogenesis </li></ul><ul><li>LPS  express RANKL = osteoclast formation </li></ul><ul><li>Final stage of osteoclast differ. NFATc1 is crucial part of osteoclast differentiation. </li></ul><ul><li>Inhibit NFATc1 pathway  inhibit bone destruction? </li></ul>
  16. 16. Materials/Methods <ul><li>Mouse hematopoetic cells  osteoclasts </li></ul><ul><li>siRNA transfection (silencing NFATc1 </li></ul><ul><li>ELISA for TNF-α/IL-6 </li></ul><ul><li>Immunocytochemistry </li></ul><ul><li>Staining of nuclei </li></ul>
  17. 17. Materials/Methods <ul><li>Primers for detection of </li></ul><ul><ul><li>Cathepsin K gene CSK </li></ul></ul><ul><ul><li>IL-6 </li></ul></ul><ul><ul><li>TNF-α </li></ul></ul><ul><li>Compare osteoclasts transfected w/control vs NFATc1 siRNA </li></ul>
  18. 18. Results
  19. 19. Conclusion <ul><li>Deliver siRNA into cytoplasm w/  efficiency </li></ul><ul><li>Significant  of TNF-α and IL-6 in response to LPS stimulation </li></ul><ul><li>Significant  in # of mature osteoclasts in response to LPS </li></ul><ul><li> in osteoclast-specific gene expression to LPS stimulation </li></ul>
  20. 20. RNAi Challenges <ul><li>Systemic delivery obstacle (for RNAi drugs) </li></ul><ul><li>How to control the amount of siRNA being delivered (above or below therapeutic levels) </li></ul><ul><li>Possible stimulation of “off target” genes </li></ul><ul><li>Long-term effects </li></ul>
  21. 21. Any Questions?

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