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Manipulating Acquired Immunity through Gene Silencing

Manipulating Acquired Immunity through Gene Silencing. Wei-Ping Min, MD.,PhD University of Western Ontario Canada. By Chiara R. and Kate . Immune System.

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Manipulating Acquired Immunity through Gene Silencing

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  1. Manipulating Acquired Immunity through Gene Silencing Wei-Ping Min, MD.,PhD University of Western Ontario Canada

  2. By Chiara R. and Kate Immune System The Immune system is a group of cells and organs that work together to fight infections in our bodies. The Immune System protects our body from pathogens and disease-causing agents, such as bacteria.  

  3. Antigen Presenting Cells • Signal 1: TCR triggering • Signal 2: Costimulation • Signal 3: Polarization

  4. Dendritic CellsFactors Contributing to Immunity • Antigen processing: • Active endocytosis • Phagocytosis • Receptor-mediated uptake • High MHC I and II expression • High costimulatory molecule expression • High production of IL-12 • Formation of large clusters with T cells

  5. Tolerance Immunity Dendritic Cell-- A Double Edged Sword

  6. Immune System Tolerance Immunity Hyper-immune responses Autoimmune diseases Allergic diseases Graft rejection Hypo-immuneresponses Infections Cancer Immune Response Immune tolerance Immune Modulation and Immune Therapy

  7. Concept of RNA Interference • Double-stranded RNA (dsRNA) is frequently produced when foreign genes (eg., viral infection or transgenes) enter animals or plants. • RNA interference (RNAi) is the process by which cells destroy dsRNA and endogenous transcripts with homology to the dsRNA. • Small interfering RNA (siRNA) is cleaved from dsRNA by Dicer RNAse III, and is the mediator of RNAi.

  8. Milestones of RNAi • 1998-First RNA interference using dsRNA in C. elegans (Fire et al, Nature 391:806) • 2001-First RNA interference using siRNA in mammalian cells (Tuschl, Nature 411:494) • 2002-Inhibition of HIV entry and replication using siRNA to silence CD4 and gag genes (Sharp, Nature Medicine 8:681) • 2002-Silencing DC genes for immune modulation (Min, Arthritis & Rheumatism 46:s563)

  9. sequence-specific, post-transcriptional gene silencing initiated by 21bp segments of dsRNA antisense oligonucleotides blocking antibodies protein inhibitors (cancer drugs) safer and more efficient, successfully used to inhibit viral infections, tumor growth RNA interference: siRNA siRNA Cell membrane Cytosol RISC Endogeneous mRNA

  10. Gene Silencing:siRNA compared to other methods • siRNA vs Antisense Oligos: • siRNA more stable and efficient in gene silencing1,2 • Gene silencing occurs at much lower concentrations1 • siRNA vs Blocking antibodies: • Blocking Abs can be toxic and induce an immune response • Abs are not long lasting • siRNA vs Protein inhibitors (cancer): • siRNA is much more specific • siRNA is longer lasting • Bertrand et al., Biochem Biophys Res Commun.(2002), 296:1000 • Thompson JD, Drug Discovery Today (2002) 7:912

  11. 7500 5000 Number of Publication 2500 0 2000 2001 2002 2003 2004 1998 2005 1999 Year Explosion of Interest in RNAi "RNAi is the most important and exciting breakthrough of the last decade, perhaps multiple decades” Phillip A. Sharp, Nobel laureate.

  12. RNA Pol promoter Sense template Anti-sense template Terminator Loop Transfection Control SEC CD40-SEC (1) CD40-SEC (2) CD40-SEC (3) CD40-SEC (4) 57.1% 29.8% 36.3% 53.9% 64.7% CD40 siRNA Method (1)siRNA-expressing Cassette (SEC)

  13. EcoRI Hind III Control DC 61.5% Hin Mun I (6042)* d III (379)* CMV CMV forward VP22 Ap MHC II myc tag pWPM-MHC II-SEC HisTag siRNA-silenced DC PolyA 6404 bp 17.2% pUC ORI SV40 SV40 pA NEO MHC II siRNA Method (2)SEC-expressing Vector

  14. siRNA Method (3)Chemically Synthesized siRNA Untransfected GenePorter Genesilencer 65.4% 47.4% 16.3% IL-12

  15. In vivo siRNA Delivery methods (1)Viral Methods • Adenoviral /retroviral/ lentiviral vectors • Have tissue-specificity, high in vivo transduction, stable expression • Pre-existing immunity, may cause inflammation, cannot control site of integration Pictures adapted from http://www.rkm.com.au/biograph.html

  16. Electroporation Hydrodynamic injection Injecting gene Pulsing Tissue (electrical current) Liposomes • large volume of saline • containing nucleic acid • systemic distribution of • nucleic acid • transitory heart failure Genes surround Cells Cells reseal with Gene inside Cell Poration • efficient in muscle tissue • method is exclusive but • not specific • systemic delivery • not possible • small vesicles • lipid bilayer enclosing • aquesous compartment • “nanocontainer” Pictures adapted from http://www.rkm.com.au/biograph.html In vivo siRNA Delivery methods (2)Non-Viral Methods • Hydrodynamic system • Electroporation • DNA cationic polymers • Liposomes

  17. In vivo siRNA Delivery methods (3)Immunoliposomes Immunoliposomes • Small vesicle • Lipid bilayer • Aqueous interior: • siRNA encapsulation • PEG strands: • Immune camaflauge • Long circulation time • Attached antibodies: • Cell-specific targeting

  18. CD11c Dendritic cell In vivo siRNA Delivery methods (3)Immunoliposomes CD11c specific antibody

  19. Immune Response Immune tolerance Therapeutic Application of Gene Silencing • Down-regulation of Immunity • Transplant tolerance • Autoimmune disease • Allergic disease • Upregulation of Immunity • Cancer therapy • Vaccine • Infectious diseases

  20. 100 Percent Survival 50 No transfusion Control DC Gene-silenced DC 0 0 10 20 30 40 50 60 70 80 90 100 Days after transplantation Down-Immune Regulation by siRNA Preventing graft rejection in transplantation 3 days Allogeneic heart transplantation treat recipient with siRNA-silenced DC

  21. DBA/1LacJ Arthritis Onset 1 week 2 Weeks Collagen II sc, 25 mg Collagen II-pulsed siRNA-silenced DC 5x106 Collagen II sc, 10 mg 4 3 Intact No DC Arthritis Score Index 2 Control DC IL-12siRNA DC 1 0 0 2 4 7 9 11 14 16 18 21 Days after arthritis onset Down-Immune Regulation by siRNA Treatment of Autoimmune Arthritis

  22. Allow tumour formation IDO-siRNA treatment Up-Immune Regulation by siRNA Enhancing Cancer Vaccine Tumor Ag-pulsed DC Tumor Ag-pulsed DC 7 days 7 days B16 i.p. s.c i.v IDO-siRNA treated Untreated control

  23. Immune System Tolerance Immunity Autoimmune diseases Allergic diseases Misuse or Over-Regulation of Immune Responses Over-Immune Response Over-Immune Suppression Cancer Infections

  24. Summary • siRNA is a useful tool for gene-specific inhibition for manipulating immune system. • Up-regulating Immune responses is achievable by silencing immune suppressive genes, which can be used for anti-cancer therapy, vaccine development. • Down-regulating immune responses through silencing immune responsive genes possesses therapeutic potential in treatments of autoimmune and allergic diseases as well as graft rejection in transplantation. • Misuse of siRNA and over-manipulation of immune system may cause hyper- or hypo-immune responses, which may lead to various diseases.

  25. Acknowledgement • Canadian Institutes of Health Research • Roche Organ Transplant Research Foundation • Heart and Stroke Foundation of Canada • Kidney Foundation of Canada • The Physicians’ Services Incorporated Foundation • Multi-Organ Transplant Program Research Fund, LHSC

  26. Acknowledgement Jacob Igor Costin Francis Mu Ying Cecilia Xusheng Weiping Xiufen Jessica

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