Manipulating Acquired Immunity through Gene Silencing

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

• Signal 1 TCR triggering
• Signal 2 Costimulation
• Signal 3 Polarization

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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

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Dendritic Cell-- A Double Edged Sword
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Immune Modulation and Immune Therapy
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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.

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Milestones of RNAi

• 1998-First RNA interference using dsRNA in C.
  elegans (Fire et al, Nature 391806)

• 2001-First RNA interference using siRNA in
  mammalian cells (Tuschl, Nature 411494)

• 2002-Inhibition of HIV entry and replication
  using siRNA to silence CD4 and gag genes (Sharp,
  Nature Medicine 8681)

• 2002-Silencing DC genes for immune modulation
  (Min, Arthritis Rheumatism 46s563)

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RNA interference siRNA

• 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

siRNA
Cell membrane
Cytosol
RISC
Endogeneous mRNA
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Gene SilencingsiRNA 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), 2961000
• Thompson JD, Drug Discovery Today (2002) 7912

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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.
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siRNA Method (1)siRNA-expressing Cassette (SEC)
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siRNA Method (2)SEC-expressing Vector
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siRNA Method (3)Chemically Synthesized siRNA
Untransfected
GenePorter
Genesilencer
65.4
47.4
16.3
IL-12
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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/biogr
aph.html
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In vivo siRNA Delivery methods (2)Non-Viral
Methods

• Hydrodynamic system
• Electroporation
• DNA cationic polymers
• Liposomes

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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

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In vivo siRNA Delivery methods (3) Immunoliposomes
CD11c specific antibody
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Therapeutic Application of Gene Silencing

• Down-regulation of Immunity
• Transplant tolerance
• Autoimmune disease
• Allergic disease

• Upregulation of Immunity
• Cancer therapy
• Vaccine
• Infectious diseases

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Down-Immune Regulation by siRNA Preventing graft
rejection in transplantation
3 days
Allogeneic heart transplantation
treat recipient with siRNA-silenced DC
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Down-Immune Regulation by siRNA Treatment of
Autoimmune Arthritis
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Up-Immune Regulation by siRNA Enhancing Cancer
Vaccine
Tumor Ag-pulsed DC
Tumor Ag-pulsed DC
7 days
B16
7 days
i.v
i.p.
s.c
IDO-siRNA treated
Untreated control
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Misuse or Over-Regulation of Immune Responses
Over-Immune Response
Over-Immune Suppression
Cancer Infections
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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.

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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

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