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Manipulating Acquired Immunity through
Gene Silencing
Wei-Ping Min, MD.,PhD
University of Western Ontario
Canada
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.
Antigen Presenting Cells
•Signal 1: TCR triggering
•Signal 2: Costimulation
•Signal 3: Polarization
Dendritic Cells
Factors 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
Dendritic Cell-- A Double Edged Sword
Tolerance
Immunity
Immune Modulation and Immune Therapy
Immune System
Immunity
Hyper-immune responses
Autoimmune diseases
Allergic diseases
Graft rejection
Immune
tolerance
Tolerance
Hypo-immune responses
Infections
Cancer
Immune
Response
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.
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)
RNA interference:
siRNA
siRNA
Cell membrane
Cytosol
RISC
• sequence-specific, post-transcriptional
gene silencing
• initiated by 21bp segments of dsRNA
• antisense oligonucleotides
• blocking antibodies
• protein inhibitors (cancer drugs)
Endogeneous mRNA
• safer and more efficient, successfully
used to inhibit viral infections, tumor
growth
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
1.
2.
Bertrand et al., Biochem Biophys Res Commun.(2002), 296:1000
Thompson JD, Drug Discovery Today (2002) 7:912
Explosion of Interest in RNAi
Number of Publication
"RNAi is the most important and exciting breakthrough
of the last decade, perhaps multiple decades”
Phillip A. Sharp, Nobel laureate.
7500
5000
2500
0
Year
siRNA Method (1)
siRNA-expressing Cassette (SEC)
RNA Pol
promoter
Sense
template
Loop
Anti-sense
template
Terminator
Transfection
Control SEC
57.1%
CD40-SEC (1)
29.8%
CD40-SEC (2)
36.3%
CD40
CD40-SEC (3)
53.9%
CD40-SEC (4)
64.7%
siRNA Method (2)
SEC-expressing Vector
EcoRI
Hind III
Control DC
Mun I (6042)*
CMV
Hin d III (379)*
61.5%
CMV forward
VP22
Ap
pWPM-MHC II-SEC
6404 bp
myc tag
HisTag
PolyA
MHC II
siRNA-silenced DC
17.2%
pUC ORI
SV40
SV40 pA
NEO
MHC II
siRNA Method (3)
Chemically Synthesized siRNA
Untransfected
65.4%
GenePorter
47.4%
IL-12
Genesilencer
16.3%
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
In vivo siRNA Delivery methods (2)
Non-Viral Methods
•
•
•
•
Hydrodynamic system
Electroporation
DNA cationic polymers
Liposomes
Electroporation
Hydrodynamic
injection
Injecting gene
Liposomes
Pulsing Tissue
(electrical current)
Cells reseal withCell PorationGenes surround
Cells
Gene inside
• efficient in muscle tissue
• small vesicles
• method is exclusive but
• lipid bilayer enclosing
not specific
aquesous compartment • systemic delivery
• “nanocontainer”
not possible
Pictures adapted from http://www.rkm.com.au/biograph.html
• large volume of saline
containing nucleic acid
• systemic distribution of
nucleic acid
• transitory heart failure
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
In vivo siRNA Delivery methods (3)
Immunoliposomes
CD11c specific antibody
CD11
c
Dendritic
cell
Therapeutic Application of Gene Silencing
•
Down-regulation of Immunity
1.
2.
3.
Transplant tolerance
Autoimmune disease
Allergic disease
Immune
Response
•Upregulation of Immunity
1.Cancer therapy
2.Vaccine
3.Infectious diseases
Immune
tolerance
Down-Immune Regulation by siRNA
Preventing graft rejection in transplantation
3 days
Allogeneic heart
transplantation
treat recipient with
siRNA-silenced DC
Percent Survival
100
50
No transfusion
Control DC
Gene-silenced DC
0
0
10
20
30
40
50
60
70
Days after transplantation
80
90 100
Down-Immune Regulation by siRNA
Treatment of Autoimmune Arthritis
DBA/1LacJ
1 week
2 Weeks
Collagen II sc,
25 mg
Arthritis Score Index
Arthritis
Onset
Collagen II-pulsed
siRNA-silenced DC
5x106
Collagen II sc,
10 mg
4
3
Intact
No DC
Control DC
IL-12siRNA DC
2
1
0
0
2
4
7
9
11 14 16 18 21
Days after arthritis onset
Up-Immune Regulation by siRNA
Enhancing Cancer Vaccine
Tumor Ag-pulsed DC
Tumor Ag-pulsed DC
7 days
7 days
i.v
IDO-siRNA treated
i.p.
B16
s.c
Untreated control
Allow tumour
formation
IDO-siRNA
treatment
Misuse or Over-Regulation of Immune Responses
Immune System
Immunity
Over-Immune Response
Autoimmune diseases
Allergic diseases
Tolerance
Over-Immune Suppression
Cancer
Infections
Summary
1. siRNA is a useful tool for gene-specific inhibition for
manipulating immune system.
2. Up-regulating Immune responses is achievable by
silencing immune suppressive genes, which can be
used for anti-cancer therapy, vaccine development.
3. 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.
4. Misuse of siRNA and over-manipulation of immune
system may cause hyper- or hypo-immune
responses, which may lead to various diseases.
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
Acknowledgement
Jacob
Mu
Ying
Cecilia
Xusheng
Igor
Costin
Weiping
Francis
Xiufen
Jessica