Manipulating Acquired Immunity through Gene Silencing Wei-Ping Min, MD.,PhD University of Western...
-
Upload
edith-strickland -
Category
Documents
-
view
216 -
download
3
Transcript of Manipulating Acquired Immunity through Gene Silencing Wei-Ping Min, MD.,PhD University of Western...
Manipulating Acquired Immunity through Manipulating Acquired Immunity through Gene SilencingGene Silencing
Wei-Ping Min, MD.,PhDWei-Ping Min, MD.,PhD
University of Western OntarioUniversity of Western OntarioCanadaCanada
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.
Immune SystemImmune System
Antigen Presenting CellsAntigen Presenting Cells
•Signal 1: TCR triggering
•Signal 2: Costimulation
•Signal 3: Polarization
Dendritic CellsDendritic CellsFactors Contributing to ImmunityFactors Contributing to Immunity
• Antigen processing: Antigen processing: • Active endocytosisActive endocytosis• PhagocytosisPhagocytosis• Receptor-mediated uptakeReceptor-mediated uptake
• High MHC I and II expressionHigh MHC I and II expression
• High costimulatory molecule High costimulatory molecule expressionexpression
• High production of IL-12High production of IL-12
• Formation of large clusters with T Formation of large clusters with T cells cells
Immunity Tolerance
Immune System
Hyper-immune responses Autoimmune diseasesAllergic diseasesGraft rejection
Immune tolerance
Immune Response
Hypo-immune responses Infections
Cancer
Immune Modulation and Immune Therapy
Concept of RNA Interference Concept of RNA Interference
• Double-stranded RNA Double-stranded RNA (dsRNA)(dsRNA) is frequently is frequently produced when foreign genes (eg., viral infection produced when foreign genes (eg., viral infection or transgenes) enter animals or plants.or transgenes) enter animals or plants.
• RNA interference RNA interference (RNAi)(RNAi) is the process by which cells is the process by which cells destroy dsRNA and endogenous transcripts with destroy dsRNA and endogenous transcripts with homology to the dsRNA.homology to the dsRNA.
• Small interfering RNA Small interfering RNA (siRNA)(siRNA) is cleaved from dsRNA by is cleaved from dsRNA by Dicer RNAse III, and is the mediator of RNAi.Dicer RNAse III, and is the mediator of RNAi.
Milestones of RNAiMilestones of RNAi
• 19981998-First RNA interference using dsRNA in -First RNA interference using dsRNA in C. C. eleganselegans (Fire et al, (Fire et al, NatureNature 391:806) 391:806)
• 20012001-First RNA interference using siRNA in -First RNA interference using siRNA in mammalian cells (Tuschl, mammalian cells (Tuschl, NatureNature 411:494) 411:494)
• 20022002-Inhibition of HIV entry and replication -Inhibition of HIV entry and replication using siRNA to silence CD4 and gag genes using siRNA to silence CD4 and gag genes (Sharp, (Sharp, Nature MedicineNature Medicine 8:681) 8:681)
• 20022002-Silencing DC genes for immune modulation -Silencing DC genes for immune modulation (Min, (Min, Arthritis & RheumatismArthritis & Rheumatism 46:s563) 46:s563)
siRNA
Cell membrane
Cytosol
Endogeneous mRNA
RISC
RNA interferenceRNA interference: : siRNAsiRNA
• sequence-specific, post-transcriptional sequence-specific, post-transcriptional gene silencing gene silencing
• initiated by 21bp segments of dsRNAinitiated by 21bp segments of dsRNA
• antisense oligonucleotides antisense oligonucleotides • blocking antibodiesblocking antibodies• protein inhibitors (cancer drugs)protein inhibitors (cancer drugs)
• safer and more efficient, successfully safer and more efficient, successfully used to inhibit viral infections, tumor used to inhibit viral infections, tumor growthgrowth
Gene Silencing:Gene Silencing:siRNA compared to other methodssiRNA compared to other methods• siRNA vs Antisense Oligos:siRNA vs Antisense Oligos:
• siRNA more stable and efficient in gene silencingsiRNA more stable and efficient in gene silencing1,21,2
• Gene silencing occurs at much lower concentrationsGene silencing occurs at much lower concentrations11
• siRNA vs Blocking antibodies:siRNA vs Blocking antibodies:• Blocking Abs can be toxic and induce an immune responseBlocking Abs can be toxic and induce an immune response• Abs are not long lasting Abs are not long lasting
• siRNA vs Protein inhibitors (cancer):siRNA vs Protein inhibitors (cancer):• siRNA is much more specific siRNA is much more specific • siRNA is longer lasting siRNA is longer lasting
1. Bertrand et al., Biochem Biophys Res Commun.(2002), 296:10002. Thompson JD, Drug Discovery Today (2002) 7:912
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.
19981999
20002001
20022003
200420050
2500
5000
7500
Year
Nu
mb
er
of
Pu
blic
ati
on
siRNA Method (1)siRNA Method (1)siRNA-expressing Cassette (SEC)siRNA-expressing Cassette (SEC)
RNA Polpromoter
Sensetemplate
Anti-sensetemplate
TerminatorLoop
CD40-SEC (1) CD40-SEC (2) CD40-SEC (3) CD40-SEC (4)
29.8% 36.3% 53.9% 64.7%
Control SEC
57.1%
CD40
Transfection
EcoRI Hind III
pWPM-MHC II-SEC
6404 bp
NEO
Ap
SV40 pA
myc tagHisTagPolyA
CMV forward
CMV
SV40
pUC ORI
VP22
Hin d III (379)*Mun I (6042)*
61.5%
Control DC
17.2%
siRNA-silenced DC
MHC II
MHC II
siRNA Method (2)siRNA Method (2)SEC-expressing VectorSEC-expressing Vector
65.4% 47.4% 16.3%
Untransfected GenePorter Genesilencer
IL-12
siRNA Method (3)siRNA Method (3)Chemically Synthesized siRNAChemically Synthesized siRNA
In vivoIn vivo siRNA Delivery methods (1) siRNA Delivery methods (1)Viral MethodsViral Methods
• Adenoviral /retroviral/ lentiviral vectorsAdenoviral /retroviral/ lentiviral vectors• Have tissue-specificity, high in vivo transduction, Have tissue-specificity, high in vivo transduction,
stable expressionstable expression• Pre-existing immunity, may cause inflammation, Pre-existing immunity, may cause inflammation,
cannot control site of integrationcannot control site of integration
Pictures adapted from http://www.rkm.com.au/biograph.html
In vivoIn vivo siRNA Delivery methods (2) siRNA Delivery methods (2)Non-Viral MethodsNon-Viral Methods
• Hydrodynamic systemHydrodynamic system• ElectroporationElectroporation• DNA cationic polymersDNA cationic polymers• LiposomesLiposomes
Pictures adapted from http://www.rkm.com.au/biograph.html
Liposomes
• small vesicles• lipid bilayer enclosing aquesous compartment• “nanocontainer”
Injecting gene Pulsing Tissue(electrical current)
Genes surroundCells
Cell PorationCells reseal withGene inside
• efficient in muscle tissue• method is exclusive but not specific• systemic delivery not possible
Electroporation
Hydrodynamic injection
• large volume of saline containing nucleic acid• systemic distribution of nucleic acid• transitory heart failure
Immunoliposomes
• Small vesicle • Lipid bilayer• Aqueous interior:
• siRNA encapsulation
• PEG strands:• Immune camaflauge• Long circulation time
• Attached antibodies:• Cell-specific targeting
In vivoIn vivo siRNA Delivery methods (3) siRNA Delivery methods (3) Immunoliposomes
CD11c specific antibody
CD11c
Dendritic cell
In vivoIn vivo siRNA Delivery methods (3) siRNA Delivery methods (3) Immunoliposomes
• Down-regulation of ImmunityDown-regulation of Immunity1.1. Transplant toleranceTransplant tolerance
2.2. Autoimmune diseaseAutoimmune disease
3.3. Allergic diseaseAllergic disease
Therapeutic Application of Gene SilencingTherapeutic Application of Gene Silencing
Immune Response
Immune tolerance
•Upregulation of ImmunityUpregulation of Immunity1.1.Cancer therapyCancer therapy2.2.VaccineVaccine3.3.Infectious diseasesInfectious diseases
Down-Immune Regulation by siRNAPreventing graft rejection in transplantation
0 10 20 30 40 50 60 70 80 90 1000
50
100
No transfusionControl DCGene-silenced DC
Days after transplantation
Per
cen
t S
urv
ival
treat recipient with siRNA-silenced DC
Allogeneic heart transplantation
3 days
0
1
2
3 IntactNo DCControl DCIL-12siRNA DC
Art
hri
tis
Sco
re I
nd
ex
0 2 4 7 9 11 14 16 18 21Days after arthritis onset
Collagen II-pulsed siRNA-silenced DC 5x106
Collagen II sc, 25 g
Collagen II sc, 10 g
DBA/1LacJ
2 Weeks1 week
4
Arthritis Onset
Down-Immune Regulation by siRNATreatment of Autoimmune Arthritis
Tumor Ag-pulsed DC
B16
i.v
Tumor Ag-pulsed DC
7 days 7 days
i.p. s.c
IDO-siRNA treatment
Allow tumour formation
IDO-siRNA treated Untreated control
Up-Immune Regulation by siRNAEnhancing Cancer Vaccine
Immunity Tolerance
Immune System
Autoimmune diseasesAllergic diseases
Over-Immune Response
CancerInfections
Misuse or Over-Regulation of Immune Responses
Over-Immune Suppression
SummarySummary
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.
AcknowledgementAcknowledgement
•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