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

ImmunityTolerance

Dendritic Cell-- A Double Edged Sword

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

AcknowledgementAcknowledgement

IgorJacob

Xusheng JessicaWeiping XiufenMu

FrancisCostin

CeciliaYing