Transcript First line of the title Subhead goes here

Gene Silencing Strategies for
Dissecting Disease Pathways
Victoria Rusakova
Senior Scientist
Sigma-Aldrich Corporation
Agenda
• Introduction to RNAi
• shRNA
• Lentiviral Transduction System
• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent
prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell
survival
• siRNA
• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
2
Modulation of Gene Expression
Central Dogma of Molecular Biology
DNA
• Zinc finger nucleases
3
RNA
• siRNA
• shRNA
Protein
• Small molecules
• mAbs
• Aptamers
Areas Using RNAi Technology
• Gene function analysis
• Testing or verifying predicted gene function
• Pathway analysis
• Target the expression of a given gene in a pathway
and monitor the expression of other genes to
identify those genes associated with the target
gene
• Target identification and validation
• Identification of potential drug targets, at the gene
or protein level
• Drug discovery
• Develop potential therapeutic compounds based
on identified targets
4
2006: The Nobel Prize in Physiology
and Medicine awarded to Andrew Z.
Fire and Craig C. Mello
RNAi: Types of Interfering RNAs
• Synthetic based
• Small or short interfering RNAs (siRNA)
– Transfected directly into cells as oligonucleotides
– Do not perpetuate as vectors
• dsRNA molecules (duplexes) shorter than 30bp
• Silencing duration and effectiveness mainly regulated
by transfection efficiency
• Clone based
5
• Short hairpin RNAs (shRNA)
– Give rise to siRNA after processing by Dicer protein
• Encoded by DNA vectors allowing multiple delivery
methods
– Standard transient transfection
– Stable transfections
– Delivery by virus
RNAi Delivery to the Cell
6
Agenda
• Introduction to RNAi
• shRNA
• Lentiviral Transduction System
• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent
prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell
survival
• siRNA
• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
7
Recombinant Lentiviral Life Cycle
8
Viral Transduction Laboratory Workflow
9
Viral Titer and MOI (Multiplicity of Infection)
• Viral titer is a very important factor
• Allows determination of the correct experimental conditions using MOI
– MOI (Multiplicity of Infection) used for desired transduction efficiency
– The number of transducing lentiviral particles per cell
• When transducing a cell line for the first time, a range of MOI should be
tested
– Most successful screen require an MOI of 0.5 to 5.0
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Lentiviral-mediated Gene Transfer in
Different Cell Lines
• Significance of controlled conditions in lentiviral vector titration
• Use MOI for predicting gene transfer events
Efficiency of lentiviral-mediated gene
transfer to commonly used cell lines
under different MOI
Genet. Vaccines Ther. 2(1):6 (2004)
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Zhang B., et al., Department of Medicine, University of
Queensland, Prince Charles Hospital, Brisbane, Australia
Enhancing Transduction Efficiency
• Magnetic transduction
• Applying magnetic fields during
transduction to potentiate cell
targeting and binding
• Serial transductions
• Allow the cells to recover for 1 day
after initial transduction and follow
with a second round
• Infecting cells with a higher titer
virus
• VSV-G envelope protein allows for
concentration via ultracentrifugation
and ultrafiltration
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VSV-G envelope
protein
Enhancing Transduction of Primary
Cells
TurboGFP particles + polybrene
TurboGFP particles + ExpressMag
Human keratinocytes transduced at a MOI of 1,
incubated for 45 hours
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Viral Transduction Laboratory Workflow
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Transient versus Stable Transduction
•
Time and cell division affects gene
expression
• Allow to establish clonal stable cell
lines
Gives immediate assessment of the
system’s efficiency
• Provides a system for long-term
gene silencing and phenotypic
observation
HT-29 cells
MOI 5
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CHO-K1 cells
MOI 1
Agenda
• Introduction to RNAi
• shRNA
• Lentiviral Transduction System
• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent
prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell
survival
• siRNA
• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
16
Modifier Screen
• Objective: Identify genes that, when silenced, can either
enhance or suppress a given phenotype
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LentiExpress Plates
• Optimization Plate
• Pre-arrayed aliquots of TurboGFP
particles and controls
• Ideal for determination of optimal cell
number and MOI for LentiExpress
assays
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• Human Kinase Plate
• A quick method for carrying out kinase
screens
• 3109 pre-arrayed lentiviruses
– shRNAs targeting 673 human kinase
genes and controls
– A total of 41 96-well plates
– Up to 80 shRNAs per plate
Prostate Cancer is the Most Frequently
Diagnosed Cancer in American Men
Cancer Incidence (per 100K)
Prostate cancer
19
Year
Transition to Metastatic Disease
Progression
normal
20
PIN
cancer
metastases
androgen
independence
death
Experiment – Knockdown Genes in an
Androgen-dependent Cell Line
Gene knockdown
LNCaP cells
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+
-
Validation of shRNA Clones in LNCaP
Cells
Androgen Receptor Knockdown Normalized to Untreated
Cells and Cyclophilin
120
Percent Expression
100
80
60
40
20
0
Untreated
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H2
H3
H5
H6
LNCaP Cells Treated with AR shRNA
LNCaP cells transduced with
non-targeting shRNA
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LNCaP cells transduced with
androgen receptor shRNA
Androgen Receptor Knockdown
120
% Expression
100
80
60
40
Androgen
Receptor
20
Non-Target
0
4 days
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5 days
Time
7 days
Androgen receptor knockdown is stable under experimental
conditions of the assay
Modifier Screen
Lentiviral shRNA particles targeting kinases
LNCaP Cells
Puromycin selection
Split 1:2
shRNA
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shRNA +
androgen
Viability assay
Viability assay
% Growth Relative to Control – Androgen
shRNA Kinome Screen – LNCaP
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% Growth Relative to Control -- Vehicle
Agenda
• Introduction to RNAi
• shRNA
• Lentiviral transduction system
• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent
prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell
survival
• siRNA
• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
27
Hypothesis
• Overexpression and activation of specific kinases occurs during
growth of osteosarcoma cells
• Disruption of specific kinases will cause osteosarcoma cell death or
apoptosis
• These kinases have the potential to be drug targets for sarcoma
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Determining Optimal Transduction
Conditions in KHOS
10,000
20,000
40,000
80,000
160,000
Various seeding densities (cells/mL) were plated in wells containing tGFP positive control particles
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Courtesy of Zhenfeng Duan, M.D.
Negative Controls Used in the
Optimization Plate
pLKO.1 Control Particles (C)
Non-Target shRNA
Control Particles (N)
Control Media (M)
1 µg/ml of puromycin causes complete cell death of KHOS, U-2OS and UCH1 in 5 days
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Courtesy of Zhenfeng Duan, M.D.
Protocol for shRNA Kinase Screen in
Human Osteosarcoma Cells
Analyze results with
a cell proliferation
assay kit
Dispense KHOS
cells into 96 well
lentiviral shRNA
kinase plates
Remove plates
from incubator
Replace wells with
fresh media
Add puromycinsupplemented
media at 1µg/mL
7 days
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overnight
overnight
Change media every 2
days with puromycin
Incubate plate at
37 °
C, 5% CO2
Incubate plate at
37 °
C, 5% CO2
Incubate plate at
37 °
C, 5% CO2
Courtesy of Zhenfeng Duan, M.D.
Positive Hits from Screen
C*
A7
A8
C
N
C2
C3
C4
A9
A10
A11
C
B11
C
N
C5
N
N
M
M
M
M
M
M
M
M
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Courtesy of Zhenfeng Duan, M.D.
M
Absorbance (490 nM)
Positive Hit 1: PLK1
Reduced Viability Upon Silencing
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
G7
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G8
G9
G10 A12 C12 H12
(C) (N) (M)
pLKO.1
particles
Non target
particles
Media
control
Absorbance (490 nM)
Positive Hit 2: ROCK1
Reduced Viability Upon Silencing
1.00
0.75
0.50
0.25
0.00
B11
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C2
C3
C4
C5
A12
(C)
C12 H12
(N) (M)
pLKO.1 Non target
particles particles
Media
control
LentiExpress Kinase Screen Summary
• Identified 4 gene candidates as potential therapeutic targets in
osteosarcoma cells, including PLK1 and ROCK1
• KHOS osteosarcoma cells exhibited decreased cell proliferation upon
knockdown of these genes
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Agenda
• Introduction to RNAi
• shRNA
• Lentiviral transduction system
• Arrayed Kinome shRNA Library
– Identifying gene targets contributing to androgen independent
prostate cancer cell growth
– Identifying novel human kinases essential for osteosarcoma cell
survival
• siRNA
• Endoribonuclease-prepared siRNA (esiRNA) Screening Library
– Discovering modulators of embryonic stem cell identity
36
RNAi: Types of Interfering RNAs
• Synthetic based
• Small or short interfering RNAs (siRNA)
– Transfected directly into cells as oligonucleotides
– Do not perpetuate as vectors
• dsRNA molecules (duplexes) shorter than 30bp
• Silencing duration and effectiveness mainly regulated by
transfection efficiency
• Clone based
37
• Short hairpin RNAs (shRNA)
– Give rise to siRNA after processing by Dicer protein
• Encoded by DNA vectors allowing multiple delivery
methods
– Standard transient transfection
– Stable transfections
– Delivery by virus
MISSION esiRNA Technology
“Super-pool” of hundreds of
siRNAs against 1 target gene
Transfect into cell
Assembly into RISC
Targeting of single mRNA
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mRNA cleavage and degradation
Generation of esiRNA
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MISSION esiRNA
1 esiRNA super-pool targeting one gene per well
esiRNA
Gene #1
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esiRNA
Gene #2
esiRNA
Gene #3
esiRNA
Gene #4
etc.
Discovering Modulators of Embryonic
Stem Cell Identity
• Objective
• Obtain a systematic understanding of the genes associated with ESC identity
• Approach
• Perform a genome-scale RNAi screen to identify genes regulating ESC identity
using an Oct4 reporter assay
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Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
Oct4 Assay
• Oct4 expression can be used to monitor the differentiation status of ESC
• Screen performed in an Oct4 reporter mouse embryonic stem cell line
(Oct4-Gip)
• GFP expression is controlled by Oct4 regulatory elements
• Transfect cells with esiRNA and monitor changes in GFP expression
• Quantification of GFP fluorescence faithfully reflects the self-renewal and
differentiation status in individual cells
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Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
Oct4 Assay: Proof of Principle
• Individual wells transfected with
GFP Expression
• Control luciferase esiRNA
• esiRNA to known pluripotency
regulators
– Sox2
– Oct4
– Stat3
• Visualized GFP by microscopy or
FACS analysis
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Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
Overview of Oct4 High-throughput
Assay
Transfect Oct4-Gip ESC
with control or genomescale esiRNA library
High-throughput GFP
fluorescence readout to
identify primary hits
Readout
No hit or negative
control (cells have
high GFP Expression)
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Primary hit or
positive control
(cells have reduced
GFP Expression)
Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
Negative control
(Luciferase esiRNA)
Primary hit
(cells have reduced GFP)
Summary of Oct4 High-throughput
Assay
• 259 known and novel candidate pluripotency genes identified
• Secondary screen performed using individual esiRNAs synthesized for
the 21 strongest candidates
• 16 genes were confirmed
• Validated targets included components of the of the Pol II-associating
factor 1 complex (Paf1C)
• Paf1C contains Paf1, Ctr9, Cdc73, Rtf1, and Leo1
• Regulates transcription initiation, elongation, and start site selection
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Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
Paf1C Affects the Expression of
Pluripotency and Lineage-marker Genes
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Ding, L. et al., Cell Stem Cell. 9:403-15 (2009)
Summary of Study
• siRNA (esiRNA) is an effective tool for modulating gene function in stem
cells
• A screen using esiRNA identified 259 known and novel candidate
pluripotency genes
• Validated targets included components of the of the Pol II-associating
factor 1 complex (Paf1C)
• Paf1C affects the expression of pluripotency and lineage-marker genes
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48
Review of RNAi Effectors
siRNA
• Benefits
•
•
•
•
•
Simple
Titratable
Modifications available
Pooling is straightforward
Efficiently transfected
– Easy to transfect cell lines
• Disadvantages
• Hard to transfect cells
• Transient knockdown
• Non-renewable
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shRNA
• Benefits
• Renewable resource
• Transient or stable knockdown
• Transfection or viral delivery
– Viral delivery to most cells
• In vivo use potential
– Knockdown mice
• Disadvantages
• Design rules less understood
• Transfection less efficient
The RNAi Consortium (TRC)
• Goals
• Create a lentiviral based shRNA libraries targeting human and mouse genes
• Make clones available to researchers worldwide for the study of disease and gene
function
• Academic Laboratories
• Broad Institute, MIT/Harvard, Massachusetts General Hospital, Dana Farber Cancer
Institute, Whitehead Institute, Washington University and Columbia University
• Life Science Organizations
• Sigma-Aldrich, Novartis, Eli Lilly, Bristol-Myers Squibb and Academia Sinica in Taiwan
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TRC1 shRNA Transfer Vector
• Transfer vector
• pLKO.1-puro
• Lentiviral-based (HIV derived) Vector
• shRNA Promoter
• U6 (human)
• Design
• Broad Institute algorithm
• 21 bp stem
• 6 bp loop
• 5 clones per target gene
• High gene coverage
• Multiple knockdown levels
• Verification of phenotype
–Different shRNA produces same
result
• 3' UTR clone for cDNA rescue
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TRC2
• TRC2 Goals
• KD evaluation for 150,000 clones
by qRT-PCR
• Optimize vector elements
• Consider and evaluate special
purpose vectors
• Develop new and improved
screening methods
– Pooled libraries
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TRC2 shRNA Transfer Vector
• Sigma uses 3rd generation
safety & design
• SIN vector (self inactivating
vector)
• Replication incompetent
lentiviral particles
• Recommended biosafety level:
BSL-2
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Woodchuck hepatitis post-transcriptional
regulatory element (WPRE)