Gene Silencing Strategies for Dissecting Disease Pathways

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Gene Silencing Strategies for Dissecting Disease
Pathways

• Victoria Rusakova
• Senior Scientist
• Sigma-Aldrich Corporation

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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

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Modulation of Gene Expression
Central Dogma of Molecular Biology
RNA
Protein
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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

2006 The Nobel Prize in Physiology and Medicine
awarded to Andrew Z. Fire and Craig C. Mello
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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
• 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

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RNAi Delivery to the Cell
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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

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Recombinant Lentiviral Life Cycle
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Viral Transduction Laboratory Workflow
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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)
Zhang B., et al., Department of Medicine,
University of Queensland, Prince Charles
Hospital, Brisbane, Australia
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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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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

• Allow to establish clonal stable cell lines
• Provides a system for long-term gene silencing
  and phenotypic observation

• Time and cell division affects gene expression
• Gives immediate assessment of the systems
  efficiency

HT-29 cells
CHO-K1 cells
MOI 5
MOI 1
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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

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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

• 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

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Prostate Cancer is the Most Frequently Diagnosed
Cancer in American Men
Prostate cancer
Cancer Incidence (per 100K)
Year
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Transition to Metastatic Disease
Progression
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Experiment Knockdown Genes in an
Androgen-dependent Cell Line

Gene knockdown
-
LNCaP cells
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Validation of shRNA Clones in LNCaP Cells
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LNCaP Cells Treated with AR shRNA
LNCaP cells transduced with non-targeting shRNA
LNCaP cells transduced with androgen receptor
shRNA
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Androgen Receptor Knockdown
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Modifier Screen
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shRNA Kinome Screen LNCaP
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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

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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 TransductionConditions in
KHOS
10,000
40,000
80,000
160,000
20,000
Various seeding densities (cells/mL) were plated
in wells containing tGFP positive control
particles
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Courtesy of Zhenfeng Duan, M.D.
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Negative Controls Used in the Optimization Plate
Non-Target shRNA Control Particles (N)
pLKO.1 Control Particles (C)
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.
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Protocol for shRNA Kinase Screen in Human
Osteosarcoma Cells
Courtesy of Zhenfeng Duan, M.D.
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Positive Hits from Screen
C
C
A7
A8
A9
A10
A11
C
C
B11
N
N
C2
C3
C4
C5
N
N
M
M
M
M
M
M
M
M
M
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Courtesy of Zhenfeng Duan, M.D.
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Positive Hit 1 PLK1Reduced Viability Upon
Silencing
pLKO.1 particles
Non target particles
Media control
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Positive Hit 2 ROCK1Reduced Viability Upon
Silencing
pLKO.1 particles
Non target particles
Media control
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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

37
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
• 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

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MISSION esiRNA Technology
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Generation of esiRNA
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MISSION esiRNA
1 esiRNA super-pool targeting one gene per well
esiRNA Gene 1
esiRNA Gene 2
esiRNA Gene 3
esiRNA Gene 4
etc.
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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

Ding, L. et al., Cell Stem Cell. 9403-15 (2009)
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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

Ding, L. et al., Cell Stem Cell. 9403-15 (2009)
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Oct4 Assay Proof of Principle
GFP Expression

• Individual wells transfected with
• Control luciferase esiRNA
• esiRNA to known pluripotency regulators
• Sox2
• Oct4
• Stat3
• Visualized GFP by microscopy or FACS analysis

Ding, L. et al., Cell Stem Cell. 9403-15 (2009)
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Overview of Oct4 High-throughput Assay
Ding, L. et al., Cell Stem Cell. 9403-15 (2009)
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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

Ding, L. et al., Cell Stem Cell. 9403-15 (2009)
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Paf1C Affects the Expression of Pluripotency and
Lineage-marker Genes
Ding, L. et al., Cell Stem Cell. 9403-15 (2009)
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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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Review of RNAi Effectors
siRNA
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

• 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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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

Woodchuck hepatitis post-transcriptional
regulatory element (WPRE)