gene silencing strategies for dissecting disease pathways

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

33

Modulation of Gene Expression

• Small molecules• mAbs• Aptamers

Central Dogma of Molecular Biology

• siRNA• shRNA

RNA Protein

• Zinc finger nucleases

DNA

4

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

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 virus5

6

RNAi Delivery to the Cell

Agenda







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Recombinant Lentiviral Life Cycle

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

VSV-G envelope protein

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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

Time and cell division affects gene expression

• Gives immediate assessment of the system’s efficiency

MOI 5

HT-29 cells

• Allow to establish clonal stable cell lines

• Provides a system for long-term gene silencing and phenotypic observation

CHO-K1 cells

MOI 1

15

Agenda







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• Objective: Identify genes that, when silenced, can either enhance or suppress a given phenotype

Modifier Screen

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• 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

LentiExpress Plates

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Can

cer I

ncid

ence

(per

100

K)

Year

Prostate cancer

Prostate Cancer is the Most Frequently Diagnosed Cancer in American Men

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normal PIN cancer metastases androgen deathindependence

Progression

Transition to Metastatic Disease

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LNCaP cells

Gene knockdown + -

Experiment – Knockdown Genes in an Androgen-dependent Cell Line

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Perc

ent E

xpre

ssio

n

120

100

80

60

40

20

0Untreated H2 H3 H5 H6

Androgen Receptor Knockdown Normalized to Untreated Cells and Cyclophilin

Validation of shRNA Clones in LNCaP Cells

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LNCaP cells transduced with non-targeting shRNA

LNCaP cells transduced with androgen receptor shRNA

LNCaP Cells Treated with AR shRNA

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0

20

40

60

80

100

120

4 days 5 days 7 daysTime

% E

xpre

ssio

n

Androgen Receptor

Non-Target

Androgen Receptor Knockdown

Androgen receptor knockdown is stable under experimental conditions of the assay24

Lentiviral shRNA particles targeting kinases

LNCaP Cells

Puromycin selection

Split 1:2shRNA shRNA +

androgen

Viability assay Viability assay

Modifier Screen

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% Growth Relative to Control -- Vehicle

% G

row

th R

elat

ive

to C

ontr

ol –

And

roge

nshRNA Kinome Screen – LNCaP

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Agenda


• shRNA• Lentiviral transduction system• Arrayed Kinome shRNA Library





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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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10,000 40,00020,000 80,000 160,000

Various seeding densities (cells/mL) were plated in wells containing tGFP positive control particles

Courtesy of Zhenfeng Duan, M.D.

Determining Optimal TransductionConditions in KHOS

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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


Negative Controls Used in the Optimization Plate

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Protocol for shRNA Kinase Screen in Human Osteosarcoma Cells

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Replace wells with fresh media

Incubate plate at 37 °C, 5% CO2

overnight

Add puromycin- supplemented

media at 1µg/mL

Dispense KHOS cells into 96 well lentiviral shRNA

kinase platesRemove plates from incubator

Analyze results with a cell proliferation

assay kit


overnight


7 daysChange media every 2

days with puromycin


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C*C C

C

N

N

N

N

M

M

M

M

M

M

M

M

A7 A8 A9 A10 A11

B11

C2 C3 C4 C5

M


Positive Hits from Screen

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Positive Hit 1: PLK1Reduced Viability Upon Silencing

pLKO.1 particles

Non target particles

Media control

00.10.20.30.40.50.60.70.80.9

G7 G8 G9 G10 A12(C)

C12(N)

H12(M)

Abs

orba

nce

(490

nM

)

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Positive Hit 2: ROCK1Reduced Viability Upon Silencing

0.00

0.25

0.50

0.75

1.00

B11 C2 C3 C4 C5 A12(C)

C12(N)

H12(M)

Abs

orba

nce

(490

nM

)

pLKO.1 particles

Non target particles

Media control34

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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


• shRNA• Lentiviral transduction system• Arrayed Kinome shRNA Library





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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 virus37

MISSION esiRNA Technology

Transfect into cell

“Super-pool” of hundreds of siRNAs against 1 target gene

Assembly into RISC

Targeting of single mRNA

mRNA cleavage and degradation38

Generation of esiRNA

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MISSION esiRNA

esiRNA Gene #1

esiRNA Gene #2

esiRNA Gene #3

esiRNA Gene #4

etc.

1 esiRNA super-pool targeting one gene per well

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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. 9:403-15 (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


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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


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Overview of Oct4 High-throughput Assay

Transfect Oct4-Gip ESC with control or genome-

scale esiRNA library

High-throughput GFP fluorescence readout to

identify primary hits

Negative control (Luciferase esiRNA)

Primary hit (cells have reduced GFP)

No hit or negative control (cells have

high GFP Expression)

Primary hit or positive control

(cells have reduced GFP Expression)

Readout


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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


Paf1C Affects the Expression of Pluripotency and Lineage-marker Genes

46Ding, 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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Review of RNAi Effectors

siRNA shRNA

• Benefits• Simple • Titratable• Modifications available• Pooling is straightforward• Efficiently transfected

– Easy to transfect cell lines

• Disadvantages• Hard to transfect cells• Transient knockdown• Non-renewable

• 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

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• 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

The RNAi Consortium (TRC)

http://hms.harvard.edu/hms/home.asp


http://www.lilly.com/index.html

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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

Woodchuck hepatitis post-transcriptional regulatory element (WPRE)

• Sigma uses 3rd generation safety & design

• SIN vector (self inactivating vector)

• Replication incompetent lentiviral particles

• Recommended biosafety level: BSL-2

gene silencing strategies for dissecting disease pathways

Documents

given gene

used cell lines

gene silencing strategies

initial transduction

rnai delivery

range of moi

dicer protein

roundinfecting cells