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Cellecta, Inc.Cellecta, Inc.Targets & ToolsTargets & Tools
March 20/21, 2012March 20/21, 2012
Paul Diehl, Ph.D.Director of Business
Development
Cellecta, Inc.Cellecta, Inc.Targets & ToolsTargets & Tools
March 20/21, 2012March 20/21, 2012
Paul Diehl, Ph.D.Director of Business
Development
RNAi Genetic Screening for Drug Target Discovery RNAi Genetic Screening
for Drug Target Discovery
Cellecta Overview
• Founded: April 2006
• Located: Mountain View, CA
• 13 NIH grants (10 direct SBIR and 3 sub-contractor)
• Network of collaborators (primarily cancer-related)
• Focus: Flexible and scalable broad-based screening and analysis assays to expedite the discovery and characterization of novel therapeutic targets and drugs.
– Genome-scale RNAi-based genetic screening
– High-throughput functional-peptide screening
• Also provide supporting services, including customized lentiviral-based shRNA libraries and related shRNA and lentiviral services
Main Applications for RNAi Screens
• Find and prioritize drug targets• Understand disease progression and gene-disease associations• Connect expression profiling data with functionality• Analyze signal transduction pathways • Characterize the mechanisms for compounds• Find synergistic targets
Research areas…• Oncology and Cancer Biology• Infectious Diseases• Inflammation• Other diseases with cell-culture model systems
RNAi Screening
> 10 m
0.1 - 1 m
0.01 - 0.5 m
TWO miRNA PROCESSING STEPS
APPROXIMATE INTRACELLULAR
CONCENTRATIONS
RNAi Screening Formats
1 2 3 1 2 3
TargetCells
Functional Assay Functional Assay / Selection
Pooled FormatshRNA Library
shRNAconstructs
Arrayed FormatCollection of siRNAs/shRNA
• Time and Resource Intense• Assay to assay variability• Fixed
• Routine Cell Culture• One Assay All Results• Flexible
shRNA Expression Library Construction
• Oligo Pool Synthesis– Agilent Arrays (unique)
• Defined complex pools• High yield long oligos• Very low mutation rate
• Cloning– Optimized Cloning
• High complexity• >99% representation
• Packaging– Optimized Large Scale
• Maintain representation• High Titer
Pooled shRNA Library Screening
Types of Screens
• Rescue Screens (Positive Selection):
Find gene required to produce a response to added factors or compound, for example, genes necessary for trigging apoptosis or cell death in response to FAS, PUMA or other effectors.
• Viability Screens (Negative Selection):
Identify essential genes in a specific population of cells (e.g., prostate, blood, mammary cancer cells)
• Selected Populations (e.g., FACS):
Look for modulators of NF-κB, p53, c-myc, HSF-1, HIF-1α using fluorescent reporter cell line, or cells expressing specific Ab-detectable marker, such as specific receptors.
TGF-β Screen
TGF-β is an extracellular cytokine that inhibits cell proliferation by arresting cell cycle progression and inducing apoptosis.
Library: Pooled 27K shRNA library targeting 8,000 genes with 3-4 shRNAs per gene.
Screening: In duplicate, 2,500,000 Hep3B cells (approximately 100-times the number of constructs in the library) were transduced at a multiplicity of infection (MOI) of 0.3.
TGF-β HT Sequencing ResultsThe figure plots the abundance of each shRNA in each of the populations
x-axis: after transduction
y-axis: after selection
•If shRNA does not affect a critical genes for the response…
• shRNA disappears from population from apoptosis.
•If shRNA does target a critical gene for apoptosis signaling…
• shRNA is represented in the treated population (cell survives).
shRNA to about 100 genes are significantly enriched
shRNAs highly represented in the treated population presumably target genes involved in TGF-beta apoptosis signaling
TGF-β Findings• Hits around PI3K and TGF-β signaling pathways
• Genes identified with the TGF-β screen starred
Validation:
18 of 19 selected TGF-β hits confirmed to knockdown target and conferred TGF-β resistance.
TGFR1 OASLID3 G6PD
DTX1 LTB4DHCASP1 TRIM59
GABPB2 MMP9TCN1 EYA2MAF TGFB3
DMRTA1 RHOBTB1DAP SMAD4
shRNA Structures
shRNA Design
~70%of shRNA
exhibit >70%knockdown
Optimized Vector Design
• Optimized, validated shRNA structure for pooled format screens
• shRNA-specific 18-nt bar-codes compatible with Illumina-Solexa HT sequencing
• High knockdown level by U6-constitutive or U6Tet-regulated expression of shRNAs
• RFP reporter for monitoring transduced cells
• PuroR for selection transduced cells
HT Sequencing of shRNA Libraries
Good Library (DECIPHER)Poor Libraries
~90% inserts within 10-fold abundance range (i.e. narrow distribution)
~95% of shRNA constructs without mutations/deletions
~98% shRNA insert rate
>20-30% of shRNA constructs are poorly represented (i.e. wide distribution)
>30% of shRNA constructs have mutations/deletions
Plasmid library 27K shRNAs Plasmid library 27K shRNAs
Library QC …
•Virtually all shRNA inserts are present in relatively equal abundance
•Clones are randomly pulled and sequenced to check for mutation and insert rate
Viability Screen for Blood Neoplasia
Biological Triplicates
K562 Cells
Cellecta Human 27K Library
Targeting Signaling Pathway Genes
Viability Hits Associated with K562 Cells
* Overlap with previous study: Luo B, et al. Highly parallel identification of essential genes in cancer cells. Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20380-5.
*
*
Validation of Predicted Essential Genes in K562 cells with Small Molecule Drugs
R115777
ZM33637
PD198306U0126
SX011SB203580SB302190
C401
NSC625987
Top Canonical PathwaySIGNALLING TO RAS
E2F REGULATION OF DNA REPLICATION
PYRUVATE METABOLISM AND TCA CYCLE
REGULATION OF RHEB GTPASE ACTIVITY BY AMPK
ACTIVATION OF ATR IN RESPONSE TO REPLICATION STRESS
MTOR SIGNALLING
SIGNALLING TO ERKS
SHC MEDIATED SIGNALLING
GRB2 EVENTS IN EGFR SIGNALING
SOS MEDIATED SIGNALLING
SHC RELATED EVENTS
G PROTEIN BETA GAMMA SIGNALLING
GLUCOSE REGULATION OF INSULIN SECRETION
Viability Gene Signatures in Cancer Cells
K562 HL60 BJ-TERT Jurkat Raji HeLa DU145 A549 H1437
P-value0 127K Decipher module 1
General Viability
Genotype-specific
Disease-specific
K562 Viability Protein-Protein Interaction Network
General Viability - Proteasome Complex
0 1
K5
62
HL6
0
BJ
Jurk
at
Raji
HeLa
DU
14
5
A5
49
H1
43
7
Ribosome & Elongation Factor Complex
K5
62
HL6
0
BJ
Jurk
at
Raji
HeLa
DU
14
5
A5
49
H1
43
7
FAS Apoptosis Regulator Screen
Library: 27K shRNA Library targeting about 5,000 annotated genes—mostly signaling pathway genes (DECIPHER I Module)
Vector: pRSI-U6wt-Ubi-RFP-2A-Puro lentiviral
Cell Treatment: 7 days
Puro added at 2 days.
FAS ligand at 4 days, grown for 3 more days.
Genomic DNA was isolated 7 days.
HT Seq Data: FAS Screening
245 shRNAs Targeting 157 Genes
shRNA in target
Plasmid Library
FAS Pathway Hits
Several Hits Confirmed Inhibit NFkB
** *
0
5
10
15
20
25
30
35
40
LUC C
ONTR
OL RELA
IKBKB
IKBKG
IKBKG
TNFR
SF1A
TNFR
SF1B IL1
R1M
YD88
IRAK
1
CSNK
1G2
IL13R
A2 APCS
CCR4
WAS
F1TN
FRSF
8CA
SRIL1
RL1
TNFR
SF25
GALN
SGC
AT MTR
UBE2
E1PR
KCE
SHOX
2TN
FRSF
18TN
FSF1
1GA
TA3
LAM
P2M
AP3K
12
RELA
TIVE
FLUO
RESC
ENCE
293-TLR5-NF-kB-GFP FLAGELLIN
0
10
20
30
40
50
60
70
80
LUC C
ONTR
OL RELA
IKBKB
IKBKG
IKBKG
TNFR
SF1A
TNFR
SF1B IL1
R1M
YD88
IRAK
1
CSNK
1G2
IL13R
A2 APCS
CCR4
WAS
F1TN
FRSF
8CA
SRIL1
RL1
TNFR
SF25
GALN
SGC
AT MTR
UBE2
E1PR
KCE
SHOX
2TN
FRSF
18TN
FSF1
1GA
TA3
LAM
P2M
AP3K
12
RELA
TIVE
FLUO
RESC
ENCE
293-NF-kB-GFP IL-1b
0
20
40
60
80
100
120
140LU
C CON
TROL RE
LAIKB
KBIKB
KGIKB
KGTN
FRSF
1ATN
FRSF
1B IL1R1
MYD
88IR
AK1
CSNK
1G2
IL13R
A2 APCS
CCR4
WAS
F1TN
FRSF
8CA
SRIL1
RL1
TNFR
SF25
GALN
SGC
AT MTR
UBE2
E1PR
KCE
SHOX
2TN
FRSF
18TN
FSF1
1GA
TA3
LAM
P2M
AP3K
12
RELA
TIVE
FLUO
RESC
ENCE
293-NF-kB-GFP TNF293 Cells stably expressing NFkB responsive GFP reporter.
Canonical Non-Canonical
Mouse Rescue from FAS Hepatitis% Survival of NIH Swiss Female Mice 8 weeks Old:
Injected With siRNA and 72 Hours LaterInjected with FAS Antibody 6ug/mouse
Days
0 2 4 6 8 10 12 14
% S
urv
ival
0
20
40
60
80
100
FAS 6ug/mousesiRNA CCR4(54)+FAS (KIT)siRNA CCR4(55)+FAS (NO KIT)siRNA APCS(84)+FAS (KIT)siRNA APCS(86)+FAS (NO KIT)
% Survival of NIH Swiss Female Mice 8 weeks Old:Injected With siRNA and 72 Hours LaterInjected with FAS Antibody 6ug/mouse
DAYS
0 2 4 6 8 10 12 14%
Su
rviv
al0
20
40
60
80
100
FAS 6ug/mouseBCME+FAS(KIT)HNRNP+FAS(KIT)WASF+FAS(KIT)CASR+FAS(KIT)DEGS+FAS(NO KIT)
6 Mice injected with siRNAs targeting mouse paralogs to targets found with FAS screen: CCR4, APCS, WASF1, HNRNP, CASR, DEGS.
siRNA to BCME was used as a control.
At 72 hours after siRNA, mice were injected with FAS Jo2 antibody (induces fulminant hepatic failure)
siRNA Protects Mice from Fas-induced Fulminant Hepatic Failure
3 siRNA pools
single siRNAs
Small Molecule Inhibitors
30 targets selected with known inhibitory pharmaceuticals and tested in HeLa, A549 and HT1080 cells.
The 3 best candidates from in vitro screen were followed up in vivo. (i.e., good dosages, low toxicity, correct tissue specificity)
Protection Concentrations for FAS-Induced Hela Cells
BIM-peptide
Small Molecules in MiceI.p. injection of NEM (0.25nM/ mouse) 1h before i.p. injection
of FAS antibodies (6mg/kg) protected NIH Swiss mice from FAS-induced death
days
0 2 4 6 8 10 12
mo
use
su
rviv
al (
%)
0
20
40
60
80
100
FASNEM+FAS
I.p. injection of BIM-peptide (25mg/mouse) 1h before i.p. injection of FAS antibodies (6 g/kg)
protected NIH Swiss mice from FAS-induced death
days
0 2 4 6 8 10 12
mo
use
su
rviv
al (
%)
0
20
40
60
80
100
FAS BIM+FAS
I.p. injection of CAN (0.25mg/kg) 4h after i.p.injection of FAS antibodies (6mg/kg) protected NIH Swiss mice
from FAS-induced death
days
0 2 4 6 8 10 12
mo
use
su
rviv
al (
%)
0
20
40
60
80
100
FASCAN+FAS
N-Ethylmaleimide (NEM)KCNQ inhibitor
BIM-peptide SSTR5 inhibitor
Cantharidine PP2A inhibitor
Protection from Acute Fulminant Hepatitis
•Intraperitoneal dose with of selected compounds (PBS for controls)
•Lethal challenge with 0.6 µg/g of anti-Fas Jo2 antibody (kills 100% of mice within 20 h)
Cellecta, Inc.320 Logue Ave.
Mountain View, CA, USA1-877-938-3910
info@cellecta.com www.cellecta.com
Cellecta, Inc.320 Logue Ave.
Mountain View, CA, USA1-877-938-3910
info@cellecta.com www.cellecta.com
Thank You! Questions?
Paul DiehlDirector of Business Development
Tel: 650-938-4050E-mail: pauld@cellecta.com
Paul DiehlDirector of Business Development
Tel: 650-938-4050E-mail: pauld@cellecta.com
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