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