November 2010

Improving life through genome engineering

November 2010

This communication expressly or implicitly contains certain forward-looking statements concerning

Cellectis and its business. Such statements involve certain known and unknown risks,

uncertainties and other factors, which could cause the actual results, financial condition,

performance or achievements of Cellectis to be materially different from any future results,

performance or achievements expressed or implied by such forward-looking statements. Cellectis

is providing this communication as of this date and does not undertake to update any forward-

looking statements contained herein as a result of new information, future events or otherwise.

Disclaimer

2

3

November 30, 2010

Welcome to Cellectis first R&D Day

November 2010 4

Program

13:30 Cellectis – An Overview – André Choulika, CEO13:40 Update on the Science of Meganucleases – Philippe

Duchateau14:00 Research Tools – Christophe Delenda14:20 Genome Engineering in Plants – Dan Voytas14:50 Therapeutic Programs – Carole Desseaux15:10 An Example of Collaboration in Therapeutics – Serge Braun AFM15:45 Perspectives: potential applications of iPS cell technologies –

David Sourdive 16:00 Q&A

November 2010 5

The post-genomic era challenge

A genome is like a recipe...CTCTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGAGGCGACTGGTGAGTACGCTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGACATAGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGCGAGCCCTCAGATGCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAGGTCTCTCTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGCAAGAGGCGAGGGGAGGCGACTGGTGAGTACGCTTTGACAGCCGCCTAGCATTTCAAAGGGAAACCAGAGGAGCTCTCTCGACGCA...

...CTCTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAATACGCTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGACATAGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGCGAGCCCTCAGATGCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAGGTCTCTCTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGCAAGAGGCGAGGGGAGGCGACTGGTGAGTACGCTTTGACAGCCGCCTAGCATTTCAAAGGGAAACCAGAGGAGCTCTCTCGACGCA...

Modifying it matters

So why not use it to improve life?

November 2010 6

• The number of complete genomes sequenced is increasing more rapidly than expected

• 1995: First sequence of a living organism (Haemophilus influenzae, 1.8M bp)

• 2002: Sequence of the mouse genome (2.2 billion bp)• 2003: First precise human genome sequence (2.9 billion bp)• 2005: Sequence of the rice genome (400M bp)

A very short history of sequencing

• The cost of sequencing a complete genome is dropping rapidly• First human genome: 2.7 billion $• 2010: 10,000$• 2012: 1,000$• 2015: 300$?

November 2010 7

• Research and production• Analysis of gene functions• Production of bioproteins

Genome engineering today

• Agrobiotech• New traits that are herbicide resistant, pesticide resistant, or have better

yields of production

• Healthcare• First gene therapies for well characterized monogenic diseases

November 2010 8

Genome engineering tomorrow

• Agrobiotech• New traits with improved nutritional properties or adapted to new climate

conditions, containing little to no foreign DNA sequences

• Healthcare• Custom medicine, able to cure a patient of a disease even before the

appearance of the first symptoms• Gene therapy 2.0 with complete control of the inserted or modified gene

• Energy• Production of petrol substitutes from algae

• White biotech• Plastics, fibers… produced from living organisms• …

9

Cellectis - a leader in genome engineering

André ChoulikaCEO of Cellectis

November 2010

Cellectis in brief

World pioneer in targeted genome engineering; established in Dec 1999Listed on Paris Stock Exchange since 2007: Alternext : ALCLS

Company

Organization

Technology Proprietary platform based on meganucleases (DNA scissors)Extensive and valuable intellectual property estate

Partners More than 50 agreements since inception (€40M generated so far)Strategically positioned in core interest areas

Financials Over €25m cash and growing revenue base

HQ in Romainville, Paris - France; > 120 employees (45 PhDs)4 subsidiaries, including one located in Saint Paul, Minnesota, USADiversified business model ; key segments in tools, agrobiotech and therapeutics

10

November 2010 11

The first in vivo DNA “cut & paste”

• Naturally occurring DNA scissors found in single-celled organisms• First discovered in baking yeast in the 1980’s• Cut DNA at a unique target site (motif of 12-30 base pairs)• Create an opening in DNA to allow sequence insertion, deletion and/or repair• Stimulate homologous recombination

Meganucleases – The basics

Chromosome

Human genome size: 6.4 billion bases

(G, A, T, C).

Meganuclease

DNA repair/insertion(Paste)

DNA cut with incredible target

specificity“Cut”

November 2010 12

Genome engineering – 3 principles of action

November 2010 13

Harvard Institute of Medicine

Research Tools

Production Tools

Upstream Research TherapeuticsAgrobiotech

Multiple partnerships strongly supporting the technology

November 2010 14

A market-based organization

Therapeutics

Agrobiotech

Production tools

Research tools

Stem cells

15

Update on the science of meganucleases

Philippe DuchateauHead of Meganuclease Research Department

November 2010

The meganuclease research department

16

Meganuclease Research is a core corporate function driving innovation

It is composed of 3 groups: Protein engineering, Cell biology and Computational biology

Its key objective is to keep the meganuclease technology at its very best and continue improving it

We are developing new tools to improve meganuclease-induced HR or mutagenesis.

November 2010

1 – Targeted approaches induced by meganucleases

November 2010

Different endonucleases used for targeted recombination

18

Zinc-Finger Nucleases

Chemical endonucleases

• Natural proteins• 1st endonucleases used for genome engineering • Low apparent modularity (2 separable domains)

• Artificial protein : zinc finger protein (DNA binding domain) fused with a catalytic domain (FokI)• 1st engineered endonuclease used to edit a human gene• High modularity (6-8 separable domains “polydactyls”)

• Chemical DNA binding domain (TFO, polyamine) fused to effector (chemical or restriction enzyme)• High modularity

TALE Nucleases

Meganucleases (homing endonucleases)

• DNA binding domain from Transcription Activator Like Effectors from Xanthomonas• Very high modularity (potential code)• Early stage technology

November 2010

The four major families of nuclease-mediated genome engineering methods

19

x x

Gene correction

Targeted deletion (knock-out)

Gene inactivation

Homologous Recombination Non-HomologousEnd Joining

(NHEJ)

Gene conversion

Targeted insertion (knock-in)

November 2010

2 - Engineered meganucleases

November 2010

Omegabase (proprietary database) Evolution over time

The combinatorial approach

21

• Hit frequency: 1/350 bp; success rate ≥ 40% • Hit frequency: 1/100 bp; success rate ≥ 25% • Timeline: 10 weeks-9 month depending on difficulty (deliverable: meganuclease characterized in a cell-based assay), possible further refinement for therapeutic grade• Production capacity: 100 last year (100 different targets)

Arnould et al. (2006) J. Mol. Biol.Smith et al. (2006) Nucleic Acids Res.Arnould et al. (2007) J. Mol. Biol.Grizot et al. (2009) Nucleic Acids Res.

5’-T G T T C T C A G G T A C C T C A G C C A G-3’3’-G C A A G A G T C C A T G G A C T C G G T C-5’

Targetable DNA sequence=

patchwork of cleavable sequence

November 2010

Improving meganuclease features: expanding number of potential targets

22

Definition of a new DNA region for which the I-CreI specificity can be modified

5’ C A A A A C G T C G T A C G A C G T T T T G 3’3’ G T T T T G C A G C A T G C T G C A A A A C 5’

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1

Such a meganuclease could hitvirtually any 22bp DNA sequence

Target DNA sequence diversity up to 18/22bp

CAAAACGTCGTACGACGTTTTGI-CreI

Examples ofnew cleaved targets

14 / 22 mismatchesCTTGGACTCATAAGAGTCCAAG

CTGGCACCCGTACGGGTGCCAG

November 2010

Improving meganuclease specificity

23

Example of the SH4 meganuclease presents an intermediate toxicity profile

Based on specificity profiles, replacement of individual modules

0

0,1

0,2

0,3

0,4

0,5

0,6

SH4 SH4newnon toxic

3 days 7 days

% In

/Del

toxicSH4 SH4new

non toxictoxic

Activity at the endogenous locus

No impact of toxicity Toxicity has a long term impact

Toxicity in cell survival assay

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25

Transfected DNA (ng)

Cell

surv

ival

(%)

SH4SH4newI-SceII-CreI

November 2010

Creating new scaffolds

24

1 21 32 4 3 54 6

I-CreII-MsoII-DmoII-AniIbI3

PI-SceII-SceII-CeuII-ChuI

I-CreII-MsoII-DmoII-AniIbI3

PI-SceII-SceII-CeuII-ChuI

Domains shuffling

Hybrid meganuclease

…

- Active hybrid meganucleases can be obtained Example: DmoCre

Chevalier et al. (2002) Mol. CellEpinat et al. (2003) Nucleic Acids Res.

November 2010

3 - Characterizing the activity of engineered meganucleases

November 2010 26

1.7 Kb

2kb 1.2kb

No DNA matrix

10cells/well(293H w/o selection)

PCR Screen

FACS analysis(GFP+ CHO-K1 cells)

PCR amplification on 293H cell population

MUTAGENESIS GENE INSERTION TOXICITY

1- + 2 3 4 5 6

454 sequencing technology

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60

DNA qty (ng)

Cel

l su

rviv

al %

age

hADCY9 hCTSZ hSMC5 SC_Rag I-Cre D75

Cell survival assay

SC-RAG

I-SceI

EXTRA-CHROMOSOMALACTIVITY

SSA, CHO-K1β-Gal activity rescue

November 2010

Meganuclease-induced HR at endogenous locus

27

PCR+ clones

WT

Targeted3kb

4kb5kb

MWHE

K29

3

6-8% of gene targeting observed(> 550 clones analyzed)

5’

5’ 3’

10-1700bp

HindIII

3’

In vitro cleavage

Cloning into 96 well plates

or 10 cells/well

Amplification and Southern Blot analysis

PCR analysis

RAG1

Example of the RAG1 gene• involved in immunoglobulin and T-cell receptor recombination• mutations cause Omenn syndrome, an autosomal recessice form of SCID

November 2010

The NHEJ and HR at a same locus have correlated frequencies

28

¹ Up to 20% with other experimental design

² 6% in clonal analysis

KI frequencies normalized to plating efficiency (30%)

Transfection293H cells

gDNA extraction (day 5-7)

PCR

Deep sequencing(individual molecules)

I

Gene mutation(NHEJ)

MN InDel % RH Freq.

WAS5 1.0 11.4

DMD21 1.4 ² 8.7¹

RAG1 1.5² 8.1

SH1 1.1 5

SH2 0.6 1.2

IL2RG3 0.2 1.08

MN11 0.2 0.9

DMD31 0.2 0.6

MN5 0.5 0.6

MN6 0.4 0.5

DMD33 0.1 0.09

November 2010

Improvement of MNs efficacy in vivo (I)

29

XPC4 target:(2 methylated CpG)

Methylation (%)5azadC (µM)

Meganuclease

0,20In

Del

(%)

NANA

CAPNS1CAPNS1

0

1

2

3

4

5

6

7

8

0 0,2 1~60~60100

- XPC4 XPC4

1~40

-

0100

XPC4

CAPNS1 target:(3 unmethylated CpGs)

0

1

2

3

4

5

6

7

XPC target

Two CpGs 100% methylated in 293 cells

T C G A G A T G T C A C A C A G A G G T A C G Ametmet

Mutations within this gene cause Xeroderma pigmentosa characterized by increased sensitivity to ultraviolet (UV) irradiation and risk of skin cancer, resulting from a defect in DNA repair

November 2010

Improvement of MN-induced HR in vivo

30

Two approaches:siRNA Small compounds gene identification target unknowndifficult to deliver easy to deliver

• 290 genes identified whose down regulation led to gene targeting stimulation• 66 genes validated with secondary screening

• 19000 genes screened with 2 siRNA per gene • 18000 compounds screened• 100 compounds identified which stimulate gene targeting• 2 compounds confirmed on secondary screening

6Impact of siRNA on the GT efficiency

at the endogenous RAG1 locus.

4.3% 8%

EGFP

EGFPRecognitionsite

meganuclease

EGFP

No compound Active compound

FACS analysis

0

1

2

3

4

5

AS EP300 ATF7IPsiRNA 33nM

Fold

incr

ease

of

KI f

requ

ency

vs

cont

rol

November 2010

Improvement of MN-induced mutagenesis in vivo

31

Strategy: selected siRNA targeting genes involved in DNA repair

Meganuclease-induced mutagenesis at RAG endogenous locuswith selected siRNA

0

0,5

1

1,5

2

2,5

control Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6

siRNA 1nM

%ag

e of

mut

agen

esis

at R

AG1

locu

s

November 2010

Conclusions

32

Using a combinatorial approach, it is possible to engineer meganucleases with tailored specificities

Based on the growth of the Omegabase, it is possible today to engineer meganucleases for virtually any gene

The activity of a meganuclease is not the sole factor governing efficacy at the endogenous locus

We are developing new tools to improve meganuclease-induced HR or mutagenesis.

33

Research Tools

Christophe DelendaCSO of Cellectis bioresearch

November 2010

Different Types of Research Tools

CBR research tools and products for in cellulo applications :

• Products already commercialized in secondary cell lines• Next demonstrations to come (2011) with primary cells

• A CBR parallel objective would be to design in vitro research tools :

• Meganucleases as restriction enzymes (2012?)

34

November 2010

Different Types of Genome Modifications

Gene targeted integration (knock-in)

• shRNA targeted integration (knock-down)

• Gene deletion or disruption (knock-out)

35

Modulation of expression

Absence of expression

Drug discovery

Gene function

Protein production

ApplicationsOver-

expression

Different types of genome customization via meganuclease-driven expression

November 2010

Meganuclease-Driven Gene Knock-In

36

GOI : gene of interest

Meganucleases for DNA target site cutters

Double-strand DNA break induction

A DNA repair matrix containing the GOI to be integrated is used

as template for homologous recombination

November 2010

Two Approaches for Site-Directed Gene Knock-In

37

Pre-engineered cell linesPre-insertion of a landing pad containing

a natural meganuclease site

Any cells from a same speciesEndogenous site recognized by a

meganuclease with modified specificity

Two lines of kits

1

2 3 2 3

cGPS® : cellular Genome Positioning

System

November 2010

Example of a cGPS®-Related Product – cGPS® CHO-K1 (1)

38

cGPS® CHO-K1 Cell Line200 000 cells / 10cm dish

Co-transfection

D+1

D+8

Selection 1G418 (Neo)

D+24

Picking(96-well plate)

Selection 2G418 + Puro

NeoR PuroR cell clones ~ 200 generated

1

2 3

1 3+ 1 2+ 1 2+ + 3

LacZ Control Integration Matrix2

Without IntegrationMatrix

Without MeganucleaseExpression Vector

D+13

Fast and effortless protocol

High selection frequency (~1x10-3)

November 2010

Example of a cGPS®-Related Product – cGPS® CHO-K1 (2)

39

Integ

ration M

atrix

Targeted LacZ+ cell clones

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CHO-K1

p10.1

cGPS

CHO-K1

> 10 kb

Very efficient site-directed gene knock-in (> 95%)

With minor associated random integrations (<10%)

Genetic pattern of targeted (vs untargeted) cell clonesDetermined by using a dedicated radioactive probe (Southern blotting analysis)

Random insertions

Targeted integration

November 2010

Example of a cGPS®-Related Product – cGPS® CHO-K1 (3)

40

Transgene expression pattern of targeted cell clonesDetermined by reporter protein expression (LacZ, luciferase)

With selection Without selection

1

10

100

1000

10000

0 10 20 30 40 50

Passages

Re

lati

ve

La

cz

un

it

1

10

100

1000

10000

0 10 20 30

Passages

Re

lati

ve

La

cz

un

itPassages

1

10

100

1000

10000

0 10 20 30 40 50

Re

lati

ve

Lu

c u

nit

0 10 20 30

Passages

1

10

100

1000

10000

Re

lati

ve

Lu

c u

nit

LacZ

ge

ne m

odel

Luci

fera

sege

ne m

odel

Homegeneity and stability of expression over time(with and without selection drugs)

November 2010

Knock-In Systems (kits)

41

cGPS® CHO-K1cGPS® CHO-S CemaxcGPS® HEK-293

Hamster cell lines

Human cell line

New human cell lines

New meganuclease target sitefor human cell lines

cGPS® Custom CHO-K1 (HPRT locus)cGPS® Custom HEK-293 (RAG1 locus)cGPS® Custom Human (RAG1 locus)cGPS® Custom HCT116 (RAG1 locus)cGPS® Custom Jurkat (RAG1 locus)cGPS® Custom U2OS (RAG1 locus)cGPS® Custom K562 (RAG1 locus)cGPS® Custom MRC5 (RAG1 locus)cGPS® Custom HEK-293 (DMD locus)cGPS® Custom HCT116 (DMD locus)cGPS® Custom Jurkat (DMD locus)

cGPS® CHO-K1 Duo (cGPS® & HPRT & loci)cGPS® HEK-293 Duo (cGPS® & RAG1 loci)cGPS® HEK-293 Custom Duo (RAG1 & DMD loci)

cGPS® systems

cGPS® Custom systems

cGPS® Duo systems Each system is sold with a dedicated and adequate protocol (i.e. User Manual)

November 2010

pIM and Mega Stores

42

Integration matrices also sold separately with adequate “goodies” for various applications (i.e. markets);

Two integration matrices per cGPS® knock-in system, i.e. one for the cloning of the gene of interest and the other encoding a reporter gene (for control of integration);

Necessary reload products for cGPS®-related kits;

Meganuclease vectors also sold separately for specific gene knock-out applications, such as GS (Lonza’s service project)

Other engineered meganucleases for enlarging their usage to other model organisms, such as mouse, rat, fishes (medaka, zebrafish), xenopus, worm, drosophila…

pIM STOREIntegration matrices

MEGA STOREMeganuclease vectors

November 2010

Current Clients

43

IndustrialServier, Galapagos, Novartis, New England Biolabs, Actelion, Biogen Idec, Boehringer Ingelheim, Abbott, KTH, Euromedex, Oncomed, CSIR Biosciences, SuperGen, GSK Canada, Xention Pharmaceuticals

AcademicMount Sinai School of Medecine, University of Minnesota, Memorial Sloan-Kettering Cancer Center

Others via distributorsTebu Bio (Europe), Wako Chemicals (Japan), Cedarlane (Canada)

Kits and sub-products (pIM & MEGA) Service

Knock-outLonza

Knock-inServier, Xention Pharmaceuticals, Cytoo

44

Genome Engineering in Plants

Dan VoytasCSO of Cellectis plant sciences

November 2010

The challenge of feeding the world population

45

Arable land required to sustain a Western diet for one year

November 2010

The challenge of feeding the world population

46

Arable land currently available per person globally

November 2010

The challenge of feeding the world population

47

Arable land available per person globally in the year 2050

November 2010

The challenge of feeding the world population

48

Source: Nature 2010. 466:546

November 2010

Important Contributors to Modern Agricultural Productivity

49

Use of Plant Hybrids

Green Revolution

Transgenesis

November 2010

Important Contributors to Modern Agricultural Productivity

50

Use of Plant Hybrids

Green Revolution

Transgenesis

Genome Engineering

Genetic variability enables crop improvement

November 2010

Historical Corn Yields in the United States

51

Open-Pollinated

Double-Cross Hybrids

Single-Cross Hybrids

Transgenics

Source: USDA

November 2010

Genome Engineering is now possible in plants

• Cellectis’ technology enables precise modification of plant genomes at high efficiency

• Genomic information is now available for numerous plant species providing many target genes for modification

• New crop varieties can be developed by first selecting the desired trait and then implementing the trait through genome engineering

52

November 2010

Mission of Cellectis plant sciences

53

• Optimize methods for genome engineering of plant cells

• Provide technical support to Cellectis’ licensees to

expedite implementation of the technology

• Establish commercial relationships as outsourced R&D

or risk-shared collaboration

• Develop proprietary traits

November 2010 54

29205 kb

17463 kb

23560 kb

22140 kb

26170 kb

Chr. I

Chr. II

Chr. III

Chr. IV

Chr. V

1

3

4

5

7

8

910

11

121315

16

17

18

19

20

21

22

14

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

6

238

39

40

Hits in exons of known coding regions

Hits in exons of undefined coding regions Hits in introns of undefined coding regions

Hits in introns of known coding regions Hits within non-coding regions

Centromere

Proof of activity of QMAPs in Arabidopsis

November 2010

Generating Germinal mutations

Introduce meganuclease into plant

Induce expression of meganuclease in

seedlings

Analyze genomic DNA for meganuclease activity

Testing meganucleases in Arabidopsis

55

November 2010 56

Transformation of Mnase

MNase induction

Genomic DNA prep on Pooled samples

PCR

Deep sequencing to estimate frequency

1% mutagenesis frequency has been achieved by Arabidopsis QMAPs.

Mutations induced by Arabidopsis QMAPs

November 2010

Mission of Cellectis plant sciences

57

• Optimize methods for genome engineering of plant cells

• Provide technical support to Cellectis’ licensees to

expedite implementation of the technology

• Establish commercial relationships as outsourced R&D

or risk-shared collaboration

• Develop proprietary traits

November 2010 58

Harvard Institute of Medicine

Research Tools

Production Tools

Upstream Research TherapeuticsAgrobiotech

Principal Partnerships

November 2010

Mission of Cellectis plant sciences

59

• Optimize methods for genome engineering of plant cells

• Provide technical support to Cellectis’ licensees to

expedite implementation of the technology

• Establish commercial relationships as outsourced R&D

or risk-shared collaboration

• Develop proprietary traits

November 2010

What’s new?

60

Genome engineering can create crops with valuable traits without adding foreign DNA

Addition of bacterial gene

Targeted modification of native plant gene

Herbicide Tolerance

TraditionalTransgenesis

Genome Engineering

November 2010

Herbicide tolerance created through genome engineering

61

Unmodified PlantsPlants with

Engineered Genomes

Both groups of plants exposed to herbicide

62

Meganucleases for Therapeutic Programs

Carole DesseauxHead of Preclinical and Clinical Programs

November 2010

Cellectis genome surgery - Overview

Gene therapy trials have dramatically improved the vision of patients who suffer from Leber congenital amaurosis, an hereditary blindness.

• Marked clinical improvements in young children with Wiskott-Aldrich syndrome, a very rare but severe immunodeficiency disorder

• Gene therapy could remedy Parkinson's disease: significant improvements in motor behaviour of monkeys after two weeks, without any visible adverse effects

• Gene therapy corrects adrenoleucodystrophy in two children

63

Gene therapy recently marked successful milestones:

Our mission is to establish genome surgery as a standard in the therapeutic field

November 2010

Cellectis genome surgery’s Mission Statement

64

Cellectis genome surgery is dedicated to the development of new approaches using meganucleases

target site

degradation

sequence removal

Virus clipping Gene correction Gene insertion « safe harbor »

« cleanest » approachLimited by the length of conversion tracts

VersatileProperties of locus to be assessed

November 2010

Gene Correction and Safe Harbor Approach

65

November 2010

The antiviral approach

66

HIV

RT

Int

Viral genomic RNA is reverse transcribed in the cytoplasm and the resulting dsDNA is integrated into the host DNA

Latent infection(circular viral DNA)

Productive infection(linear viral DNA)

HSV

Viral DNA enters the nucleous and transcription starts after circularization of the DNA genome

HBV

November 2010 67

Therapeutics R&D

November 2010

The Therapeutic Development Group

68

•Cell Proof of Concept

•Animal Proof of Concept

•Vectorization studies

•Validation of optimised leads and final vectors

•Validation of model/methods•Non clinical studies

(pharmacology, toxicology, …)

•Clinical studies

•Drug Manufacturing

•Regulatory strategy

Founded in 2008

20 employees including 10PhDs

November 2010

Strategy

69

Develop new treatment approaches for current unmet clinical needs

• Establish key partnerships with « best-in-class » academic labs and industry

• Identify unsuitable therapeutic candidates and stop expensive development programmes

• Strongly evaluate and share the risks associated with development (ie regulatory strategy, clinical and manufacturing feasibility, therapy cost, …)

November 2010

Research Field

70

In-house

• Safe harbor

Homologous recombination in T cell

Homologous recombination in Hematopietic Stem Cell

• Vectorisation

Protein manufacturing set up

Cell penetrating peptides (DPV, Vectocell technology)

Electroporation (Cytopulse technology)

Mouse and cell lines testing models

Collaboration• Gene correctionPr Notarangelo (Rag1)Pr Fischer (IL2RG, Artemis)Dr Sadelain (HBB)Dr Tremblay, Dickson, Voit (DMD)Dr Sarasin (XPC)Pr Thrasher (WAS)Pr Scharenberg (CCR5, canine XSCID)• Safe harborPr VandenDriessche (Haemophilia A et B)Pr Naldini (SH)Pr Bueren/Segovia (Fanconi Anemia / PKLR)Pr Danos (SH + vectorisation)

• AntiviralHSV & HIV : Projet ACTIVE (Pr Labetoule et Dr Gabison, Pr Wain-Hobson)HBV : Pr Zoulim

November 2010

Research Field

71

In-house

• Safe harbor

Homologous recombination in T cell

Homologous recombination in Hematopietic Stem Cell

• Vectorisation

Protein manufacturing set up

Cell penetrating peptides (DPV, Vectocell technology)

Electroporation (Cytopulse technology)

Mouse and cell lines testing models

Collaboration• Gene correctionPr Notarangelo (Rag1)Pr Fischer (IL2RG, Artemis)Dr Sadelain (HBB)Dr Tremblay, Dickson, Voit (DMD)Dr Sarasin (XPC)Pr Thrasher (WAS)Pr Scharenberg (CCR5, canine XSCID)• Safe harborPr VandenDriessche (Haemophilia A et B)Pr Naldini (SH)Pr Bueren/Segovia (Fanconi Anemia / PKLR)Pr Danos (SH + vectorisation)

• AntiviralHSV & HIV : Projet ACTIVE (Pr Labetoule et Dr Gabison, Pr Wain-Hobson)HBV : Pr Zoulim

November 2010 72

Safe Harbor Meganucleases and Blood Cells: ex vivo Approach

November 2010

The Safe Harbor Approach

73

TOXICITY

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60

DNA qty (ng)

Cell

su

rviv

al

%ag

e

hADCY9 hCTSZ hSMC5 SC_Rag I-Cre D75

Cell survival assay

SC-RAG

I-SceI

EXTRA-CHROMOSOMALACTIVITY

SSA, CHO-K1β-Gal activity rescue

No DNA matrix

PCR amplification on 293H cell population

MUTAGENESIS

454 sequencing technology

1.7 Kb

2kb 1.2kb

PCR Screen

GENE INSERTION

controls

- +

CHO-K1 293H

1 - Identify a meganuclease that targets a locus defined as safe with a high specificity and activity

November 2010

The Safe Harbor Approach

74

2 – Vectorize the meganuclease to the cell of interest via prorpietary electroporation system

The pulsed electric fields transiently permeabilizes living cells for delivery of material into cells

Need to establish the conditions necessary to have a good transfection efficiency, expression level and viability of electroporated cells (ongoing).

November 2010

The Safe Harbor Approach

75

3 – Confirm expression of « gene of interest » at the locus and establish the proof of concept of efficacy and safety in partnership with worldwide experts: example of hemophilia

Meganuclease SH+

Matrix with Gene of Interest+

Cell of Interest (CD34+)

SH

FIX

FIX

FIX

FIX

FIX

Advantages of Hemophilia:

• The protein is secreted (several

tissues can be targeted)

• 1% of normal level of expression

is sufficient to obtain a therapeutic

gain

November 2010

The Safe Harbor Approach

76

To date, acceptable percentage of mutagenesis

Good transfection efficiency with acceptable viability in electroporation assay. Expression of the marker gene.

Preliminary results of recombination in CD34+

frequencies normalized to plating efficiency (30%)

MN mutagenesis Recombination

WAS5 0,9 11,4

DMD21** 1,4 8,7

RAG1 * 1,5 8,1

SH 0,6 1,2

IL2RG3 0,2 1,08

MN20 0,1 0,99

MN11 0,2 0,9

DMD31 0,2 0,6

MN5 0,5 0,6

MN6 0,4 0,5

DMD33 0,1 0,09

The strategy will be used for different pathologies by changing

• Matrix of interest (ie Fanconi or sickle cell anemia)• Cell of interest (ie T cell for adaptative cancer therapy)• Meganuclease (antiviral meganuclease for preventing infection)

November 2010 77

Meganucleases and Antiviral ex vivo Approach: Example of HSV

November 2010

The Antiviral Approach

78

The ultimate goal of our project is to prevent graft failure due to reinfection by HSV1 (herpetic keratitis) by ex vivo treatment of donor cornea graft

No effect on latent viruses

Need of chronic antiviral therapy

Current antiviral agents do not destroy viruses but only prevent population growth

November 2010

Anti-HSV Approach

79

Prevention of replication by HSV2 and HSV4

Empty vector HSV1m2 HSV1m4

I-SceI Rag1m I-CreIT48

T120

Empty vector HSV1m2 HSV1m4

I-SceI Rag1m I-CreI

0

20

40

60

80

100

120

140

T48

T120

Blu

e p

laq

ue

nu

mb

er

Transfection COS-7 cells with meganuclease plasmid

(Approx. 0.2 x 106 cells)

Infection rHSV ( MOI 10-3) X-gal staining (0,5%)

(Blue plaque number)

24h

T0 after 1h infection at 37°C Fresh medium

48/120h

November 2010

Anti-HSV Approach

80

ex-vivo PoC (rabbit cornea)

Up to 50% of protection against HSV (number and size lysis) versus infected cornea without meganuclease pre-treatment

• ex-vivo PoC (human cornea) – next steps

• The ex vivo antiviral meganuclease pre-treatment approach could be developed for any organ/cells to be grafted in order to reduce deleterious effects of viral reactivation after transplantation

Whole cornea transduced by a rAAV-GFP coding vector. Observation in confocal microscopy

November 2010

Conclusion

81

Meganucleases engineering allows the development of new potential therapeutic approaches by genome surgery

• Innovative approaches in gene therapy field

• 2 types of indications are developed by Cellectis genome surgery• Antiviral : A new class of antiviral agents, viral genome cleavage (ex: HIV)• Inherited monogenic diseases : Gene correction by HR, insertion in a safe locus

(ex: sickle cell anemia)

Many research programs are conducted in collaboration with academic teams and biotechnology companies in Europe and in the US

82

An Example of Collaboration

Serge BraunChief Scientific Officer

Association Française contre les Myopathies

83

Perspectives - Potential Applications of iPS Cell Technologies

David Sourdive Executive VP, Corporate Development

November 2010

Pluripotent stem cells

84

Potency and indefinite replication

ES EG EC

November 2010

Embryonic stem cells were the first source of pluripotent stem cells

85

Advantages: indefinite self-replication can give rise to any cell type (whole organism)

Drawbacks: requires embryonic cells very limited choice of genotype genotype not from a whole organism with known features and

phenotype differentiation may be difficult, irreproducible, incomplete, etc. not industrial

November 2010 86

Skin (or other adult) cell

ES EG EC

Pluripotent cellsCan be differentiated into any

cell type

iPS

Klf4

Sox2

Oct4C-myc

Making pluripotent stem cells directly from adult cells

Induced pluripotent stem cells

Shinya Yamanaka : 2006; 2007

November 2010

Induced pluripotent stem cells: breakthrough

87

Advantages: indefinite self-replication can give rise to any cell type (whole organism) broad choice of genotypes can be derived from a whole organism with known phenotype

Drawbacks: uses random viral transgenesis of oncogenes recent technology with many features remaining to establish differentiation may be difficult, irreproducible, incomplete, subject to

epigenetic memory etc. not (yet) industrial

November 2010

RAG1

Actin BRHE +

Repair Matrix

Repair Matrix

RHE +

Empty Vector

RHE +

Repair Matrix

Repair Matrix

RHE +

Empty Vector

2.6 kb

Lt Homology Rt Homology

F Primer

4.4 kb

Rt Homology

F Primer R Primer

Lt Homology SV40 NEO IRES MYC

SV40 NEO IRES MYCLt Homology Rt Homology

RAG1RSHE Cut site

3.3 kb

R Primer

Meganucleases induce high levels of recombination in iPS cells

November 2010 89

RAG1

Actin B

RHE +

Repair Matrix

Repair Matrix

RHE +

Empty Vector

RHE +

Repair Matrix

Repair Matrix

RHE +

Empty Vector

0

5

10

15

20

25

30

Rela

tive

exp

ress

ion

(fo

ld

incr

ease

)

Meganucleases induce high levels of recombination in iPS cells

RT-PCR

November 2010 90

Skin (or other adult) cell

ES EG EC

Pluripotent cellsCan be differentiated into any

Cell type

iPS

Klf4

Sox2

Oct4C-myc

Bringing robustness to industrialize induced pluripotent stem cells

November 2010

Rationale

• Focus on industrialization of iPS (reprogrammation, culture, differentiation, etc.)

Goals :

Making iPS safe (targeting dangerous genes, putting safety “devices”, etc.)

Making iPS robust (reproducible differentiation, more “adult” phenotypes, highly enriched (pure) cell populations, etc.)

Turning cells into assay devices

Targeted Medicine

iPS used as tools for research and industry

Regenerative Medicine

iPS used as sources of grafts

• Use genome engineering (like was successfully used to industrialize cell lines)

November 2010

Strategy: robust combination of genotype and phenotype

iPS

Reproducible and robustprocessing

Somatic cells

Samples

Differentiation

Subjects/patientsChoice of genotypes

Choice of phenotypes

Single cell type with many genotypes

in vitro models Source of grafts

Genome engineering

November 2010 93

CellMill: a large iPS cells biobank

55555555

Collectioncenter

55555555

Collectioncenter

55555555

Collectioncenter

55555555

Collectioncenter iPS cell bank with

104 to 105 entries(i.e. iPS + phenotype)

Patients and siblings cohorts

Differentiated engineered

cells/tissues from multiple donors

November 2010 94

Reduce attrition and allow targeted medicine

Develop industrial in vitro tools predicting human physiology, pathology and genetic diversity

Meet a strong industrial demand : better cell-based assays-Reduce attrition and development costs -Allow targeting drugs towards responders

Today, 10% to 20% of drugs entering clinical development reach the market The most expensive failures come late, when the drug is confronted to human genetic diversity.

Responders0% 20% 40% 60% 80% 100%

ß2-agonists (asthae)

statins (cholesterol)

SSRIs (anti-depress.)

ß-blockers (cardio)

Inhib. ACE (Hypertension)

~2 years

Preclinical

1-2 years

Phase I Phase II

2-3 years

…

Phase IVPhase III

10-12 years

20-100 patients Failure: 80-90% Cost: 10%

10 000

mole

cu

les

2505

Filing for marketCommercialization

1

100-500 patients Failure: 60% Cost: 10-15%

500 to thousands patients

Failure: 80-90% Cost: 30-35%

Cell culture animal

Cost: 30%

Pharmacovigilance Cost: 10-15%

3-5 years

IND

November 2010 95

Haplobank: a GMP biobank of haplotypically homozygous iPS cell lines

GMP iPS bank with(i.e. iPS + phenotype)

Existing Frenchregistry of volunteers

for bone marrow donation(1.7 x 105 people)

55555555

55555555

55555555

55555555 Known sub-population

of triple HLA-A B and DR homozygotes(1015 people)

555555

555555

Differentiated engineered cells/tissues of chosen

types and origins

November 2010

Conclusion

Induced pluripotent stem cells unleash the potential of stem cells

Meganucleases to leverage the opportunity lying in iPS cells: Providing control over their behavior Making them robust Making them safe

Focus on industrialization

Target two main fields Tools for research and industry, representative of human physiology,

pathology and genetic diversity Tools for regenerative medicine: source of cells compatible with clinical

applications

November 2010

Structure and partnerships

Expertise in iPS and stem cells

Strategic intellectual property Commercial license to iPS technology for tools Commercial license to iPS technology for therapeutic applications

(first world wide)

Other key players to come …

Key partners and collaborations

Dedicated subsidiary for the project

November 2010

Thank you

Parc Biocitech102, Avenue Gaston Roussel93230 RomainvilleFrance

http://www.cellectis.cominvestors@cellectis.com

Tel: +33 (0) 1 41 83 99 00Fax: + 33 (0) 1 41 83 99 03

Alternext: ALCLS.PA