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
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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• …
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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
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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
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Update on the science of meganucleases
Philippe DuchateauHead of Meganuclease Research Department
November 2010
The meganuclease research department
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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
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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
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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
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• 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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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.
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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?)
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November 2010
Different Types of Genome Modifications
Gene targeted integration (knock-in)
• shRNA targeted integration (knock-down)
• Gene deletion or disruption (knock-out)
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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
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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
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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)
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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)
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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)
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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
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1000
10000
0 10 20 30 40 50
Re
lati
ve
Lu
c u
nit
0 10 20 30
Passages
1
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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)
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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
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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
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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
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Genome Engineering in Plants
Dan VoytasCSO of Cellectis plant sciences
November 2010
The challenge of feeding the world population
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Arable land required to sustain a Western diet for one year
November 2010
The challenge of feeding the world population
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Arable land currently available per person globally
November 2010
The challenge of feeding the world population
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Arable land available per person globally in the year 2050
November 2010
The challenge of feeding the world population
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Source: Nature 2010. 466:546
November 2010
Important Contributors to Modern Agricultural Productivity
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Use of Plant Hybrids
Green Revolution
Transgenesis
November 2010
Important Contributors to Modern Agricultural Productivity
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Use of Plant Hybrids
Green Revolution
Transgenesis
Genome Engineering
Genetic variability enables crop improvement
November 2010
Historical Corn Yields in the United States
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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
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November 2010
Mission of Cellectis plant sciences
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• 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
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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
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•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://[email protected]
Tel: +33 (0) 1 41 83 99 00Fax: + 33 (0) 1 41 83 99 03
Alternext: ALCLS.PA
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