iGEM SupBiotech Paris Project2009.igem.org/files/presentation/SupBiotech-Paris.pdf · Presentation...
Transcript of iGEM SupBiotech Paris Project2009.igem.org/files/presentation/SupBiotech-Paris.pdf · Presentation...
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iGEM SupBiotech Paris Project
The Double Vector System
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iGEM SupBiotech Paris Project
1st Observation
Double targeting for gene therapy
First Observation
Genes Galenic Vectorization DVS
2
Galenic Vectorization DVSGenes
Sup’Biotech Paris DVS Project 2
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• Viral Disease Insertion of foreign genome
• Cancer Genome modification
• Genetic disease Dysfunctional genome
Identified Problem : The Genome
First Observation :
Problem identificationGalenic Vectorization DVSGenes
Sup’Biotech Paris DVS Project 3
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• Chemical drugs No specificity so Toxic
• Bio-drugs Specific but Temporary
(protein, siRNA, etc)
• Gene insert Highly specific and Permanent
Best agent : gene sequence
Galenic Vectorization DVSGenes
First Observation :
Action on the genome
Sup’Biotech Paris DVS Project 4
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• Very low toxicity
• Specificity
• Need to reach the nucleus
• Low stability outside the cell
√
√
Protection
Galenic Vectorization DVSGenes
First Observation :
Gene characteristics
Sup’Biotech Paris DVS Project 5
Specific sequence
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• Ex vivo Gene action Personal treatment
COST
• Classical galenic Low protection
DEGRADATION
• Vectorization Encapsulation
GENE PROTECTION
Ideal galenic : gene vectorization
6
Galenic Vectorization DVSGenes
First Observation :
How to protect it ?
Sup’Biotech Paris DVS Project
Galenic
6
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• Components:– Reservoir– Targeting system– Stealth system
• Types of vectors :– Virus– Phages– Bacteria– Lipidic nanoparticles– Polymer nanoparticles
Galenic DVSGenes
First Observation :
What is a vector ?
Sup’Biotech Paris DVS Project
Vectorization
7
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Stability Toxicity TargetPassage through
membranesImmune system
resistanceIndustrialization
Virus Good High Cell Very good Null Difficult
Phages Good Null Cell Very low Null Easy
Bacteria Good Low/High Tissue Low Null/ High Easy
Lipidic NPs
Low Low Cell Good Low Medium
polymerNPs
Good Medium Cell Low Low Medium
• The 6 major issues of vectorization
Technological barrier
Galenic DVSGenes
First Observation :
Vectorization issues
Sup’Biotech Paris DVS Project
Vectorization
8
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• Each issue can be resolved by a vector
• Three types of vectors have a genome
• Synthetic Biology :
Possible to mix genomes
Solution : the Double Vector System (DVS)
Galenic Genes
First Observation :
Solution !
Sup’Biotech Paris DVS Project
Vectorization DVS
9
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Characteristics :- Tissue targeting- Immune system resistance - Produces phages under control
Characteristics :- Cell targeting- Passage through membranes- Encapsidates an
exogenous plasmid
Tissue Vector : Specific bacterium
Cell Vector : Recombinant phage
+
10
Galenic Genes
First Observation :
Double Vector System
Sup’Biotech Paris DVS Project
Vectorization
10
DVS
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Tissue Vector :
– Mycobacterium avium subspecies avium
– (a) Phage genome
– (b) System of phage production control
(a) (b)
tetP/O RBS Terminator
Ampicillin
ORI Tetracyclin cassette
pSBA3T5
TetR LacIRepressor
Phage genome
LacP/O
Galenic Genes
First Observation :
Double Vector System
Sup’Biotech Paris DVS Project
Vectorization
11
RBS
DVS
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Doxycyclin
inhibition
tetP/O RBS
LacITetR
Lac P/O
cI repressor
cI synthesis
inhibition
No Doxycyclin
tetP/O RBS
LacITetR
Lac P/O
cI repressor
cI synthesis
inhibition
cI inhibition Production of recombinant phages
Galenic Genes
First Observation :
Control of the Cell vector synthesis
Sup’Biotech Paris DVS Project
Vectorization
12
RBS
RBS
DVS
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Cell Vector :– Lambda Phage
– Controlled lysogeny
– Viral Proteins on the capsid
cI repressor
Lac P/O
Protein DProtein J
Lam
bd
a ge
no
me
Lamb
da gen
om
e
Lambda genome
KanamycinCassette
pLpR
TargetingProtein
Polypeptide III
Galenic Genes
First Observation :
Double Vector System
Sup’Biotech Paris DVS Project
Vectorization
13
DVS
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Type III polypeptide
• From adenovirus penton base
• Fused with the D protein
• Contain RGD motives recognize integrins
• Clustering of integrins
• Internalization of the cell vector into the eukaryotic cell
Galenic Genes
First Observation :
Protein of cell internalization
Sup’Biotech Paris DVS Project
Vectorization
14
DVS
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Therapeutic Plasmid:
– COS sequence for encapsidation
– DTS sequence for nucleus targeting
– Sequence of therapeutic aim
Sequence cos
DTS
Therapeutic sequence
AzithromycineCassette
Galenic Genes
First Observation :
Double Vector System
Sup’Biotech Paris DVS Project
Vectorization
15
DVS
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• Double targeting Highly specific
• Penetration into tissues then into cells Vectorized gene
• Encapsulated gene Protected
• Living organism Stability
• Prokaryotic organisms Low Cost
• Immune system resistance Low clearance
• Use of a dangerous bacterium Hazardous
Injection of doxycyclin induces bacterial lysis
Galenic Genes
First Observation :
DVS advantages
Sup’Biotech Paris DVS Project
Vectorization
16
DVS
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iGEM SupBiotech Paris Project
2nd observation
Cancer is only wrong information !
Sup’Biotech Paris DVS Project
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• Cell containing an issue
• Problem of genetic information
• Wrong information blocking a system
• Often apoptosis system
Solution : To bring the right information !
Sup’Biotech Paris DVS Project 18
GenePresentation
Second Observation :
CancerPromoter
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• Right information is the non-mutated version of a gene.
What happens if you bring this information ?
• Cell can activate the gene pathway.
Anticancer Solution : To provide the missing genetic information
Sup’Biotech Paris DVS Project 19
Presentation Gene
Second Observation :
What is right information ?Promoter
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• Promoters control genetic information response.
• Genetic information is expressed only if required.
Providing wild type promoter allows cells tochoose the right regulation
Sup’Biotech Paris DVS Project 20
Presentation Gene
Second Observation :
How to control the genetic information?Promoter
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iGEM SupBiotech Paris Project
DVS application on Lung Cancer
Sup’Biotech Paris DVS Project
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Tissue vector:
• Pulmonary tropism
Cell vector:
• Unspecific targeting
Therapeutic plasmid :
• « Wild type » version of tumor suppressor gene + « wild type » promoter
Sup’Biotech Paris DVS Project 22
Tissue Presentation
Implementation:
DVS versus Lung CancerCell Apoptosis
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Penetration into the lung
Blood vessel
Sup’Biotech Paris DVS Project 23
Tissue Vector
Macrophage
Macrophage infected by Tissue Vector
Lung
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Blood vessel
Dispersion in the Lung
Sup’Biotech Paris DVS Project 24
Lung
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• 3 murine models: immunodeficient, normal, cancerous.
• Cell suspension is analyzed by flow cytometry. Size and granularity: eukaryotic murine cells ≠ M.avium
Day -7 Day 0 Day 7
-Inoculation of fibroblastic cancer cells - subcutaneously - normal mice
Inoculation of 10^6 CFU.ml-1 of M.avium,-IV route in the tail -3 mice models
Mice sacrifice, organs extraction and cell suspension from the lungs, tumors
Sup’Biotech Paris DVS Project 25
Tissue Presentation
Implementation:
Tissue TargetingCell ModelingTissue Presentation Cell Apoptosis
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Sup’Biotech Paris DVS Project 26
Implementation:
Tissue TargetingTissue Presentation Cell ModelingTissue Presentation Cell Apoptosis
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• Our
experiments
• Literature
Too short to prove the presence
of M. avium in lung
Proven many times
DVS can be used on lung cancer
Sup’Biotech Paris DVS Project 27
Tissue Presentation
Implementation:
Tissue TargetingCell ModelingTissue Presentation Cell ModelingTissue Presentation Cell Apoptosis
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Cell Vector Release
Sup’Biotech Paris DVS Project 28
Cell Vector
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Cell Vector Dispersion and Cancer Cells Targeting
Sup’Biotech Paris DVS Project 29
Cell Vector
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Membrane Receptor Targeting
Cancer Cell
Cell Vector
30Sup’Biotech Paris DVS Project 30
Integrin
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Cell Vector Internalization
Sup’Biotech Paris DVS Project 31
Nucleus
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Nucleus
Insertion of the gene into the target cell
Paradigm of therapeuticplasmid release is not
elucidated
Sup’Biotech Paris DVS Project 32
acidification
acidification
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• Wild Type Lambda phage
• Recombined Lambda phage with penton bases on D proteins
Source: Stefania Piersanti et al., 2004
Sup’Biotech Paris DVS Project 33
Tissue Presentation
Implementation:
Cell TargetingCell ModelingTissue Presentation Cell ModelingTissue Presentation Cell Apoptosis
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• Fusion of adenovirus 5 penton base with D protein
From Lambda
phage genome
From a plasmid
coding for the virus
Sup’Biotech Paris DVS Project 34
Tissue Presentation
Implementation:
Cell TargetingCell ModelingTissue Presentation Cell ModelingTissue Presentation Cell Apoptosis
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• Fusion protein not built in time.
• Capacity to infect eukaryotic cells and to quit endosomes.
• RGD fragment alone: higher efficiency of interaction with integrinsof eukaryotic cells.
• In our application: use of the complete sequence
Possible to use a recombined Lambda phage to insert therapeutic genes
Sup’Biotech Paris DVS Project 35
Tissue Presentation
Implementation:
Cell TargetingCell ModelingTissue Presentation Cell ModelingTissue Presentation Cell Apoptosis
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Nucleus
NLS recrutement by DTS sequence
Gene insert
36Sup’Biotech Paris DVS Project 36
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Nucleus
Nuclear insertion37
Sup’Biotech Paris DVS Project 37
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Chromosome
Therapeutic gene expression
Nucleus
p53 wt
Apoptosis pathwayinduction
38Sup’Biotech Paris DVS Project 38
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• Cell population: Prostatic cancer mutated p53 DU-145
• Kinetic monitoring of apoptosis induction : annexin V assay every 6 hours for 48 hours after electroporation
– Control population
– pcDNA3 CMV+p53wt
Sup’Biotech Paris DVS Project 39
Tissue Presentation Cell ModelingTissue Presentation Cell ModelingTissue Presentation Cell
Implementation:
Apoptosis InductionApoptosis
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• Apoptosis detection
Specific fixation of the annexin V coupled with a fluorophore and analysis by flow cytometry.
Source: Chunlin Yang et al Adenovirus-mediated Wild-Type p53 Expression Induces Apoptosis and
Suppresses Tumorigenesis of Prostatic Tumor Cells 1995
Sup’Biotech Paris DVS Project 40
Tissue Presentation Cell ModelingTissue Presentation Cell ModelingTissue Presentation Cell
Implementation:
Apoptosis InductionApoptosis
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Application of DVS in non small cell lung cancer is confirmed
p53
wild-
type
p53
mutated cell
population
- Apoptosis
- Decreased
tumor
population
Sup’Biotech Paris DVS Project 41
Tissue Presentation Cell ModelingTissue Presentation Cell ModelingTissue Presentation Cell
Implementation:
Apoptosis InductionApoptosis
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Blood vessel
Apoptosis Induction 42
Sup’Biotech Paris DVS Project 42
Lung
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Lung: natural tropism of Mycobacterium avium
DVS: reactivation of apoptotic pathway in tumor cells by bringing the wild-type version of tumor suppressor genes.
DVS is a straight alternative to current treatments.
Sup’Biotech Paris DVS Project 43
Conclusion
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To confirm the concept on lung cancer
To adapt it on other diseases
Sup’Biotech Paris DVS Project 44
Perspectives
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Our compilation system is not easy but really useful!
This new concept brings new outlooks for synthetic biology
Sup’Biotech Paris DVS Project 45
iGEM innovation
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Sup’Biotech Paris DVS Project 46
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• Our instructors:• Pierre Ougen, Project director at Sup’Biotech Paris
• Gavin Browne, in charge of international relationship
• Scientists: Our sponsors:• Lluis M Mir
• Karim Benhioud
• Bassim Al-sakere
• Brian D Roberston
• Nicolas Veziris
• Srinivas Kaveri
• Vladimir Lazar
• Benyoussef Naimi
• Franck Griceli
• Claudie Bourgaux
• Claudia Nobrega
• Jean-Yves Trosset
• And the others…
Sup’Biotech Paris DVS Project 47
Acknowledgements
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48Sup’Biotech Paris DVS Project 48
Annexs
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Doxycyclin
inhibition
tetP/O RBS
LacITetR
Lac P/O
cI repressor
cI synthesis
inhibition
No Doxycyclin
tetP/O RBS
LacITetR
Lac P/O
cI repressor
cI synthesis
inhibition
cI inhibition Production of recombinant phages
Galenic Genes
First Observation :
Control of the Cell vector synthesis
Sup’Biotech Paris DVS Project
Vectorization
49
RBS
RBS
DVS
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50
Galenic Genes
About:
Mycobacterial membrane
Sup’Biotech Paris DVS Project
Vectorization DVS
50
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51
Galenic Genes
About:
Side effects of bacterial lysis
Sup’Biotech Paris DVS Project
Vectorization DVS
51
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• Insertion sequences: insertion of both the phage genome and the control plasmid into the bacterialgenome
• Just one plasmid, the therapeutic one, replicated and transmitted to the daughter bacterial cells
52
Galenic Genes
About:
Plasmid repartition after cell division
Sup’Biotech Paris DVS Project
Vectorization DVS
52
Repressor
Phage genome
LacP/O tetP/O RBS TerminatorAmpicillin
ORI Tetracyclin cassette
pSBA3T5
TetR LacI
tetP/O RBS Terminator
Ampicillin
ORI Tetracyclin cassette
pSBA3T5
TetR LacIRepressor
Phage genome
LacP/O RBSInt
attP
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Galenic Genes
About:
Encapsidation mechanism of COS sequences
Sup’Biotech Paris DVS Project
Vectorization DVS
53
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• Bioluminescence protocol, which allows the real-time monitoring.
• Good reporter system to analyze the mycobacterial implantation and the clearance in vivo.
Sup’Biotech Paris DVS Project 54
Tissue Presentation
Experimental perspective
Tissue targetingCell ModelingTissue Presentation Cell ModelingPresentation Cell Apoptosis
Luciferase
Kanamycin
resistance
int
attP
High
Promoter
Provided by Dr.Brian D.Robertson
Tissue
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Sup’Biotech Paris DVS Project 55
Tissue Presentation
Experimental perspective
Tissue targetingCell ModelingTissue Presentation Cell ModelingPresentation Cell Apoptosis
• Exemple of obtained images by the CCD camera:
Tropisms: Implantation in different tissue Quantification:
Extracted from: Bioluminescent Monitoring of In Vivo Colonization and Clearance Dynamics by Light-Emitting Bacteria, Brian D.Robertson
Tissue
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• Introduction of the fusion protein in a BioBrick plasmid.
+ Antibiotic resistance
to confirm the transfection into bacteria
+ GFP reporter gene with CMV promoter
to confirm the transfection into eukaryotic cells
Sup’Biotech Paris DVS Project 56
Tissue Presentation
About:
Cell TargetingCell ModelingTissue Presentation Cell ModelingTissue Presentation Cell Apoptosis
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Sup’Biotech Paris DVS Project 57
Tissue Presentation
About:
Therapeutic plasmidCell ModelingTissue Presentation Cell ModelingTissue Presentation Cell Apoptosis
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Sup’Biotech Paris DVS Project 58
Tissue Presentation
About:
Treatment efficiency ModelingCell ModelingTissue Presentation Cell ModelingTissue Presentation Cell Modeling
With :
-Nc (t), the number of cancer cells depending time,- V (t), tumor volume,- V1 and V2, two tumors volumes respectively times t1 and t2, - Vcc, the volume of a cancer cell,- Nbi, the number of injected tissue vectors,- Pp, the lung percentage of tissue vectors relative to the injected dose,- DTB, the doubling time of tissue vector,- tinj, injection time of the tissue vectors],- Npl, the number of cell vectors released by bacteria.- λ, phage efficiency
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Sup’Biotech Paris DVS Project 59
Tissue Presentation
iGEM for us !
Cell ModelingTissue Presentation Cell Modeling