iGEM SupBiotech Paris Project The Double Vector System First - - PowerPoint PPT Presentation

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iGEM SupBiotech Paris Project The Double Vector System First Observation Genes Galenic Vectorization DVS Genes Galenic Vectorization DVS iGEM SupBiotech Paris Project 1 st Observation Double targeting for gene therapy SupBiotech

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SLIDE 1

iGEM SupBiotech Paris Project

The Double Vector System

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SLIDE 2

iGEM SupBiotech Paris Project

1st Observation Double targeting for gene therapy

First Observation

Genes Galenic Vectorization DVS

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Galenic Vectorization DVS Genes Sup’Biotech Paris DVS Project

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SLIDE 3
  • Viral Disease

 Insertion of foreign genome

  • Cancer

 Genome modification

  • Genetic disease

 Dysfunctional genome

Identified Problem : The Genome

First Observation :

Problem identification

Galenic Vectorization DVS Genes Sup’Biotech Paris DVS Project

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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 DVS Genes

First Observation :

Action on the genome

Sup’Biotech Paris DVS Project

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  • Very low toxicity
  • Specificity
  • Need to reach the nucleus
  • Low stability outside the cell

√ √ Protection

Galenic Vectorization DVS Genes

First Observation :

Gene characteristics

Sup’Biotech Paris DVS Project

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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

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Galenic Vectorization DVS Genes

First Observation :

How to protect it ?

Sup’Biotech Paris DVS Project Galenic

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SLIDE 7
  • Components:

– Reservoir – Targeting system – Stealth system

  • Types of vectors :

– Virus – Phages – Bacteria – Lipidic nanoparticles – Polymer nanoparticles

Galenic DVS Genes

First Observation :

What is a vector ?

Sup’Biotech Paris DVS Project Vectorization

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Stability Toxicity Target Passage through membranes Immune system resistance Industrialization 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 polymer NPs Good Medium Cell Low Low Medium

  • The 6 major issues of vectorization

 Technological barrier

Galenic DVS Genes

First Observation :

Vectorization issues

Sup’Biotech Paris DVS Project Vectorization

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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

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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

+

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Galenic Genes

First Observation :

Double Vector System

Sup’Biotech Paris DVS Project Vectorization

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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 LacI

Repressor

Phage genome LacP/O Galenic Genes

First Observation :

Double Vector System

Sup’Biotech Paris DVS Project Vectorization

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RBS DVS

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SLIDE 12

Doxycyclin

inhibition tetP/O RBS

LacI TetR

Lac P/O

cI repressor

cI synthesis inhibition

No Doxycyclin

tetP/O RBS

LacI TetR

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

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RBS RBS DVS

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Cell Vector :

– Lambda Phage – Controlled lysogeny – Viral Proteins on the capsid

cI repressor

Lac P/O

Protein D Protein J

Lambda genome Lambda genome Lambda genome

Kanamycin Cassette pL pR

Targeting Protein Polypeptide III

Galenic Genes

First Observation :

Double Vector System

Sup’Biotech Paris DVS Project Vectorization

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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

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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

Azithromycine Cassette Galenic Genes

First Observation :

Double Vector System

Sup’Biotech Paris DVS Project Vectorization

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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

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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

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Gene Presentation

Second Observation :

Cancer

Promoter

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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

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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 to choose the right regulation

Sup’Biotech Paris DVS Project

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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

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Tissue Presentation

Implementation:

DVS versus Lung Cancer

Cell Apoptosis

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Penetration into the lung

Blood vessel

Sup’Biotech Paris DVS Project

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Tissue Vector Macrophage Macrophage infected by Tissue Vector

Lung

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Blood vessel

Dispersion in the Lung

Sup’Biotech Paris DVS Project

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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,

  • rgans extraction and

cell suspension from the lungs, tumors Sup’Biotech Paris DVS Project

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Tissue Presentation

Implementation:

Tissue Targeting

Cell Modeling Tissue Presentation Cell Apoptosis

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SLIDE 26

Sup’Biotech Paris DVS Project

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Implementation:

Tissue Targeting

Tissue Presentation Cell Modeling Tissue Presentation Cell Apoptosis

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  • Our

experiments

  • Literature

Too short to prove the presence

  • f M. avium in lung

Proven many times

 DVS can be used on lung cancer

Sup’Biotech Paris DVS Project

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Tissue Presentation

Implementation:

Tissue Targeting

Cell Modeling Tissue Presentation Cell Modeling Tissue Presentation Cell Apoptosis

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Cell Vector Release

Sup’Biotech Paris DVS Project

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Cell Vector

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Cell Vector Dispersion and Cancer Cells Targeting

Sup’Biotech Paris DVS Project

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Cell Vector

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Membrane Receptor Targeting

Cancer Cell Cell Vector

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Sup’Biotech Paris DVS Project

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Integrin

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Cell Vector Internalization

Sup’Biotech Paris DVS Project

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Nucleus

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Nucleus

Insertion of the gene into the target cell

Paradigm of therapeutic plasmid release is not elucidated

Sup’Biotech Paris DVS Project

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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

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Tissue Presentation

Implementation:

Cell Targeting

Cell Modeling Tissue Presentation Cell Modeling Tissue 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

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Tissue Presentation

Implementation:

Cell Targeting

Cell Modeling Tissue Presentation Cell Modeling Tissue 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 integrins
  • f 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

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Tissue Presentation

Implementation:

Cell Targeting

Cell Modeling Tissue Presentation Cell Modeling Tissue Presentation Cell Apoptosis

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Nucleus

NLS recrutement by DTS sequence

Gene insert

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Sup’Biotech Paris DVS Project

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Nucleus

Nuclear insertion

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Sup’Biotech Paris DVS Project

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Chromosome

Therapeutic gene expression

Nucleus

p53 wt

Apoptosis pathway induction

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Sup’Biotech Paris DVS Project

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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

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Tissue Presentation Cell Modeling Tissue Presentation Cell Modeling Tissue Presentation Cell

Implementation:

Apoptosis Induction

Apoptosis

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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

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Tissue Presentation Cell Modeling Tissue Presentation Cell Modeling Tissue Presentation Cell

Implementation:

Apoptosis Induction

Apoptosis

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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

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Tissue Presentation Cell Modeling Tissue Presentation Cell Modeling Tissue Presentation Cell

Implementation:

Apoptosis Induction

Apoptosis

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Blood vessel

Apoptosis Induction

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Sup’Biotech Paris DVS Project

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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

  • f tumor suppressor genes.

DVS is a straight alternative to current treatments.

Sup’Biotech Paris DVS Project

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Conclusion

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To confirm the concept on lung cancer To adapt it on other diseases

Sup’Biotech Paris DVS Project

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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

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iGEM innovation

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Sup’Biotech Paris DVS Project

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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

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Acknowledgements

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SLIDE 48

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Sup’Biotech Paris DVS Project

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Annexs

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Doxycyclin

inhibition tetP/O RBS

LacI TetR

Lac P/O

cI repressor

cI synthesis inhibition

No Doxycyclin

tetP/O RBS

LacI TetR

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

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RBS RBS DVS

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Galenic Genes

About:

Mycobacterial membrane

Sup’Biotech Paris DVS Project Vectorization DVS

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Galenic Genes

About:

Side effects of bacterial lysis

Sup’Biotech Paris DVS Project Vectorization DVS

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  • Insertion sequences: insertion of both the phage

genome and the control plasmid into the bacterial genome

  • Just one plasmid, the therapeutic one, replicated and

transmitted to the daughter bacterial cells

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Galenic Genes

About:

Plasmid repartition after cell division

Sup’Biotech Paris DVS Project Vectorization DVS

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Repressor

Phage genome

LacP/O

tetP/O RBS Terminator Ampicillin ORI Tetracyclin cassette pSBA3T5

TetR LacI

tetP/O RBS Terminator Ampicillin ORI Tetracyclin cassette pSBA3T5

TetR LacI

Repressor

Phage genome LacP/O RBS Int attP

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Galenic Genes

About:

Encapsidation mechanism of COS sequences

Sup’Biotech Paris DVS Project Vectorization DVS

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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

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Tissue Presentation

Experimental perspective

Tissue targeting

Cell Modeling Tissue Presentation Cell Modeling Presentation 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

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Tissue Presentation

Experimental perspective

Tissue targeting

Cell Modeling Tissue Presentation Cell Modeling Presentation 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.Rob

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

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Tissue Presentation

About:

Cell Targeting

Cell Modeling Tissue Presentation Cell Modeling Tissue Presentation Cell Apoptosis

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SLIDE 57

Sup’Biotech Paris DVS Project

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Tissue Presentation

About:

Therapeutic plasmid

Cell Modeling Tissue Presentation Cell Modeling Tissue Presentation Cell Apoptosis

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Sup’Biotech Paris DVS Project

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Tissue Presentation

About:

Treatment efficiency Modeling

Cell Modeling Tissue Presentation Cell Modeling Tissue 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

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Tissue Presentation

iGEM for us !

Cell Modeling Tissue Presentation Cell Modeling