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

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 Sup’Biotech Paris DVS Project 2 2

First Observation : Problem identification Genes Galenic Vectorization DVS • Viral Disease  Insertion of foreign genome • Cancer  Genome modification • Genetic disease  Dysfunctional genome Identified Problem : The Genome Sup’Biotech Paris DVS Project 3

First Observation : Action on the genome Genes Galenic Vectorization DVS • Chemical drugs  No specificity so Toxic • Bio-drugs  Specific but Temporary (protein, siRNA, etc) • Gene insert  Highly specific and Permanent Best agent : gene sequence Sup’Biotech Paris DVS Project 4

First Observation : Gene characteristics Genes Galenic Vectorization DVS • Very low toxicity √ • Specificity √ Specific • Need to reach the nucleus sequence • Low stability outside the cell Protection Sup’Biotech Paris DVS Project 5

First Observation : How to protect it ? Genes Galenic Galenic Vectorization DVS • Ex vivo Gene action  Personal treatment COST • Classical galenic  Low protection DEGRADATION • Vectorization  Encapsulation GENE PROTECTION Ideal galenic : gene vectorization Sup’Biotech Paris DVS Project 6 6

First Observation : What is a vector ? Genes Galenic Vectorization DVS • Components: – Reservoir – Targeting system – Stealth system • Types of vectors : – Virus – Phages – Bacteria – Lipidic nanoparticles – Polymer nanoparticles Sup’Biotech Paris DVS Project 7

First Observation : Vectorization issues Genes Galenic Vectorization DVS • The 6 major issues of vectorization Passage through Immune system Stability Toxicity Target Industrialization membranes resistance 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 Low Low Cell Good Low Medium NPs polymer Good Medium Cell Low Low Medium NPs  Technological barrier Sup’Biotech Paris DVS Project 8

First Observation : Solution ! Genes Galenic Vectorization DVS • 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) Sup’Biotech Paris DVS Project 9

First Observation : Double Vector System Genes Galenic Vectorization DVS Cell Vector : Recombinant phage Tissue Vector : Specific bacterium + Characteristics : Characteristics : - Cell targeting - Tissue targeting - Passage through membranes - Immune system resistance - Encapsidates an - Produces phages under control exogenous plasmid 10 Sup’Biotech Paris DVS Project 10

First Observation : Double Vector System Genes Galenic Vectorization DVS Tissue Vector : – Mycobacterium avium subspecies avium – (a) Phage genome – (b) System of phage production control Repressor TetR LacI RBS RBS LacP/O tetP/O Terminator (a) (b) Ampicillin pSBA3T5 ORI Tetracyclin cassette Phage genome Sup’Biotech Paris DVS Project 11

First Observation : Control of the Cell vector synthesis Genes Galenic Vectorization DVS inhibition No Doxycyclin cI synthesis TetR LacI RBS RBS tetP/O Lac P/O cI repressor inhibition inhibition Doxycyclin cI synthesis TetR LacI RBS RBS tetP/O Lac P/O cI repressor cI inhibition  Production of recombinant phages Sup’Biotech Paris DVS Project 12

First Observation : Double Vector System Genes Galenic Vectorization DVS Cell Vector : – Lambda Phage – Controlled lysogeny – Viral Proteins on the capsid cI repressor Kanamycin Lac P/O pL Lambda genome Lambda genome Cassette pR Targeting Polypeptide Lambda genome Protein J Protein D Protein III Sup’Biotech Paris DVS Project 13

First Observation : Protein of cell internalization Genes Galenic Vectorization DVS 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 Sup’Biotech Paris DVS Project 14

First Observation : Double Vector System Genes Galenic Vectorization DVS Therapeutic Plasmid: – COS sequence for encapsidation – DTS sequence for nucleus targeting – Sequence of therapeutic aim Therapeutic sequence Azithromycine DTS Cassette Sequence cos Sup’Biotech Paris DVS Project 15

First Observation : DVS advantages Genes Galenic Vectorization DVS • 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 Sup’Biotech Paris DVS Project 16

iGEM SupBiotech Paris Project 2 nd observation Cancer is only wrong information ! Sup’Biotech Paris DVS Project

Second Observation : Cancer Presentation Gene Promoter • 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

Second Observation : What is right information ? Presentation Gene Promoter • 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

Second Observation : How to control the genetic information? Presentation Gene Promoter • 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 20

iGEM SupBiotech Paris Project DVS application on Lung Cancer Sup’Biotech Paris DVS Project

Implementation: DVS versus Lung Cancer Presentation Tissue Cell Apoptosis 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

Lung Macrophage infected by Tissue Vector Macrophage Tissue Vector Blood vessel Penetration into the lung Sup’Biotech Paris DVS Project 23

Lung Blood vessel Dispersion in the Lung Sup’Biotech Paris DVS Project 24

Implementation: Tissue Targeting Presentation Presentation Tissue Tissue Cell Cell Modeling Apoptosis • 3 murine models: immunodeficient, normal, cancerous. Day -7 Day 0 Day 7 -Inoculation of Inoculation of 10^6 Mice sacrifice, CFU.ml -1 of M.avium , fibroblastic cancer cells organs extraction and - subcutaneously -IV route in the tail cell suspension - normal mice -3 mice models from the lungs, tumors • Cell suspension is analyzed by flow cytometry. Size and granularity: eukaryotic murine cells ≠ M.avium Sup’Biotech Paris DVS Project 25

Implementation: Tissue Targeting Presentation Presentation Tissue Tissue Cell Cell Modeling Apoptosis Sup’Biotech Paris DVS Project 26

Implementation: Tissue Targeting Presentation Presentation Presentation Tissue Tissue Tissue Cell Cell Cell Modeling Modeling Apoptosis • Our Too short to prove the presence experiments of M. avium in lung • Literature Proven many times  DVS can be used on lung cancer Sup’Biotech Paris DVS Project 27

Cell Vector Cell Vector Release Sup’Biotech Paris DVS Project 28

Cell Vector Cell Vector Dispersion and Cancer Cells Targeting Sup’Biotech Paris DVS Project 29

Integrin Cancer Cell Cell Vector Membrane Receptor Targeting 30 Sup’Biotech Paris DVS Project 30

Nucleus Cell Vector Internalization Sup’Biotech Paris DVS Project 31

acidification Nucleus Paradigm of therapeutic plasmid release is not elucidated acidification Insertion of the gene into the target cell Sup’Biotech Paris DVS Project 32