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Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health

PolExGene. Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health. UGent contribution to PolExGene: Development of cationic polymers as synthetic vectors for gene delivery. PolExGene technical meeting Paris, 08-09/02/2007.

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Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health

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  1. PolExGene Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health UGent contribution to PolExGene: Development of cationic polymers as synthetic vectors for gene delivery PolExGene technical meeting Paris, 08-09/02/2007 V. Vermeersch,P. Dubruel, E. Schacht

  2. Summary UGhent tasks Development of CPP-containing polymers (WP2) Development of CIP-containing polymer membranes by solvent casting and electrospinning (WP3) Preparation of plasmids and CPP-containing polyplexes(WP4)

  3. WP 2 Development of CPP-containing polymers WP 2.1 Synthesis and characterisation of cationic polymers and peptides  biodegradable poly--amino acids •Synthesis: Pathway pDMAEG: 4 steps

  4. WP 2 Development of CPP-containing polymers WP 2.1 Synthesis and characterisation of cationic polymers and peptides  biodegradable poly--amino acids •Synthesis: pKa ≈ 10 Analog procedure for poly-g-trichloroethyl-L-glutamate Dissolved in 0,1 N HCl polycation

  5. WP 2 Development of CPP-containing polymers WP 2.1 Synthesis and characterisation of cationic polymers and peptides  biodegradable poly--amino acids •Synthesis: Polymerization g-benzyl-L-glutamate B pDMAEG: 100 % conversion through NMR confirmation A •Charachterization: A B 1H-NMR Infra-red GPC 1 6

  6. WP 2 Development of CPP-containing polymers WP 2.1 Synthesis and characterisation of cationic polymers and peptides  biodegradable poly--amino acids Infra-red polymerization aminolysis BG-NCA pBG pDMAEG

  7. WP 2 Development of CPP-containing polymers WP 2.2 Synthesis and characterisation of cationic polymers and peptides  reference polymers •Synthesis & charachterization: Commercially available Mw = 25.000 (gr/mol) Intendend Mw 200.000 gr/mol 1H-NMR GPC

  8. WP 2 Development of CPP-containing polymers WP 2.3 Synthesis and characterisation of cationic polymers and peptides  biodegradable co-poly--amino acids •Synthesis (in progress): n/m ratio e.g. 9/1 Adjustable Variable amines possible: pKa adjustable! Also: Poly-L-arginine Tertiary amines: DNA coupling Primary amines: coupling(e.g. cell penetrating peptides)(e.g. fluorescent markers)

  9. WP 4 Preparation of DNA & CPP-containing polyplexes WP 4.1 DNA condensing Polycations •Polyplex formation: Polyanion.(X+) + Polycation (Y-) Polyanion.polycation + (X+Y-) (DNA) (polymer) (polyplex) (counterions) +(physiological pH) Calf Thymus (23 kbase pairs) Electrostatic forces Degree of protonation: Amine dependant Prior to PEC formation the molecular weight per charge is calculated

  10. WP 4 Preparation of DNA & CPP-containing polyplexes WP 4.2 Phisical evaluation of polyplexes •EtBr exclusiontests: Determining of charge ratio at which condensation starts Fluorescence light absorption (l = 510 nm), emission (l = 590 nm) EtBr is expelled out of DNA at polyplex formation ( intensity) pDMAEG:Start condensation 0.8/1 charge ratio Mw increase pDMAEG Minor decrease in cr (±0.1)

  11. WP 4 Preparation of DNA & CPP-containing polyplexes WP 4.2 Phisical evaluation of polyplexes •Gelelectrophoresis: Agarose gels 1% – Electric field (90 V) for 1 hour Free DNA migrates to anode (+) pEI pDMAEMA 79k pDMAEG 250k anode anode

  12. WP 4 Preparation of DNA & CPP-containing polyplexes WP 4.2 Phisical evaluation of polyplexes •Light scattering: Determining size of polyplexes All polyplexes < 200 nm(maximum size for receptor mediated endocytosis) •Zeta-potential measurments: experiments will start on short notice

  13. WP 2 & 4 Future plans •Continued physical evaluation of polyplexes: Zeta-potential measurementsSurface plasmon resonance •Complexstability: Stability towards salt (NaCl)Stability towards proteines (Albumine) •Continued Synthesis: Synthesis of copolymers containing tertiary & primary aminesLinkage of CPP & fluorescent groups to primary amines •Preparation of Terplexes: Initial work on the adding of DNA & CPP to polymer solutions in different order pDMAEG chemically charachterized - DNA interaction confirmed starting up biological evaluation samples will be sent to partners

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