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PolExGene technical meeting

PolExGene technical meeting. Astrid Subrizi, CDR, University of Helsinki Prague, 22-23 Mai 2008. Work packages for HY. WP 4: Preparation of plasmids and CPP-containing polyplexes. Objective: to develop therapeutic plasmids for the ocular and the cardiovascular aspect of the project.

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PolExGene technical meeting

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  1. PolExGene technical meeting Astrid Subrizi, CDR, University of Helsinki Prague, 22-23 Mai 2008

  2. Work packages for HY WP 4: Preparation of plasmids and CPP-containing polyplexes. Objective: to develop therapeutic plasmids for the ocular and the cardiovascular aspect of the project. • Plasmids with marker genes will be used for performing a detailed physicochemical characterization of CPP-containing polyplexes. • The coating of polymer membranes and vascular grafts with CPP-containing polyplexes will be studied in detail. WP 5: Characterization of polyplex-cell and polymer membrane-cell interactions. Objective: to study the interaction of different cell types, including RPE cells, vascular endothelial cells and smooth muscle cells, with the polymer materials. • Both the interaction between CPP-containing polyplexes and cells and the interaction between CIP-containing polymer membranes and cells will be investigated.

  3. Achievements by month 18 • Polymer membrane (methacrylamide modified gelatin) – to solve the “cracking problems”, decision to switch to glass slides with a spincoated layer of gelatin (60 nm). • EBNA plasmid with RPE-specific promoter – promoters hTyr(-462).luc and hTyr(-2525)+E.luc (at Ark). • RCS rat RPE cells (from UAT) – development of a purification protocol. • Optimized transfection protocol – to be used at ENS, HY and UKU.

  4. Planned activities (months 19-24) • Biocompatibility of spincoated gelatine membrane with cells (proliferation, differentiation, toxicity). • Purification of RCS rat RPE cells. • Transfection of ARPE19 using optimized protocol.

  5. Results by month 24 • Cell viability of ARPE 19 cells grown on spincoated gelatine membrane, compared to cells grown on tissue-culture treated surface • Final optimization of the transfection protocol

  6. A. Viability study

  7. B. Transfection protocol

  8. Cell density 4’000, 8’000 or 20’000 ARPE19 cells/well • highest protein production with 20’000 cells/well Charge ratio 4/1, 2/1 or 1/1 (PEI/DNA) • best results (efficiency vs. toxicity) with 2/1 • might depend on the polymer used, therefore test all 3 ratios Diluted vs. concentrated polyplex preparation conditions • no substantial difference in most cases • diluted conditions allow better mixing Polyplex prep. buffer mqH2O or Mes-Hepes buffered saline • almost no protein production with water-prepared polyplexes • Mes-Hepes buffered saline as buffer of choice Measurement time after transfection 24h, 48h, 72h • Measure always all 3 timepoints in order to get AUC-type data

  9. Incubation time of polyplexes with cells 30 min, 1h, 2h, 5h, 12h, 24h

  10. PolExGene Transfection protocol Cell density 20’000 cells/well Measurement at 24h, 48h and 72h Incubation time 1h or 2h Mes-Hepes buffer Diluted conditions Charge ratio 4/1, 2/1 and 1/1

  11. Plans for months 25-30 • Compare expression of biochemical markers CRALBP and RPE65 of gelatine-cultured vs. TC-cultured ARPE19 cells with PCR • Purification of RCS rat RPE cells. • Testing of transfection efficiency / toxicity of cationic carriers (UGent) with new PolExGene transfection protocol

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