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

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Presentation on theme: "PolExGene technical meeting Astrid Subrizi, CDR, University of Helsinki Prague, 22-23 Mai 2008."— Presentation transcript:

1 PolExGene technical meeting Astrid Subrizi, CDR, University of Helsinki Prague, 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 A.Cell viability of ARPE 19 cells grown on spincoated gelatine membrane, compared to cells grown on tissue- culture treated surface B.Final optimization of the transfection protocol

6 A. Viability study

7 B. Transfection protocol Transfection Matrix Cell density 4’000 8’000 20’000 Charge ratio 4/1 2/1 1/1 Diluted vs. concentrated conditions Buffer Water Mes-Hepes saline Incubation time 30min, 1h, 2h, 5h, 12h, 24h Measurement 24h 48h 72h

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

11 Plans for months 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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