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 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 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