PolExGene ( Month 24-30)

1. PolExGene(Month24-30) Thomas Wirth University of Kuopio (UKU) Participant #: 9

2. Work packages • WP4: • 4.1. Development of plasmids for polyplex incorporation. • 4.4. Functionalisation of polymer membrane with polyplexes • WP5: • 5.1. Interaction between CPP-containing polyplexes and cells • 5.2. Interaction between CIP-containing polymer membranes and cells. • WP7: • 7.2. Implantation of polymer membranes for cardiovascular diseases.

3. Where are we now ? • WP4: • 4.1. Development of plasmids for polyplex incorporation. • VEGF • VEGF mutants • IK-17 • SEAP • 4.4. Functionalisation of polymer membrane with polyplexes • Production of recombinant proteins • WP5: • 5.1. Interaction between CPP-containing polyplexes and cells • Tested transfection efficiencies of different polyplexes • Tested different protocols • compared transfection efficiencies of polyplexes to viral vectors • 5.2. Interaction between CIP-containing polymer membranes and cells. • WP7: • 7.2. Implantation of polymer membranes for cardiovascular diseases.

4. What was planned for months 24-30 • Evaluation of new polyplexes received from UGent • Testing of transfection protocol (UH.FP) • Sending stents to UGent • Evaluation of cell growth on polymers

5. Evaluation of VT01, VT02-2 and VT09 polyplex-transfection Polyplex formation protocol Transfectionprotocol • Different charge ratio’s were prepared with DNA (EPI-SEAP plasmid) and VT01, VT02-2 and VT09 polyplexes • Charge ratios 4/1, 2/1 and 1/1 were tested • Diluted with Cationic Polymers Stock Solution (2,95 mM) • Polyplexes were prepared 2h before transfection • Total 600ng DNA was used in each well • 15000 cells were planted to 96-well plate • The following day cells were washed with 150µl PBS • 100µl serum-free medium was added to each well • 50µl polyplex-DNA solution was added to the cells • Incubate for 1h and 2h • Wash with 150µl PBS • Add 100µl full growth medium (F12/DMEM + serum + penstrep) • Incubate for 72h before measurement

6. Measurement: Chemiluminescent SEAP Assay (Great EscAPe SEAP, Clonetech) • Start with 50µl cell culture medium (72h incubation after transfection) • briefly centrifuge and take supernatant to measurement (15µl) • Add 45µl 1x Dilution Buffer • Incubate 30min at +65ºC -> cool samples by placing on ice 2-3min • Add 60µl Assay Buffer and incubate 5min, RT • Add 60µl fresh CSPD Substrate working dilution and incubate for 10min • Measure with VectorII Luminometer

7. VT01 polyplex • 2 Different time points and 3 different charge ratios were tested. • Charge ratio 4/1 didn’t give any effect on transfection • Charge ratio 2/1 worked with 2h transfection but not with 1h • Charge ratio 1/1 gave best transfection. • 1h transfection gave better transfection efficacy than 2h

8. VT02-2 • Only 4/1 charge ratio showed any transfection efficiency. • 2h transfection time was superior compared to 1h.

9. VT09 • Transfection efficiency was poor using VT09. • Only low levels of SEAP was measured from cell medium under any condition used.

10. Conclusions • VT01 • Transfection was efficient with 1h transfection and 1/1 charge ratio • VT02-2 • Transfection was efficient with 2h transfection and 4/1 charge ratio • VT09 • Poor transfection using VT09

11. What we will do • WP4: • 4.1. Development of plasmids for polyplex incorporation. • VEGF • VEGF mutants • IK-17 • SEAP • 4.4. Functionalisation of polymer membrane with polyplexes • Production of recombinant proteins • WP5: • 5.1. Interaction between CPP-containing polyplexes and cells • Tested transfection efficiencies of different polyplexes • Tested different protocols • compared transfection efficiencies of polyplexes to viral vectors • 5.2. Interaction between CIP-containing polymer membranes and cells. • WP7: • 7.2. Implantation of polymer membranes for cardiovascular diseases.