24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group ENS Paris

Work package 1:Selection of CIP and CPP Work package 2:Development of CPP-containing polymers Work package 4:Preparation of plasmids and CPP-containing polyplexes Work package 5:Characteriation of polyplex-cell and polymer membrane-cell interactions ENS partner workpackages contribution:

Last 6 months tasks: Test the transfection efficiencies of new polymers synthetized by UGent (VT0 and V0 series) Study the intracellular behaviours of fluorescent polymers and fluorescently labelled plasmid by confocal microscopy on live transfected cells

Test new polymers CV1 cells 5µL complex 24hrs incubation RucHA plasmid VT01 and VT02-2 are active and efficient

Test new polymers ARPE19 cells 10µL complex 24hrs incubation RucHA plasmid VT02-2 is confirmed to be active also on ARPE VT09 is active too

Test new polymers ARPE19 cells 5µL complex 24hrs incubation RucHA plasmid VT02-2 is confirmed to be active also on ARPE VT09 is active too

Conclusions on new polymers: All the members of the VO serie of polymers tested in the last 12 months are inactive VT01 and VT09 are (biodegradable?) guanylated variants of PEI and are active VT02-2 represents a new biodegradable active polymer

Aims of this technique: Compare the cellular behaviour of Oregon Green labelled PEI (active) and V0 polyplexes (inactive) Follow the intracellular localization of fluorescent polymers and fluorescent plasmids Correlate intracellular polyplex behaviours with reporter protein expression Confocal microscopy studies of fluorescent polyplexes intracellular behaviours

Transfection activity of fluorescent polyplexes 0,250,3750,50,75 11,5230,250,3750,50,75 11,523 PEI-Oregon Green PEI-fluo UGent PEI 0,25/1 0,5/1 1/1 DNA

Transfection activity of fluorescent polyplexes DNA 0,25 0,5 1/1 0,25 0,32 0,5 0,75 1/1 1,5 2 PEIPEI-Oregon DNA 0,25 0,5 1/1 0,25 0,32 0,5 0,75 1/1 1,5 2 PEIPEI-Oregon control plasmidsytox labelled plasmid

Compare internalization efficiency and intracellular behaviours of Oregon Green labelled PEI and V0 polymers (inactive)

PEI-Oregon Green without DNA imaged at 3h add Blue Trypan Cell surface fluorescent staining is quenched by Blue Trypan Distinguish between extra and intracellular PEI

PEI-Oregon Green complexed with labelled DNA 1h4hrs24hrs +BT PEI DNA WGA

V07 complexed complexed with labelled DNA 1h4hrs24hrs +BT PEI DNA WGA

PEI and V07 complexes have different sizes PEIV07

Follow the intracellular localization of fluorescent polymers and fluorescent plasmids separately

PEI-Oregon complexed with labelled DNA 1h 4hrs 24hrs +BT mergePEIplasmid Dead cells

Correlate intracellular polymer/plasmid behaviours with reporter protein expression Early protein expression (4hrs) after transfection could help us in correlating polymer/DNA intracellular localization with transfection activity!

To do in the next 6 months: Using the optimized conditions tested by UHFP we want to: Test the correlation between transfection activity of the polyplexes and their intracellular behaviours at different time points (expression of GFP or HaloTagged proteins) Use high PEI n.p. ratios to understand if toxicity affects all the treated cells or if it is induced particularly in the transfected ones Test the intracellular distributions of new biodegradable polymers synthetized by Ugent Test the effects on polyplex behaviours and on kinetics of internalization and intracellular distribution of CPP-linked polymers