24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and...
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![Page 1: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/1.jpg)
24 months Prague meeting
Sixth Framework Programme Priority 1
Life Sciences, Genomics and Biotechnology for Health
Lorenzo TibaldiAlain Joliot’s groupENS Paris
![Page 2: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/2.jpg)
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:
![Page 3: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/3.jpg)
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
![Page 4: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/4.jpg)
Test new polymers
CV1 cells5µL complex24hrs incubationRucHA plasmid
VT01 and VT02-2 areactive and efficient
Ruc activity 5µL
-5,0E+04
0,0E+00
5,0E+04
1,0E+05
1,5E+05
2,0E+05
2,5E+05
VT01 VT02-2 VT03 VT04 VT05 VT07-1 pLL V11c V12 V13a V13b PEI
RLU
n.p. 8/1
n.p. 4/1
n.p. 2/1
n.p. 1/1
Viability 5µL
-20%
0%
20%
40%
60%
80%
100%
120%
140%
VT01 VT02-2 VT03 VT04 VT05 VT07-1 pLL V11c V12 V13a V13b PEI
cell viability
n.p. 8/1
n.p. 4/1
n.p. 2/1
n.p. 1/1
![Page 5: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/5.jpg)
Test new polymers
ARPE19 cells10µL complex24hrs incubationRucHA plasmid
VT02-2 is confirmed to beactive also on ARPE
VT09 is active too
Ruc activity 10µL
-5,0E+04
0,0E+00
5,0E+04
1,0E+05
1,5E+05
2,0E+05
2,5E+05
3,0E+05
3,5E+05
4,0E+05
4,5E+05
5,0E+05
VT02-2 VT07-2 VT07-3 VT07-4 VT09 VT10 PEI
RLU
n.p. 8/1
n.p. 4/1
n.p. 2/1
n.p. 1/1
Viability 10µL
-20%
0%
20%
40%
60%
80%
100%
120%
140%
160%
VT02-2 VT07-2 VT07-3 VT07-4 VT09 VT10 PEI
Viability
n.p. 8/1
n.p. 4/1
n.p. 2/1
n.p. 1/1
![Page 6: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/6.jpg)
Test new polymers
ARPE19 cells5µL complex24hrs incubationRucHA plasmid
VT02-2 is confirmed to beactive also on ARPE
VT09 is active too
Ruc activity 5µL
-1,0E+05
0,0E+00
1,0E+05
2,0E+05
3,0E+05
4,0E+05
5,0E+05
6,0E+05
VT02-2 VT07-2 VT07-3 VT07-4 VT09 VT10 PEI
RLU
n.p. 8/1
n.p. 4/1
n.p. 2/1
n.p. 1/1
Viability 5µL
-20%
0%
20%
40%
60%
80%
100%
120%
140%
VT02-2 VT07-2 VT07-3 VT07-4 VT09 VT10 PEI
Viability
n.p. 8/1
n.p. 4/1
n.p. 2/1
n.p. 1/1
![Page 7: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/7.jpg)
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
![Page 8: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/8.jpg)
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
![Page 9: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/9.jpg)
Transfection activity of fluorescent polyplexes
sRuc SN supernatant 10µL
0,0E+00
5,0E+04
1,0E+05
1,5E+05
2,0E+05
2,5E+05
3,0E+05
3,5E+05
4,0E+05
np 3/1 np 2,5/1 np 2/1 np 1,5/1 np 1/1
PEI
PEI-fluo UGent
PEI-Oregon Green
QuickTime™ et undécompresseur TIFF (LZW)
sont requis pour visionner cette image.
QuickTime™ et undécompresseur TIFF (LZW)
sont requis pour visionner cette image.
0,25 0,375 0,5 0,75 1 1,5 2 3 0,25 0,375 0,5 0,75 1 1,5 2 3
PEI-Oregon Green PEI-fluo UGentPEI0,25/1 0,5/1 1/1 DNA
![Page 10: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/10.jpg)
Transfection activity of fluorescent polyplexes
sRuc Supernatant 10µL
0,0E+00
2,0E+05
4,0E+05
6,0E+05
8,0E+05
1,0E+06
1,2E+06
np 4/1 np 3/1 np 2,5/1 np 2/1 np 1,5/1
PEIPEI-DNAsytoxG 5µMPEI-Oregon
DNA 0,25 0,5 1/1 0,25 0,32 0,5 0,75 1/1 1,5 2
PEI PEI-Oregon
DNA 0,25 0,5 1/1 0,25 0,32 0,5 0,75 1/1 1,5 2
PEI PEI-Oregon
control plasmid sytox labelled plasmid
![Page 11: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/11.jpg)
Compare internalization efficiency and intracellular behavioursof Oregon Green labelled PEI and V0 polymers (inactive)
Ruc activity 24hrs after transfection
0,0E+00
2,0E+05
4,0E+05
6,0E+05
8,0E+05
1,0E+06
1,2E+06
1,4E+06
1,6E+06
1,8E+06
PEI LF2000 V07
RLU
![Page 12: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/12.jpg)
QuickTime™ et undécompresseur TIFF (LZW)
sont requis pour visionner cette image.
QuickTime™ et undécompresseur TIFF (LZW)
sont requis pour visionner cette image.
PEI-Oregon Green without DNA
imaged at 3h
add Blue Trypan
Cell surface fluorescentstaining is quenched byBlue Trypan
Distinguish between extraand intracellular PEI
![Page 13: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/13.jpg)
PEI-Oregon Green complexed with labelled DNA
1h 4hrs 24hrs
+BT
PEIDNAWGA
![Page 14: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/14.jpg)
V07 complexed complexed with labelled DNA
1h 4hrs 24hrs
+BT
PEIDNAWGA
![Page 15: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/15.jpg)
PEI and V07 complexes have different sizes
PEI V07
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Follow the intracellular localization of fluorescent polymers and fluorescent plasmids separately
![Page 17: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/17.jpg)
PEI-Oregon complexed with labelled DNA
1h
4hrs
24hrs
+BT
merge PEI plasmid
Dead cells
![Page 18: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/18.jpg)
Correlate intracellular polymer/plasmid behaviours with reporter protein expression
Early protein expression (4hrs)after transfection could help usin correlating polymer/DNA intracellular localization withtransfection activity!
Ruc Expression (dose 10µL)
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5 6 7
hours polyplex incubation
RLU
PEI-Oregon 2,5/1
![Page 19: 24 months Prague meeting Sixth Framework Programme Priority 1 Life Sciences, Genomics and Biotechnology for Health Lorenzo Tibaldi Alain Joliot’s group.](https://reader035.fdocuments.in/reader035/viewer/2022070308/551b9a79550346942b8b54b6/html5/thumbnails/19.jpg)
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