PolExGene technical meeting

Astrid Subrizi, CDR, University of Helsinki

Prague, 22-23 Mai 2008

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.

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.

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.

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

A. Viability study

50,00 %

60,00 %

70,00 %

80,00 %

90,00 %

100,00 %

110,00 %

1 2 3 13

Days after seeding

Via

bil

ity

gel

atin

e-g

row

n v

s. T

C-g

row

n

cell

s

10'000 cells 50'000 cells 100'000 cells

B. Transfection protocol

Measurement24h48h72h

Incubation time30min, 1h,

2h, 5h, 12h, 24h

BufferWater

Mes-Hepes saline

Diluted vs.concentrated

conditions

Charge ratio4/12/11/1

Cell density4’0008’000

20’000

TransfectionMatrix

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 mqH2O 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

Incubation time of polyplexes with cells 30 min, 1h, 2h, 5h, 12h, 24h

24h 48h 72h

-0,4

-0,2

0

0,2

0,4

0,6

0,8

ng/ ml

Mes-Hepes, 30 m in, 20'000 ce lls /w ell

Free DNA

PEI 4/1 diluted

PEI 4/1 conc.

PEI 2/1 diluted

PEI 2/1 conc.

PEI 1/1 diluted

PEI 1/1 conc.

24h 48h 72h

-2

0

2

4

6

8

10

12

14

ng/ ml

Mes-Hepes, 1h, 20'000 ce lls /w ell

Free DNA

PEI 4/1 diluted

PEI 4/1 conc.

PEI 2/1 diluted

PEI 2/1 conc.

PEI 1/1 diluted

PEI 1/1 conc.

24h 48h 72h

-5

0

5

10

15

20

25

ng/ ml

Mes-Hepes, 2h, 20'000 ce lls /w ell

Free DNA

PEI 4/1 diluted

PEI 4/1 conc.

PEI 2/1 diluted

PEI 2/1 conc.

PEI 1/1 diluted

PEI 1/1 conc.

24h 48h 72h

0,0 %

20,0 %

40,0 %

60,0 %

80,0 %

100,0 %

120,0 %

140,0 %

Mes-Hepes, 30 m in, 20'000 ce lls /w ell

Free DNA

P EI 4/1 diluted

P EI 4/1 conc.

P EI 2/1 diluted

P EI 2/1 conc.

P EI 1/1 diluted

P EI 1/1 conc.

P EI alone 1

24h 48h 72h

0,0 %

20,0 %

40,0 %

60,0 %

80,0 %

100,0 %

120,0 %

140,0 %

Mes-Hepes, 1h, 20'000 ce lls /w ell

Free DNA

P EI 4/1 diluted

P EI 4/1 conc.

P EI 2/1 diluted

P EI 2/1 conc.

P EI 1/1 diluted

P EI 1/1 conc.

P EI alone 1

24h 48h 72h

0,0 %

20,0 %

40,0 %

60,0 %

80,0 %

100,0 %

120,0 %

140,0 %

Mes-Hepes, 2h, 20'000 ce lls /w ell

Free DNA

P EI 4/1 diluted

P EI 4/1 conc.

P EI 2/1 diluted

P EI 2/1 conc.

P EI 1/1 diluted

P EI 1/1 conc.

P EI alone 1

Cell density20’000 cells/well

PolExGene Transfection protocol

Charge ratio4/1, 2/1 and 1/1

Diluted conditions

Mes-Hepesbuffer

Incubation time1h or 2h

Measurement at24h, 48h and 72h

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