PolExGene 12 th month technical meeting

1. PolExGene 12th month technical meeting Helsinki, 23-24 August 2007

2. Work packages for UH 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. Achievments by month 6 • Characterization of the human RPE cell line (ARPE19) with regard to the expression of specific biochemical markers (CRALBP and RPE65). • Determination of the paracellular permeability of ARPE19 monolayers by measuring the apical to basolateral movement of 6-CF over a time course of 3 hours. • Transfections were performed using 2 different carriers (PEI n/p 8 and 10, DOTAP/DOPE/PS). • Recording of the transepithelial resistance of ARPE19 monolayers to determine the time course and extent of tight junction formation. • Visualization of the intracellular distribution of Alexa 488-Tat peptide by CLSM after incubation in dividing ARPE19 cells at 37 °C. • A preliminary experiment with hydrogel (crosslinked gelatine) coated Transwell inserts was performed. The gel showed large cracks after one day incubation at 37 °C.

4. Planned activities (months 7-12) • Inspection of the hydrogels by microscope to confirm that they are intact, study of their permeability to fluorescent molecules with different molecular weights. • Demonstration of the biocompatibility of the hydrogels by studying ARPE19 cell proliferation, differentiation and toxicity. • Testing of the functionality of the hydrogels on Transwell inserts by mean of paracellular permeability and transfection. • Testing of the efficacy of EBNA plasmid, a self-replicating plasmid for prolonged transfection, and comparison with results obtained with pCMV-SEAP2.

5. Results by month 12 • Polymer membrane (methacrylamide modified gelatin) – “cracking” problem not solved • EBNA plasmid – transfection of ARPE19 cells • Poly(dimethylaminoethyl-L-glutamine) PDMAEG (V01 and V03) – DNA complexation and transfection efficiency tested • Tat peptide and analogues as CPPs – cell uptake studies with ARPE19, CHO wt and mutant cell line

6. Gelatin hydrogels Problem: • Hydrogels “crack” after incubation in cell medium (contains 1% FBS). Cracks form after hours (7h) or days (3d). Tentative solution: • Preparation of hydrogels in Helsinki, following protocol used in Ghent. • 50 mg modified gelatine dissolved in 500 μl mqH2O, at 40°C. • 20 mg Irgacure 2959 dissolved in 2,5 ml mqH2O, at 50°C. • 7 μl Irgacure 2959 solution were added to the gelatine, at 40°C. • 50 μl hydrogel were pipetted onto chamber (Ø 8 mm). • Gels cured with UV (7,33 mW/cm2, 365 nm) for 1 and 1,5 h. • Gels obtained were about 1 mm thick, transparent, intact. BUT • Thickness is not uniform (thinner in the middle and thicker on the sides). • After 1h incubation in 1x PBS at 37°C, hydrogel is dissolved.

7. Permeation of fluorescent markers • Permeability of different fluorescent markers (10 μM) tested with an intact batch. Experiment lasts 3 hours. • 6-Carboxyfluorescein (MW: 376) : 13% permeated to acceptor, 53% still in donor. • FITC-dextran (MW 10’000 and 70’000) did not permeate within the experimental timetable Human SEAP exists in several molecular species (MWmonomer 58’000). • Dextrans might need longer time to permeate the gel (lag phase). Experiment time should be increased. • Hydrogel thickness should be decreased • to mimic physiological conditions (Bruch’s membrane < 10 μm) • to facilitate basolateral transfection of cells

8. EBNA plasmid pCMV-SEAP2 EBNA plasmid – pEpi-SEAP • Responsible for extrachromosomal maintenance • Latent replication origin (OriP) • EBV nuclear antigen 1 (EBNA-1) Ensure replication of plasmid once per cell cycle during S phase and segregation into the daughter cells.

9. EBNA plasmid pCMV-SEAP2 pCMV-SEAP2 EBNA plasmid

10. EBNA plasmid No significant benefit observed • Transfected cells were differentiated and non dividing (3 weeks growth on filter) The same experiment will be repeated with non differentiated, dividing cells.

11. Triton X-100 (10%) Dextran sulfate (3x) Dextran sulfate (3x) Triton X-100 (10%) Polycations – PDMAEG V01 and V032 secondary amines and 1 tertiary amine • V01 and V03 (n/p ratios 4 and 8) were also tested for transfection efficiency with pCMVß (ß-Gal) plasmid (1,5 μg/well) in ARPE19 cells. Polyplexes did not transfect the cells. • PEI8 and 10 used as positive controls gave a ß-Gal activity of 3 mU/ml resp. 3,3 mU/ml supernatant. • Similar results obtained earlier with PDMAEG of different MW (Biomacromolecules 2003). • Modification of the polycation with CPP may enhance transfection efficiency.

12. CPPs – Tat peptide and analogues • Tat peptide and 16 analogues were tested for cell uptake experiments (flow cytometry) with 3 different cell lines (ARPE 19, CHO wt and CHO mutant).

13. Plans for months 13-18 • Biocompatibility of improved biopolymer membrane with cells (proliferation, differentiation, toxicity) • Transfection of dividing ARPE19 with EBNA plasmid • Transfection of ARPE19 with plasmid containing a RPE-specific promoter (tyrosinase promoters) • Comparison of transfection efficiencies of polyplexes non-covalently linked to CPP (penetratin and Tat peptide) (protocol?)

14. From UGent: PDMAEG V01 and V03 Gelatin coated transwell Modified gelatine From Ark: pEPI-SEAP EBNA plasmid with RPE-specific promoter (in future) From CNRS: Penetratin To UGent, IMIC and IBMT: pCMVß Exchange products