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PolExGene Midterm technical meeting

PolExGene Midterm technical meeting. Astrid Subrizi, DDTC, University of Helsinki Gent, 17 December 2007. 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.

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PolExGene Midterm technical meeting

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  1. PolExGene Midtermtechnical meeting Astrid Subrizi, DDTC, University of Helsinki Gent, 17 December 2007

  2. 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.

  3. Achievements by month 12 • Polymer membrane (methacrylamide modified gelatin) – permeation of fluorescent markers with different MW through the hydrogel. • 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.

  4. Planned activities (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?).

  5. Results by month 18th • 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.

  6. Purification protocol for RCS rat RPE cells – Protocol 1 • Plate the cells in a flask and let them attach for 5-15 minutes. • Remove the supernatant and transfer it to another flask. • Repeat 1.-2. for 3 times. Fibroblasts stay in the flask and RPE cells remain in the supernatant because fibroblasts attach faster than RPE cells.

  7. Purification protocol for RCS rat RPE cells – Protocol 2 Selective trypsinization • Trypsinize the cells for 1-2 minutes. • Remove trypsin and wash the cells. Fibroblasts detach faster than RPE cells.

  8. Purification protocol for RCS rat RPE cells – Protocol 3 Cloning • Prepare a very diluted cell suspension. • Triturate carefully to get a “single cell” suspension. • Plate the cells suspension in a 96 wp in order to get 1 cell per well. • Select a well having a pure RPE culture.

  9. Optimized transfection protocol

  10. Plans for 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.

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