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PolExGene Midterm technical meeting

nevan avatar
nevan
10 diapositives · 219 Views · Aug 21, 2014

This project focuses on preparation and characterization of CPP-containing polyplexes for ocular and cardiovascular therapies, studying cell interactions and transfection efficiencies. Achievements and planned activities are detailed, including biocompatibility testing and transfection protocols. Results include solving membrane cracking issues and optimized transfection protocols. Purification protocols for RCS rat RPE cells are outlined for efficient cell selection. Future plans involve biocompatibility studies, further purification of RPE cells, and continued transfection experiments.

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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.