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WP4 progress and planning University of Helsinki

WP4 progress and planning University of Helsinki. Astrid Subrizi, 18 December 2007. 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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WP4 progress and planning University of Helsinki

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  1. WP4 progress and planningUniversity of Helsinki Astrid Subrizi, 18 December 2007

  2. 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.

  3. RPE cells pigmented monolayer of cells that form the outer blood/retina barrier have unique morphologic and functional polarity properties cubical (cross-section), hexagonal (viewed from above) monolayer of cells joined apically by tight junctions (zonula occludens). ARPE 19 cell line human RPE cell line established and characterised by K.C. Dunn in 1996 arose spontaneously, highly epithelial, strong growth potential characterization: morphology expression of biochemical markers specific for the retina barrier properties established in vitro model for the outer blood-retinal barrier Characterization of ARPE 19 cell line Differentiation markers (RT-PCR) CRALBP and RPE65 Barrier properties TEER: 65 Ω cm2 Paracellular permeability (6-CF): Papp = 76,6 x 10-7 cm/s Porcine RPE tissue sheets: Papp Na-Fluorescein = 1,8 x 10-7 cm/s (with attached retina) Steuer H, Brain Res. Prot. 13 (2004) Bovine RPE-choroid specimens: Papp 6-CF = 23,3 x 10-7 cm/s Pitkänen L, IOVS 46 (2005)

  4. Plasmids Non-secreted reporter protein: pCMVβ (7.2 kb) Used for testing DNA-binding properties of polymers developed in Gent. Secreted reporter protein: pCMV-SEAP2 (5747 bp) and pEpi-SEAP (11’857 bp, Ark) Used for transfection of differentiated ARPE 19 cells. pCDNA7sRucHA (6152 bp, ENS) Soon in use for transfection experiments with non differentiated ARPE19 cells EBNA plasmid with RPE-specific promoter With promoters hTyr(-462).luc and hTyr(-2525)+E.luc (under devel. Ark).

  5. 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. • Modification of the polycation with CPP may enhance transfection efficiency.

  6. Hydrogel coated Transwell inserts (1) Problem: Hydrogels “crack” after incubation in cell medium (contains 1% FBS). Cracks form after hours (7h) or days (3d). 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. Hydrogel thickness should be decreased In future, use of spincoated • to mimic physiological conditions (Bruch’s membrane 1 μm) gelatine. • to facilitate basolateral transfection of cells (min. thickness 60 nm) Hydrogel coated Transwell inserts (2) • 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).

  8. Achievements by month 18th • Characterization of the human RPE cell line (ARPE19) with regard to the expression of specific biochemical markers (CRALBP and RPE65) and the paracellular permeability (barrier properties) of ARPE19 monolayers. • Preliminary testing of polycations PDMAEG V01 and V03 (DNA condensation and transfection efficiency). • Testing of methacrylamide modified gelatine hydrogels, including permeation of fluorescent markers with different molecular weights. Choice of improved polymer membrane (spincoated hydrogel) for further biocompatibility testing.

  9. Planned activities (months 19-36) • Testing of cell specific and therapeutic plasmids with ARPE19 cells and RCS rat RPE cells. • Biocompatibility testing of improved polymer membranes. • Transfection efficiency of polymer membranes functionalized with polyplexes.

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