Presentation on theme: "PolExGene Meeting in Paris 8 February 2007 Astrid Subrizi, DDTC, University of Helsinki."— Presentation transcript:
PolExGene Meeting in Paris 8 February 2007 Astrid Subrizi, DDTC, University of Helsinki
Introduction RPE and its functions ARPE19 cell line as model for RPE Objectives of UH.FP Current experiments Transfection with pCMV-SEAP Intracellular distribution of Tat peptide and list of Tat analogues to be tested Gelatin coated transwell system Future plans
Physiologic roles of the RPE Regeneration of bleached visual pigments (rhodopsin) Formation and maintenance of the interphotoreceptor matrix and Bruch’s membrane Transport of fluids and ions between photoreceptors and the choriocapillaris Phagocytosis of shed photoreceptor outer segments Absorption of light and dissipation of heat energy … The RPE is a central element in the pathogenesis of AMD
Retinal Pigment Epithelium and ARPE 19 cell line RPE cells are a pigmented monolayer of cells that form the outer part of the blood/retina barrier have unique morphologic and functional polarity properties cuboidal (cross-section), hexagonal (viewed from above) monolayer of cells joined apically by tight junctions (zonula occludens). RPE cells are small (10 μm) in the macular region, but become flatter and broader (up to 60 μm) toward the periphery. average of 45 photoreceptors overly each RPE cell ARPE 19 cell line is a human RPE cell line established and characterised by K.C. Dunn in 1996 arose spontaneously and was purified until a highly epithelial culture of RPE cells with a strong growth potential was obtained characterization: morphology expression of biochemical markers specific for the retina (CRALBP and RPE65) barrier properties (TER and permeability of paracellular barrier markers) is an established in vitro model for the outer blood-retinal barrier
Objectives of UH.FP WP 4 Preparation of plasmids and CPP-containing polyplexes Development of plasmids and CPP-containing polyplexes Therapeutic plasmids EBNA plasmid Functionalisation of polymer membranes with polyplexes WP 5 Characterisation of polyplex-cell and polymer membrane-cell interactions Interaction between CPP-containing polyplexes and cells Interaction between CIP-containing polymer membranes and cells
Transfection A secreted reporter gene (SEAP) was used in order to evaluate duration and direction of the secretion of the expressed gene product. From Mannermaa E, Curr Eye Res. 30:345–353, 2005 Summed-up SEAP secretion (μg/well): Peak transgene expression differences: DOTAP/DOPE/PS ~ 65 x PEI n/p 8 DOTAP/DOPE/PS ~ 25 x PEI n/p 10 Summed-up SEAP secretion (total amount): DOTAP/DOPE/PS ~ 18 x PEI n/p 8 DOTAP/DOPE/PS ~ 12,5 x PEI n/p 10 PEI 8PEI 10DOTAP Apical11,6016,36272,41 Basolateral13,0119,10171,07 Total24,6135,45443,47 For each carrier tested, gene expression lasted for more than 40 days.
Intracellular distribution of Tat Intracellular distribution of Alexa 488-Tat in dividing ARPE19 cells at 37 °C. ARPE19 cells were incubated with 1 μM or 500 nM Alexa 488-tagged Tat peptide respectively (green). Nucleus was stained with 1 μg/ml Hoechst 33258 (blue).
Tat-related CPPs to be checked (Dr. Maxim Antopolsky) No.CodeSequenceHPLC purity %MW calculatedMW measuredAmount mg 1Tat[C 1 ]H-CGRKKRRQRRRPPQ-OH951821.31822.115 2Tat[C 1 W 7,8 ]H-CGRKKRWWRQRRRPPQ-OH93.52193.82194.15 3Tat[C 1 W 11,12 ]H-CGRKKRRQRRWWRPPQ-OH96.22193.82194.07 4Tat[C 1 W 10,11 ]H-CGRKKRRQRWWRRPPQ-OH92.32193.82194.058 5Tat[C 1 W 7,8,12, 13, 15, 16 ]H-CGRKKRWWRQRWWRWWRPPQ-OHTobeSynthesized 6Tat[C 1 A 7,8 ]H-CGRKKRAARQRRRPPQ-OH98.21963.51964.1210 7Tat[C 1 A 11,12 ]H-CGRKKRRQRRAARPPQ-OH97.11963.51964.158 8Tat[C 1 A 10,11 ]H-CGRKKRRQRAARRPPQ-OH95.11963.51964.1611 9Tat[C 1 A 7,8,12, 13, 15, 16 ]H-CGRKKRAARQRAARAARPPQ-OHTobeSynthesized 10Tat[C 1 L 7,8 ]H-CGRKKRLLRQRRRPPQ-OHTobeSynthesized 11Tat[C 1 L 11,12 ]H-CGRKKRRQRRLLRPPQ-OHTobeSynthesized 12Tat[C 1 L 10,11 ]H-CGRKKRRQRLLRRPPQ-OHTobeSynthesized 13Tat[C 1 L 7,8,12, 13, 15, 16 ]H-CGRKKRLLRQRLLRLLRPPQ-OHTobeSynthesized 14Tat[C 1 Ahx 11 ]H-CGRKKRRQRR-Ahx-RPPQ-OHTobeSynthesized 15Tat[C 1 Ahx 7 ]H-CGRKKR-Ahx-RQRRRPPQ-OHTobeSynthesized 16Tat[C 1 Ahx 10 ]H-CGRKKRRQR-Ahx-RRPPQ-OHTobeSynthesized 17Tat[C 1 Ahx 7,11,13 ]H-CGRKKR-Ahx-RQR-Ahx-R-Ahx-RPPQ- OH TobeSynthesized All peptides are synthesized employing standard Fmoc- solid phase peptide synthesis on Wang resin, purified by RP C18 semi-preparative HPLC, characterized by MS-spectrometry and available at 5-10 mg scale.
Gelatin coated transwell system (from UGent) ARPE19 cells were seeded (750’000 cells/filter) on gelatin coated transwells Hydrogel is composed of 10 wt% gelatin type B Synthesis: polymerization of methacrylamide modified gelatin using e-beam Sample thickness is 2-3 mm Gels cracked. Possible under- cooling during transport.
Future plans Characterization of ARPE19 cells grown on polymer matrix Biochemical differentiation (CRALBP and RPE65) Barrier properties (TER and paracellular permeability) Transfection of ARPE19 cells EBNA plasmid (self-replicating plasmid for prolonged transfection), from Ark Therapeutics Comparison of transfection efficiency in dividing, non differentiated cells, vs. differentiated cells