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PolExGene Meeting in Paris8 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
Transfection with PEI n/p 8
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
0 10 20 30 40
Days in culture after transfectionSE
AP
[ng/
h fil
ter]
Apical
Basolateral
Transfection with PEI n/p 10
0,0
50,0
100,0
150,0
200,0
250,0
0 10 20 30 40
Days in culture after transfection
SEA
P [n
g/h
filte
r]
Apical
Basolateral
Transfection with DOTAP/DOPE/PS
0,0
1000,0
2000,0
3000,0
4000,0
5000,0
6000,0
0 10 20 30 40
Days in culture after transfection
SE
AP
[n
g/h
filt
er]
Apical
Basolateral0,0
100,0
200,0
300,0
400,0
500,0
0 20 40
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 8 PEI 10
DOTAP
Apical 11,60
16,36 272,41
Basolateral
13,01
19,10 171,07
Total 24,61
35,45 443,47
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. Code Sequence HPLC purity % MW calculated MW measured Amount mg
1 Tat[C1] H-CGRKKRRQRRRPPQ-OH 95 1821.3 1822.1 15
2 Tat[C1W7,8] H-CGRKKRWWRQRRRPPQ-OH 93.5 2193.8 2194.1 5
3 Tat[C1W11,12] H-CGRKKRRQRRWWRPPQ-OH 96.2 2193.8 2194.0 7
4 Tat[C1W10,11] H-CGRKKRRQRWWRRPPQ-OH 92.3 2193.8 2194.05 8
5 Tat[C1W7,8,12, 13, 15, 16] H-CGRKKRWWRQRWWRWWRPPQ-OH To be Synthesized
6 Tat[C1A7,8] H-CGRKKRAARQRRRPPQ-OH 98.2 1963.5 1964.12 10
7 Tat[C1A11,12] H-CGRKKRRQRRAARPPQ-OH 97.1 1963.5 1964.15 8
8 Tat[C1A10,11] H-CGRKKRRQRAARRPPQ-OH 95.1 1963.5 1964.16 11
9 Tat[C1A7,8,12, 13, 15, 16] H-CGRKKRAARQRAARAARPPQ-OH To be Synthesized
10 Tat[C1L7,8] H-CGRKKRLLRQRRRPPQ-OH To be Synthesized
11 Tat[C1L11,12] H-CGRKKRRQRRLLRPPQ-OH To be Synthesized
12 Tat[C1L10,11] H-CGRKKRRQRLLRRPPQ-OH To be Synthesized
13 Tat[C1L7,8,12, 13, 15, 16] H-CGRKKRLLRQRLLRLLRPPQ-OH To be Synthesized
14 Tat[C1Ahx11] H-CGRKKRRQRR-Ahx-RPPQ-OH To be Synthesized
15 Tat[C1Ahx7] H-CGRKKR-Ahx-RQRRRPPQ-OH To be Synthesized
16 Tat[C1Ahx10] H-CGRKKRRQR-Ahx-RRPPQ-OH To be Synthesized
17 Tat[C1Ahx7,11,13] H-CGRKKR-Ahx-RQR-Ahx-R-Ahx-RPPQ-OH
To be Synthesized
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 matrixBiochemical differentiation (CRALBP and RPE65)Barrier properties (TER and paracellular
permeability)
• Transfection of ARPE19 cellsEBNA plasmid (self-replicating plasmid for
prolonged transfection), from Ark TherapeuticsComparison of transfection efficiency in dividing,
non differentiated cells, vs. differentiated cells