polexgene meeting in paris 8 february 2007 astrid subrizi, ddtc, university of helsinki
TRANSCRIPT
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PolExGene Meeting in Paris8 February 2007
Astrid Subrizi, DDTC, University of Helsinki
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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
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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
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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
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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
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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.
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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).
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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.
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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.
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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