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