Polexgene 12 th month technical meeting
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PolExGene 12 th month technical meeting. Helsinki, 23-24 August 2007. Work packages for UH. 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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Polexgene 12 th month technical meeting

PolExGene 12th month technical meeting

Helsinki, 23-24 August 2007


Work packages for uh
Work packages for UH

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.

    WP 5: Characterization of polyplex-cell and polymer membrane-cell interactions

    Objective: to study the interaction of different cell types, including RPE cells, vascular endothelial cells and smooth muscle cells, with the polymer materials.

  • Both the interaction between CPP-containing polyplexes and cells and the interaction between CIP-containing polymer membranes and cells will be investigated.


Achievments by month 6
Achievments by month 6

  • Characterization of the human RPE cell line (ARPE19) with regard to the expression of specific biochemical markers (CRALBP and RPE65).

  • Determination of the paracellular permeability of ARPE19 monolayers by measuring the apical to basolateral movement of 6-CF over a time course of 3 hours.

  • Transfections were performed using 2 different carriers (PEI n/p 8 and 10, DOTAP/DOPE/PS).

  • Recording of the transepithelial resistance of ARPE19 monolayers to determine the time course and extent of tight junction formation.

  • Visualization of the intracellular distribution of Alexa 488-Tat peptide by CLSM after incubation in dividing ARPE19 cells at 37 °C.

  • A preliminary experiment with hydrogel (crosslinked gelatine) coated Transwell inserts was performed. The gel showed large cracks after one day incubation at 37 °C.


Planned activities months 7 12
Planned activities (months 7-12)

  • Inspection of the hydrogels by microscope to confirm that they are intact, study of their permeability to fluorescent molecules with different molecular weights.

  • Demonstration of the biocompatibility of the hydrogels by studying ARPE19 cell proliferation, differentiation and toxicity.

  • Testing of the functionality of the hydrogels on Transwell inserts by mean of paracellular permeability and transfection.

  • Testing of the efficacy of EBNA plasmid, a self-replicating plasmid for prolonged transfection, and comparison with results obtained with pCMV-SEAP2.


Results by month 12
Results by month 12

  • Polymer membrane (methacrylamide modified gelatin) – “cracking” problem not solved

  • EBNA plasmid – transfection of ARPE19 cells

  • Poly(dimethylaminoethyl-L-glutamine) PDMAEG (V01 and V03) – DNA complexation and transfection efficiency tested

  • Tat peptide and analogues as CPPs – cell uptake studies with ARPE19, CHO wt and mutant cell line


Gelatin hydrogels
Gelatin hydrogels

Problem:

  • Hydrogels “crack” after incubation in cell medium (contains 1% FBS). Cracks form after hours (7h) or days (3d).

    Tentative solution:

  • 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.


  • Permeation of fluorescent markers
    Permeation of fluorescent markers

    • 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).

    • Dextrans might need longer time to permeate the gel (lag phase). Experiment time should be increased.

    • Hydrogel thickness should be decreased

      • to mimic physiological conditions (Bruch’s membrane < 10 μm)

      • to facilitate basolateral transfection of cells


    Ebna plasmid pepi seap

    EBNA plasmid

    pCMV-SEAP2

    EBNA plasmid – pEpi-SEAP

    • Responsible for extrachromosomal maintenance

      • Latent replication origin (OriP)

      • EBV nuclear antigen 1 (EBNA-1)

        Ensure replication of plasmid once per cell cycle during S phase and segregation into the daughter cells.


    EBNA plasmid

    pCMV-SEAP2

    pCMV-SEAP2

    EBNA plasmid


    Ebna plasmid
    EBNA plasmid

    No significant benefit observed

    • Transfected cells were differentiated and non dividing (3 weeks growth on filter)

      The same experiment will be repeated with non differentiated, dividing cells.


    Polycations pdmaeg v01 and v03 2 secondary amines and 1 tertiary amine

    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.

      • Similar results obtained earlier with PDMAEG of different MW (Biomacromolecules 2003).

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


    Cpps tat peptide and analogues
    CPPs – Tat peptide and analogues

    • Tat peptide and 16 analogues were tested for cell uptake experiments (flow cytometry) with 3 different cell lines (ARPE 19, CHO wt and CHO mutant).


    Plans for months 13 18
    Plans for months 13-18

    • Biocompatibility of improved biopolymer membrane with cells (proliferation, differentiation, toxicity)

    • Transfection of dividing ARPE19 with EBNA plasmid

    • Transfection of ARPE19 with plasmid containing a RPE-specific promoter (tyrosinase promoters)

    • Comparison of transfection efficiencies of polyplexes non-covalently linked to CPP (penetratin and Tat peptide) (protocol?)


    Exchange products

    From UGent:

    PDMAEG V01 and V03

    Gelatin coated transwell

    Modified gelatine

    From Ark:

    pEPI-SEAP

    EBNA plasmid with RPE-specific promoter (in future)

    From CNRS:

    Penetratin

    To UGent, IMIC and IBMT:

    pCMVß

    Exchange products


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