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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.

slide9

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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