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PolExGene MIDTERM MEETING GHENT

PolExGene MIDTERM MEETING GHENT. Biocompatible non-viral polymeric gene delivery systems for the in vivo treatment of ocular and cardiovascular diseases with high unmet medical need. Sylvie Julien Tobias Peters.

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PolExGene MIDTERM MEETING GHENT

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  1. PolExGene MIDTERM MEETING GHENT Biocompatible non-viral polymeric gene delivery systems for the in vivo treatment of ocular and cardiovascular diseases with high unmet medical need Sylvie Julien Tobias Peters Tuebingen group: Prof. E. Zrenner, Prof. U. Schraermeyer, Prof. M. Seeliger, PD Dr. Szurman, Dr. E. Fahl, Dr. S. Julien, T. Peters PolExGene Midterm Meeting Ghent December 2007

  2. The Tübingen Contributions • WP 7.1: Implantation of polymer membranes for ocular diseases (30 months) • WP 6.2:In vivo immunological properties of polyplexes and polymer membranes (6 months) PolExGene Midterm Meeting Ghent December 2007

  3. Implantation of polymer membranes for ocular diseases Feasibility of eye surgery 18 eyes are implanted: 6 implants intraocular, 12 implants subretinal → first implantations succeeded WP 7.1 (Implantation) PolExGene Midterm Meeting Ghent December 2007

  4. The principle of subretinal surgery • Subretinal implantation of the polymer complex: • -anaesthesia, • -lateral sclerotomy, • -insertion of the polymer complex funduscopically controlled. • SLO examination PolExGene Midterm Meeting Ghent December 2007

  5. Position of implant in rat eyes by using SLO • The HRA I features 2 argon wavelengths (488 nm and 514 nm) and 2 infrared diodes lasers (795 nm and 830 nm). • The short wavelenght range provides higher contrast images of the retina but can not penetrate the RPE/choroid. • The long wavelenght range permits to see through the choroid and the sclera. • The 488 nm and 795 nm lasers are used for fluorescein (FA) and indocyanine green (ICG) angiographies and offer an access to vascular structures associated with each layer. • Duration of SLO imaging: 10-20 min (one eye) PolExGene Midterm Meeting Ghent December 2007

  6. Position of implant in rat eyes (example) AF 20° AF 10° RF 20° RF 10° Rat 3RF20 PolExGene Midterm Meeting Ghent December 2007

  7. Preservation of retinal and choroidal vessels integrity after surgery FA 20° FA 10° ICG 20° ICG 10° Rat 3FA 10 PolExGene Midterm Meeting Ghent December 2007

  8. Development of implant tool, prototype: Surgical instruments PolExGene Midterm Meeting Ghent December 2007

  9. Electrophysiology (ERG): function of rods: - scotopic protocol: - condition dark adapted - increasing stimulus luminance up to 100 cds/m² - increasing interstimulus intervals Function testing from first rats (example) PolExGene Midterm Meeting Ghent December 2007

  10. scotopic ERG after implantation (example) Control Rat 5 OS Rat 6 OS Control II 0.0003 cd 0.003 cd 0.03 cd 0.3 cd [500 µV] per scalebar 3 cd 10 cd 30 cd 100 cd PolExGene Midterm Meeting Ghent December 2007 Latenz [ms]

  11. scotopic ERG after implantation (example) Scotopic a-wave Scotopic -wave Light intensity cd.s/m² Light intensity cd.s/m² PolExGene Midterm Meeting Ghent December 2007

  12. Function of cones: photopic Protocol: - condition light adapted - 30Hz flicker - light stimulus Examples of function testing from first rats PolExGene Midterm Meeting Ghent December 2007

  13. photopic ERG after implantation (example) Control Rat 5 OS Rat 6 OS Control II PolExGene Midterm Meeting Ghent December 2007

  14. In vivo tissue reaction Ophthalmoscopy via direct Fundusphotography, scanning laser ophthalmoscopy and fluorescence angiography out of WP 7.1 Histological exam via electron microscopy and immunohistochemistry (in progress) WP 6.2 (immunological properties ) PolExGene Midterm Meeting Ghent December 2007

  15. Mice: Various disease models Subretinal approach difficult Rats: Disease models Subretinal approach good Rabbits: No disease model surgical approach good Why do we have different animals? PolExGene Midterm Meeting Ghent December 2007

  16. Concepts for degenerated retinae (RCS rats) ONL 5 weeks 3 weeks 7 weeks inner nuclear layer disturbed Phagozytosis of outer segments via RPE outer plexiform layer outer nuclear layer inner segments debris layer retinal pigment epithelium ERG response choroid PolExGene Midterm Meeting Ghent December 2007 M Tschernutter, et al; Br J Ophthalmol 2006

  17. Does transfection work in RCS RPE? Primary RPE cell culture RPE from RCS rats (to UH.FP and ENS) • - Source: 16 eyes of 8 RCS rats, 5 month of age • incubated in a solution of 2% dispase in DMEM • opened by an incision posterior to the ora • serrata. • Cornea, lens and vitreous were removed • RPE was separated from the retina and • the Bruch‘s membrane and immersed in a • 0.125% trypsin solution in EDTA for 45 min • at 37°C. • Enzyme reaction stopped by addition of DMEM. • Cell suspension was centrifugated and • resuspended in DMEM with 0.1 mg/ml • Gentamycin, 0.25 mg/ml amphotericin B and • 2 mM L-Glutamine. Cytokeratin staining after 3 weeks of culture PolExGene Midterm Meeting Ghent December 2007

  18. How can therapeutic approach be assessed ? • Maxipreparation of MertK cDNA (gift from Douglas Vollrath) • C-mer proto oncogene receptor tyrosine kinase • Development of in vivo markers and Providing antibody for MertK-HA detection by ENS PolExGene Midterm Meeting Ghent December 2007

  19. Thank you for attention PolExGene Midterm Meeting Ghent December 2007

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