PolExGene kick-off meeting (Ghent, 2006)

1. PolExGene kick-off meeting(Ghent, 2006) Thomas Wirth M.Sci (Pharm), PhD A.I. Virtanen Institute University of Kuopio, Kuopio, Finland

2. located at the A.I. Virtanen institute the A.I. Virtanen institute harbors 12 research groups harbors the  Center of Excellence for Research in Cardiovascular Diseases and Type 2 Diabetes The UKU group

3. Cardiovascular diseases Atherosclerosis stenosis/restenosis ischemia Cancer glioma ovary kidney pancreatic The UKU group... Thomas Wirth, et al. Safety profile of plasmid/liposomes and virus vectors in clinical gene therapy. Curr Drug Safety (2006). Thomas Wirth and Seppo Ylä-Herttuala. The brain as a target for gene therapy. Review. Adv Tech Stand Neurosurg (2006).

4. Nanoparticulate drug carriers for cancer therapy for cardiovascular diseases Phage Display human IgG libraries The UKU group...

5. Gene transfer technology with nonviral vectors and with viral vectros (adenoviruses, lentiviruses, AAV, baculoviruses and SFV) Gene arrays Laser capture microscopy Small animal imaging MRI CT-SPECT general molecular biology and biochemistry techniques In vivo studies Research methods

6. Cardiovascular diseases transgenic mice rats rabbits (including transgenic rabbits) pigs Cancer mice rats Animal models

7. Objective Our objective during this project is to develop an ex vivo based gene therapy approach for the treatment of stenosis/restenosis in cardiovascular diseases.

8. Reasons for occlusion • Anastomosis = intersection of blood vessels and vascular graft (e.g.hemodialysis patients) • autologous graft • e.g. bypass surgery • artificial graft • e.g. end stage renale disease: 70% currently use artificial vascular grafts for hemodialysis • Atheroslcerosis • Restenosis • Diabetes

9. Treatment of occluded vessels • Vascular graft surgery • autologous graft • artificial grafts • stents • Percutaneous coronary intervention • balloon angioplasty • brachytherapy • bare metal stents • drug eluting stents • Gene therapy

10. Balloon angioplasty • balloon is at the end of a catherter • by inflating the balloon the atherosclerotic plaque is flattend against the arterial wall • sometimes artery collapse immediately after balloon dilatation (recoil) • sometimes arteries begin to close up within a time frame of 6 months (negative remodelling, restenosis)

11. Stents • mesh-like metal device • bare metal stent • drug eluting sten (DES) • prevents artery from immediate collapsing after angioplasty • rates of restenosis lower than with balloon angioplasty • restenosis occurs usually between 6 to 9 months after procedure

12. Brachytherapy • only utilized for cases requiring a second angioplasty • a catherter with radioactive isotopes attached is placed for 5 to 20 minutes to the previously placed stent

13. Patients undergoing percutaneous coronary intervention (PCI), reocclusion of the dilated artery might happen = Restenosis

14. Restenosis after PCI

15. Prevention of stenosis / restenosis • Preventing SMC proliferation • DES (sirolimus, paclitaxel) very effective but delayed healing of EC layer causes late thrombosis • Brachytherapy • Inducing late apoptosis • P53 overexpression effective in animal models but no long-term data. Also early apoptosis contributes to later SMC proliferation • Improving EC recovery • VEGF • Anti-inflammatory therapy • Tacrolimus seems promising

16. VEGF • vascular protection • suppression of thrombosis • suppression of SMC proliferation • anti-inflamatory • improvement of EC recovery

18. Our goal is... to develop an ex vivo based gene therapy approach for the treatment of stenosis/restenosis in cardiovascular diseases.

19. What are we going to deliver... • cell specific and therapeutic plasmids • transfected of vascular cells using polyplexes • transfected vascular cells on polymer membranes • polyplex containing polymer membranes seeded with vascular cells using marker/therapeutic genes • vascular cell seeded polymer membranes as coating for vascular prosthesis

20. How are we going to delivery • cloning of marker/therapeutic genes into plasmids • under CMV promoter • under RPE-specific tyrosinase promoter (EBNA plasmid) • evaluation of transfection efficiency of polyplexes in vitro • evaluation of expression of poymer membrane seeded with ex vivo transfected vascular cells in vitro • evaluation of expression of polyplex containing polymer membranes seeded with vascular cells in vitro

21. Continues... • evaluation of expression of polymer membrane seeded with ex vivo transfected vascular cells in vivo • evaluation of expression of polyplex containing polymer membranes seeded with vascular cells in vivo

22. + ELISA In vitro model Vascular cells polyplexes

23. In vitro model vascular cells polymer membrane with polyplexes Medium ELISA

24. New Zealand white (NZW) Atherosclerosis can be induced with diet Mostly fatty streaks in large vessels Coagulation system close to human Thrombus formation Cholesterol metabolism similar to human ApoB not edited to apoB48 in liver Cholesterol ester transfer protein as human Watanabe heritable hyperlipidemic (WHHL) LDLr mutation leading to FH-like condition Early formation of fatty streaks Complicated lesions after 1 yr No significant calcifications Lesions in large vessels Cholesterol levels beyond physiological limits St.Thomas hospital strain High VLDL, IDL and LDL due to lipoprotein synthesis Rabbit

25. Collar model