Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit... Show moreGene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over "classical" rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo. Show less
This thesis focuses on growth inhibition of small abdominal aortic aneurysms (AAA), which is a potentially lethal, pathological dilatation of 30 mm or more of the infrarenal aorta. The main... Show moreThis thesis focuses on growth inhibition of small abdominal aortic aneurysms (AAA), which is a potentially lethal, pathological dilatation of 30 mm or more of the infrarenal aorta. The main clinical challenges are currently: Can we predict aneurysm growth? Is it possible to keep small aneurysms small? Can we translate the preclinical evidence into a medical therapy? On theoretical grounds and preclinical data, doxycycline appears an excellent lead-candidate for the medical stabilization of growing AAA. However, this randomized placebo controlled multicentre trial revealed that doxycycline treatment did not inhibit aneurysm progression but increased the growth rate. These highly remarkable findings fundamentally challenge the current paradigm that inflammation per se is harmful in AAA progression. In this context, other findings of this thesis gain importance, showing that patients with diabetes mellitus have slower aneurysm growth and that aneurysm patients are more likely to develop COPD. The responsible pathophysiological mechanisms contain undoubtedly important clues, but have to be unraveled yet. Show less
