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Osteoarthritis (OA) is one of the most common musculoskeletal disorders around the globe. Affected patients suffer from a huge loss in quality of life, and the treatment of OA has a strong financial impact on society. The underlying mechanism is based on cartilage destruction accompanied by an inflammatory response that leads to chondrocyte apoptosis and matrix degradation. However, an effective causal therapy which provides long-term remedy has not been found yet.
One new promising strategy is the application of local gene therapy. In order to avoid continuous overexpression, a controlled expression of therapeutic transgenes which should be self-limiting and operate only in case of inflammation is of main interest. Therefore, a “genetically switchable” plasmid was developed by placing the Cox-2 promoter in front of the chosen transgene IL-4. Previous studies by others proved the feasibility of the new gene therapeutic approach in a canine in vitro model. The aim of this thesis was to transfer and test the inflammation-controlled and self-limiting gene therapeutic approach in an in vitro equine cell culture model. Additionally, different questions on the feasibility and state of the art in OA gene therapeutic research, the possible risk and toxicity of IL-4 as effective transgene, and the suitable animal model for OA research were addressed in a copious literature search.
The first step in the experimental work included the establishment of an inflammatory in vitro 2D equine cell culture model to create an OA-like system. Therefore, equine chondrocytes were used in passage 3 to 4 and stimulated with 100 ng/ml recombinant equine IL-1β and LPS. The successful treatment was shown by proper inflammatory increased IL-1β mRNA expression. In a further step, alignment studies on the canine and equine Cox-2 promoter were performed with the result that only 46.4% consensus positions were determined. For the cloning of equine Cox-2 promoter regulated plasmids expressing equine IL-4, pIRES2-EGFP and pN3-EGFP were used as basis plasmids. During the optimization of the transfection protocols and adjustment to equine chondrocytes, different non-viral transfection reagents were examined, that yielded transfection rates up to 21% by combining Turbofect and pIRES2-EGFP. The transfection efficiency could be raised up to 44% by delivering a plasmid without IRES (pN3-EGFP). Further investigations focused on the functionality of the equine plasmid pN3.Cox-2.IL-4. Equine chondrocytes were transfected with pN3.Cox-2.IL-4 and exogenously stimulated, which leads to a significant increase in IL-4 mRNA expression and protein translation. Simultaneously, endogenous IL-1β expression as well as expressions of specific OA markers decreased conceivably due to the presence of newly synthesized IL-4.
The literature part covers the potential risk and toxicity of an intra-articular injection and expression of IL-4. Several clinical studies were summarized, which showed the risk of adverse effects of systemic rhuIL-4 application in high doses (up to 45 μg/kg/d i.v.) and strong treatment protocols in late stage cancer patients. Nevertheless, the risk of achieving these comparable doses when applying IL-4 via gene therapy in the joint was rated as low.
In addition, the choice of a suitable animal model is of great importance for the translational process as well as the interpolation possibility to the human patient. Because of the size, joint anatomy and cartilage thickness, the horse provides a number of advantages as animal model for OA research. As natural occurring model, OA pathogenesis parallels the human disease in many facets and the translation to patient horse and afterwards to the human is accelerated. Furthermore, during the last years, many efforts have been made towards the safety and acceptation of OA gene therapy. First clinical trials have been successfully conducted and further studies are in preparation.
Nevertheless, very few researches focus on the controlled expression of therapeutic transgenes. In this thesis, the transformation of a molecular therapeutic approach to the equine system could be demonstrated. This new approach should be exploited to develop an anti-inflammation therapy initiating itself by an inflammatory microenvironment. Molecular tools such as the described therapeutic plasmid pave the way for a successful controlled, self-limiting and local gene therapy.