Fachbereich Veterinärmedizin



    Nicht-viraler Gentransfer mittels Magnetofektion bei primären Zellen des Equinen Sarkoids (2014)

    Wärther, Barbara (WE 17)
    Berlin: Mensch und Buch Verlag, 2014 — VIII, 102 Seiten
    ISBN: 978-3-86387-443-8
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000096422
    Klinik für Pferde, allgemeine Chirurgie und Radiologie

    Oertzenweg 19 b
    14163 Berlin
    +49 30 838 62299

    Abstract / Zusammenfassung

    Equine Sarcoids are the most common tumors in horses and represent more than 90% of skin tumors in this species. Although it is a non-matastasising neoplasia and not directly life-threatening, the owners of affected horses are often confronted with high costs. These are due to the high recurrence rate of Equine Sarcoids and the need for repeated and extensive treatments. In addition the frequent occurrence on sites like saddle and girth region and head lead to a restricted use and thus to a loss of value of the animals.
    An infection of horses with the Bovine Papillomavirus 1 and -2 in combination with a genetic predisposition of certain individuals, is considered as the cause of the disease. This infection leads to a reduction or even loss of MHC I molecules on the cell surface of infected skin cells, so that the immune system can not detect and fight malignant cells. There is a variety of available therapies, ranging from conventional surgery on the production of autologous vaccines to various approaches of immunotherapy. Many of these treatments require invasive procedures and the continued tendency to relapse is high. The aim of gene therapy in treating Equine Sarcoids is to stimulate the body's immune system to cure tumors and thus reduce the incidence of relapse. Gene therapy methods for the treatment of cancer and other diseases are widely used in human medicine and there are already promising results in small animal medicine.
    The aim of this work was to find an effective method of non-viral gene transfer for primary cell cultures of Equine Sarcoids, which should also be suitable for a future use in vivo. To obtain the primary cell cultures, tumor tissue from twelve animals was harvested. These horses were treated for removal of Equine Sarcoids in the Equine Clinic of the Veterinary Faculty of Ludwig-Maximilians University in Munich.
    To find an effective method of non-viral gene transfer, primary cell lines of six horses were transfected using each the standard method “lipofection” and the relatively new method “magnetofection”. Here, differences were found when using different transfection reagents and culture media. In all experiments carried out, cells transfected using the magnetofection protocol showed higher transfection efficiency, measured in nanogram luciferase per milligram of cell protein produced. In the lower dose levels used, highly significant increases could be achieved by the action of the magnetic field. These results are consistent with those of previous studies in the field of small animal and human medicine. Thus, the method is suitable for later use for non-viral gene transfer in Equine Sarcoids in vivo.
    In the second trial, the effect of heterologous feline cytokines on the presentation of MHC-I molecules on the surface of cultured tumor cells of the Equine Sarcoid was investigated. These cells were primary cells from Equine Sarcoids of six animals in which the oncoprotein E5 of Bovine Papillomavirus-1 and -2 was detected. Transfection using the magnetofection method with both the feline plasmid genes IFN-γ and GM-CSF achieved a slight increase of MHC I molecules. This increased expression of MHC-I molecules, however, was also obtained when cells were transfected with the control plasmid pBluc. The effect of the introduced plasmids on the surface properties of cultured cells of the Equine Sarcoid might therefore be the effect of nonspecific immune stimulation. This is due to the DNA backbone of the plasmids used.
    Further studies should therefore first be carried out with the corresponding equine plasmid genes. To evaluate whether the cytokines produced from these plasmids are biologically active, a proliferation assay should also be carried out.