Klonieren der Felinen Zytokin-Gene IL-2, IFNγ und GM-CSF zum Adjuvanten, Nonviralen Gentherapeutischen Einsatz beim Fibrosarkom der Katze

The feline fibrosarcoma is a spontaneously occurring malignant soft tissue sarcoma. Due to its high recurrence rate of approximately 70%, the prognosis is very poor even after radical surgical excision. So far different cytokine gene therapy protocols have been carried out adjuvant to surgical tumour excision. A nonviral gene delivery system has been developed in this project. A collagen sponge was loaded with plasmids in a copolymer-protected gene vector (COPROG) formulation. The plasmids were coding for interleukin-2 (felL-2), interferon y (felFNγ) and granulocyte-macrophage colony-stimulating factor (feGM-CSF). The aim of this gene therapeutic approach is to enhance anti-tumour immunity, extend the tumour-free survival time or to decrease the recurrence rate in fibrosarcomaaffected cats. Indeed, the final aim is the development of an immunotherapy adjuvant to the surgical excision of the feline fibrosarcoma. The initial goal reported in this paper, however, was to clone these three cytokines as well as to prove the biological activity of the recombinant proteins. The cDNA coding for felL-2, felFNγ and feGM-CSF was cloned via PCR using feline peripheral mononuclear blood cells. The recombinant proteins were expressed in a mammalian cell line. The felL-2 and feGM-CSF bioactivity was demonstrated in proliferation assays. The biological activity of felFNγ was measured by FACS analysis. Feline IFNγ induces an increased MHCI and II expression on feline fibrosarcoma cells. After plasmid complexation with the polycation polyethylenimine and protective copolymers, the collagen sponge was soaked with the gene vectors and lyophilised. It was stored under sterile conditions until implantation in the tumour bed of fibrosarcoma-affected cats. A phase 1 dose escalation study was conducted using the vector-loaded collagen sponge.