Cloning of the feline cytokines IL-2, IFN gamma and GM-CSF for an adjuvant nonviral gene therapy of feline fibrosarcoma.

摘要

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 as an adjunct to surgical excision of the tumour. A non-viral gene delivery system was developed in this study. A collagen sponge was loaded with plasmids in a copolymer-protected gene vector (COPROG) formulation. The plasmids coded for interleukin 2 (feIL-2), interferon- gamma (feIFN gamma ) 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 and decrease the recurrence rate in fibrosarcoma-affected cats. 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 feIL-2, feIFN gamma and feGM-CSF was cloned via PCR using feline peripheral mononuclear blood cells. The recombinant proteins were expressed in a mammalian cell line. The feIL-2 and feGM-CSF bioactivity was demonstrated in proliferation assays. The biological activity of feIFN gamma was measured by FACS analysis. Feline IFN gamma induced an increase in MHC I 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 lyophilized. It was stored under sterile conditions until implantation in the tumour bed of fibrosarcoma-affected cats. A phase I dose escalation study was conducted using the vector-loaded collagen sponge.