Genetically Modified Fibroblasts-Fibrin-Glue-Suspension for in vivo Drug Delivery of Epidermal Growth Factor (EGF): A Surface Gene Therapy Model not only for Wounds

Abstract

Gene transfer offers an attractive method for the localized and persistent active delivery of growth factors to healing wounds: Gene therapy allows the controlled modification of cells to secrete the continuous localized production of polypeptides into the surrounding tissues. The purpose of this experimental study was to demonstrate the successful transplantation of in vitro liposomally transfected human fibroblasts expressing the human Epidermal Growth Factor (EGF) — gene, resuspended in fibrin glue, for the in vivo production of the growth factor EGF. Liposome transfection of the human fibroblast cell line KMST-6 was performed with an human EGF-Plasmid-Construct. The most effective clones were selected and cloned. The production of human EGF was detected with a specific ELISA for human EGF. The biological effect of the secreted EGF on keratinocyte proliferation was defined in a cell-proliferation-assay. Full-thickness excisional wounds were created on the dorsum of nude mice. In the treatment group transfected and lethally irradiated fibroblasts, resuspended in fibrin glue were transplanted (Group I, n=14). Full-thickness wounds, transplanted with untransfected fibroblasts (Group II, n=14) and untreated full-thickness wounds (Group III, n=14) served as controls. On day 1, 2, 3, 4, 5, 7 and 14 wounds of two animals from each group were biopsied and the biopsies then homogenized. Human EGF-levels in the tissue were detected with an anti-human-EGF ELISA. We detected 40 ng/24 h human EGF in the supernatant of 106 transfected fibroblasts in vitro. Furthermore keratinocyte proliferation was enhanced twolfold after treatment with conditioned supernatants from EGF-transfected fibroblasts. Moreover we measured 470 pg/ml human-EGF in the wound tissue on day 1 in Group I, compared to the 18 pg/ml in Group II and 1,3 pg/ml in Group III in vivo. On days 2-7 EGF levels decreased in Group I, but were still significantly higher compared to Groups II and III. On day 14 no human EGF was detectable. In summary, human fibroblasts were successfully transfected with the human EGF gene using a liposomal genetransfer method. The transgene protein was biologically active and increased in vitro keratinocyte proliferation. After transplantation of these transfected fibroblasts resuspended in fibrin glue onto full-thickness wounds, human EGF secretion was detectable until day 7. These data suggest the possibility to use in vitro lipofected fibroblasts for the in vivo production of therapeutic proteins for therapy of chronic wounds. It may also be used as an in vivo Drug-Delivery-System for other therapeutic proteins