Ž . Molecular Brain Research 47 1997 195–201 Research report Potential effect of cytokines on transgene expression in primary fibroblasts implanted into the rat brain Malcolm Schinstine a , Jasodhara Ray b, ) , Fred H. Gage b a Department of Neurobiology and Anatomy, Medical College of PennsylÕania and Hahnemann UniÕersity, Broad and Vine Streets, Philadelphia, PA 19102, USA b Laboratory of Genetics, Salk Institute, 10010 N Torrey Pines Road, La Jolla, CA 92037, USA Accepted 23 December 1996 Abstract Fibroblasts genetically modified with retroviral vectors fail to demonstrate long-term transgene expression upon implantation into the body. Although the mechanisms behind this phenomenon have not been elucidated, one likely cause is the response of the host to the graft. For example, genetically modified fibroblasts grafted into the brain are surrounded by activated microglia and astrocytes. The apparent inflammatory response can last for several weeks. In addition, the center of the graft is typically infiltrated with macrophage-like cells that appear to reside continuously within the graft. This proximity of inflammatory cells to the graft suggests that these cells may w somehow influence transgene expression. In the current study, an in vitro model was used to test the effect cytokines transforming Ž . Ž . Ž .x growth factor-b 1 TGF-b 1 , interleukin-1 b IL-1 b and tumor necrosis factor-a TNF-a that are typically released by peripheral Ž . macrophages, activated microglia andror astrocytes have on long-terminal repeat LTR -driven transgene expression in primary fibroblasts. Our data demonstrate that these cytokines can significantly reduce the steady-state level of proviral mRNA. The amount of proviral mRNA returned to control levels within 24 h if the cytokines were removed. In addition, the down-regulation of proviral mRNA Ž . levels could be prevented if the cells were incubated with dexamethasone 25 mM concurrent with the introduction of cytokines. These data demonstrate that cytokines can down-regulate LTR-driven transgene expression in primary fibroblasts maintained in culture. This interaction may be a major reason why transduced cells do not demonstrate long-term transgene expression in vivo. Keywords: Retroviral vector; Transplantation; Drosophila choline acetyltransferase; Dexamethasone; Cytokine 1. Introduction Genetically modified fibroblasts have been proposed as one method to deliver deficient or therapeutic molecules Ž . w x into the central nervous system CNS 3,7,9,10,18,32 . Most fibroblasts used in somatic gene therapy studies have been modified via retroviral vectors. This method offers the advantages of high-transduction efficiency and ease of transduction. In addition, expression from the retroviral LTR in cultured fibroblasts is relatively high when com- pared to other viral or cellular promoters. Moreover, trans- gene expression in cultured fibroblasts is stable and can be sustained for long periods. Because of these in vitro obser- vations, infected fibroblasts were implanted into animals with the expectation that the sustained high levels of ) Ž . Corresponding author. Fax: q1 619 597-0824. transgene expression observed in culture would also be observed in vivo. Although initial findings were promis- ing, more recent data have revealed that with time the expression of transgenes in implanted fibroblasts dramati- w x cally decreases 16,20 . These studies suggest that some Ž. factor s affects the expression of LTR-driven transgenes w x in implanted fibroblasts 22,33 . One of the factors that may influence transgene expres- sion in vivo is the mitotic state of the post-implant cells. Fibroblasts implanted into the CNS are non-proliferative. Data from our laboratory have shown that the expression Ž . of LTR-driven transgenes in quiescent non-mitotic fi- broblasts gradually decreases, over a 2-week period, to w x 20–25% of the activity measured in growing cells 21 . Therefore, one of the factors that may account for the decline in transgene expression in vivo is the non-prolifer- ative state of the fibroblasts; however, this mechanism alone probably does not account for the entire decrease in transgene expression observed in vivo. 0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0169-328X 97 00049-1