Regulation of L-DOPA production by genetically modified primary fibroblasts transfected with retrovirus vector system

Abstract

Although intracerebral grafting has become established as a new strategy for the treatment of Parkinson's disease, there are many problems regarding such grafts. We focused on the grafting of primary skin fibroblasts. Rat primary skin fibroblasts were transfected with a retrovirus vector containing cDNA of human tyrosine hydroxylase (TH) type 1 (LTHSNL) or of cytomegalovirus promoter (CTHSNL) as a foreign promoter. In these genetically modified fibroblasts, L-DOPA production and release were analyzed in vitro by immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Being supplemented with the biopterin (BH4:(6R)-L-erythro-tetrahydrobiopterin) cofactors required for TH activity, these cells produced and released L-DOPA into the culture medium. When CTHSNL and BH4 were combined, L-DOPA production increased with time, regardless of the number of cell passages, or the duration of liquid nitrogen freezing. This suggests that the foreign gene (THcDNA) containing retrovirus vector integrates into the chromosomal DNA of the target cells (fibroblasts). Primary fibroblasts can be easily obtained and cultured. Thus, genetically modified primary skin fibroblasts transfected with a retrovirus vector system containing the TH cDNA may be promising grafts for transplantation and gene therapy in Parkinson's disease.