Gene therapy for malignant melanoma using bacterial cytosine deaminase gene under the control of tyrosinase promoter

Gene therapy for malignant melanoma using bacterial cytosine deaminase gene under the control of tyrosinase promoter

Objective To clone tyrosinase gene promoter and evaluate the role of the promoter in malignantmelanoma cell--specific prodrug activation gene therapy. Methods: PCR was employed to clone murine tyrosinase gene promoter from genomic DNA of B16 cells. Mutation of the promoter was analyzed by DNA scquencing. The promoter was then utilized to direct bacterial cytosine deaminase (CD) gene in a retroviral vector. In vitro gene expression and in vivo gene therapy were carried out. Results: Sequence of tyrosinase genepromoter from B16 cells showed a 98. 7 % homology to that from normal cells. The tyrosinase gene promoterconferred a selective expression of CD gene in B16 cells in vitro. The in situ produced retroviruses containingtyrosinase gene promoter drived CD gene were capable of infecting tumor cells and bone marrow cells inC57BL/6 mice. The Bl6 established tumor significantly regressed following 5-fluorocytosine (5-FC) systemicadministration. The in vivo gene therapy, unlike long terminal repeat (LTR ) universal promoter directed CDgene, did not lead to the arrest of bone marrow. Conclusion: CD gene directed by the tyrosinase gene promoters, either from melanorna cells or from normal cells, is effective and safe for the melanoma in vivo genetherapy.