Development of murine leukemia virus-based self-activating vectors that efficiently delete the selectable drug resistance gene during reverse transcription

Expression of the selectable drug resistance gene in retroviral vectors used for gene therapy can lead to a decreased expression of the gene of interest and may induce a host immune response, resulting in a decreased efficiency of gene therapy. In this study, we demonstrate that high-frequency deletion of direct repeats, an inherent property of reverse transcriptases, can be used to efficiently excise the drug resistance gene during reverse transcription. One retroviral vector containing a direct repeat deleted the neomycin resistance expres-sion cassette during a single replication cycle at>99 % efficiency. Most retroviral vectors used for gene therapy are based on murine leukemia virus (MLV) and express a gene of interest as well as a drug resistance gene. A drug resistance gene is typi-cally necessary for selection of virus-producing helper cells or cell clones so that high virus titers can be achieved. The bac-terial neomycin phosphotransferase gene (neo) is commonly used as a selectable marker in retroviral vectors (35). The expression of two genes can be achieved by using the viral long terminal repeat (LTR) promoter to express the gene of inter