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A recombination between the short homologous regions of nucleotide sequences in the retroviral vector and packaging cell line has been thought to be a major cause of the production of replication-competent retrovirus (RCR). Therefore, the removal of overlapping sequences between the vector and the packaging constructs is crucial for minimizing the possibility of homologous recombination, and therefore, the production of RCR. We have recently constructed a series of retroviral vectors that contain no viral coding sequences, but still produce high viral titer and high-level gene expression. However, many previously constructed murine leukemia viurs (MLV)-based packaging constructs contained significantly long 5 0 and/or 3 0 untranslated regions of MLV, which are also present in the retroviral vector, and as such could possibly lead to homologous recombination. To make a retroviral production system that is free from homologous recombination, we constructed expression plasmids for gag-pol and env, precisely starting from the start codon and ending at the stop codon of respective open reading frames. When the packaging function was provided from one plasmid, a vector containing bits of all three viral coding sequences produced RCR at a significant frequency, while our vector remained free of any RCR. Our retrovirus production system is anticipated to have the minimum possible frequency of RCR production due to the elimination of potential sites for homologous recombination. Based on these results, a highly efficient new packaging line Vamp that contains no overlapping sequences with our retroviral vector was also developed. Gene Therapy (2003) 10, 706-711. doi:10.1038/sj.gt.3301892Keywords: retroviral vector; packaging line; homologous recombination; RCR Murine leukemia virus (MLV)-based vectors are the most frequently used gene delivery system, employed in almost 40% of approved protocols and 50% of patients who have been subjected to or are undergoing clinical trials. However, there are many problems that need to be improved in order for MLV-based vectors to become a viable form of gene delivery vehicle in actual clinical settings. In particular, the issue of safety has often been raised because of the possibility of the production of replication-competent retrovirus (RCR). The mechanism of RCR generation still remains poorly understood, but it has been thought to result from homologous recombination between the same nucleotide sequences present in the vector and in the packaging constructs. Indeed, it has been reported that homologous recombination could occur between as very short regions of homology as 7 or 11 bp. 1-3We have recently constructed a series of retroviral vectors that are absent of any viral coding sequences without compromising viral titer or the level of gene expression. 4 These vectors are thought to be safer than other existing vectors harboring viral coding sequences because the probability of homologous recombination between the vector and the packaging genome is much lower with these newly d...

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This assay system might provide a useful tool for selecting the appropriate vector structure, as well as for studying the molecular mechanism underlying the cis-activation by the viral LTR.

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