86. Mutation Frequency of the p53 cDNA Introduced by the Retroviral Vector

Abstract

The safety and utility of the retroviral vectors must be carefully re-evaluated for further development of gene therapy. It has been well documented that retrovirus mutates at high frequency probably because of the low fidelity of reverse transcriptase. We have previously shown that more than 10 % of clones transduced with a retroviral vector carrying the HSV-TK gene have mutations in the integrated proviral genome. The base substitution rate was estimated to be approximately 1 × 10−4 per base pair. The high mutation rate of retroviral vectors may be a potential problem in certain types of gene therapy protocols. In this study, we examined the mutation rate of the retroviral vector carrying the normal p53 cDNA, which is widely used for gene therapy of cancer. We generated the retroviral vector containing the neoR selectable marker gene and the functional p53 cDNA in the sense orientation (pG1-p53S). As controls, the retroviral vectors containing the p53 cDNA in the antisense orientation (pG1-p53AS) and the mutant p53 cDNA which has a stop codon immediately after downstream of the initiation codon (pG1-mp53S) were also constructed. The vectors were introduced into the p53 deficient lung cancer cell line, H358. G418 resistant clones were isolated and the p53 sequence in the integrated retroviral vector was analyzed by PCR-SSCP followed by direct sequencing. The transduction efficiency of pG1-p53S was significantly less than that of pG1-p53AS or pG1-mp53S. Molecular analysis of clones transduced with pG1-p53S showed that 90% of clones (93/105) have mutated p53 sequences. In contrast, the p53 mutations were identified in 1/123 clones transduced with pG1-p53AS and 1/95 clones transduced with PG1-mp53S. These results indicate that the mutation rate is strongly influenced by the sequence of inserted genes. The p53 sequence is relatively stable compared to the HSV-TK sequence. However, there is still a significant chance that cancer cells could escape from p53 gene therapy because of retroviral replication errors.