376. Various Aspects of the Single-Stranded DNA Fragment-Mediated Gene Correction

Abstract

The gene correction strategy, by which mutations are converted to the normal sequences in living cells, is expected as an important technology for gene therapy and biotechnology. We have developed a new gene correction method by using a sense single-stranded (ss) DNA fragment (fSense) prepared from phagemid DNA (Tsuchiya H, et al. J. Gene Med., in press). This ss DNA fragment provided 12-fold higher gene correction efficiency as compared with a double-stranded (ds) PCR fragment used in the conventional small-fragment homologous replacement method. However, at the present time, very limited information is available about factors affecting the correction efficiency and mechanisms involved in the ss DNA fragment-mediated gene correction. In the present study, we performed several experiments to reveal the factors and mechanisms how the ss DNA fragments correct mutations of target genes. Moreover, we examined the potency of new DNA fragments modified based on the outcomes of the mechanistic studies. First, we investigated the effects of the transcription of the target gene, of the DNA modifications (5'-phosphate and N6-methyladenine [mA]) of fSense and of the length of ss DNA fragments on the gene correction. Second, we examined whether fSense was integrated into the homologous strand of the target gene. It was revealed that the transcription did not affect the gene correction while the mA, but not 5'-phosphate, significantly stimulated the gene correction (2-fold increase), and that there was an optimum value in the length (400|[ndash]|1200 nt). Additionally, fSense was indeed integrated into the target gene, indicating that a homologous recombination (HR) pathway was, at least in part, involved in the gene correction process. This finding led us to further modifications of the ss DNA fragments based on the HR mechanism. Currently, the newly modified DNA fragments are under investigation. These results suggest that further improvement in the gene correction efficiency can be achieved by modification(s) of DNA fragments based on the correction mechanism, the HR pathway.