Biol. Pharm. Bull. 28(10) 1958—1962 (2005)

fragments, containing the normal sequences, have been examined for their gene correction abilities by the small fragment homologous replacement (SFHR) method. Correction of mutations in the CFTR and dystrophin genes by the SFHR method has been examined and partial gene corrections were obtained. The corrected genes should be properly expressed under the control of the original promoter. When corrected, the therapeutic effects are expected to be life-long. Gain-of-function or predominant mutations could be suitable subjects for gene correction, making SFHR a highly attractive therapeutic strategy. However, the current SFHR method yielded the low correction efficiency, and thus, the correction efficiency of SFHR must be improved. Recently, we prepared a ds DNA fragment, and singlestranded (ss) DNA fragments containing sense and antisense sequences, by restriction enzyme digestions of plasmid DNA and ss phagemid DNAs, respectively. The gene correction efficiencies of these fragments were tested with an inactivated episomal hygromycin-resistance (Hyg) and enhanced green fluorescence protein (EGFP) fusion gene containing a base substitution, as a model target (Fig. 1A). We found that the ds DNA fragment prepared by restriction enzyme digestion of plasmid DNA (dsHES, Fig. 1B) provided a 2-fold increase in the gene correction activity, as compared to the conventional PCR fragment (pcrHES). Moreover, the ss DNA fragment with the sense sequence (fSense) yielded a more than 10-fold higher gene correction frequency in comparison with pcrHES. Frameshift mutations can inactivate genes and cause disease, and thus they are interesting targets for gene correction. In this study, we examined whether these newly designed DNA fragments were also effective in correcting frameshift mutations. Hyg-EGFP fusion genes inactivated by one base deletion and insertion mutations were chosen as model targets, and sense and antisense ss DNA fragments and ds DNA fragments were co-introduced into CHO-K1 cells with a plasmid DNA carrying the target gene (Fig. 1). The gene correction efficiency was quantitatively determined by counting the EGFP-positive and hygromycin-resistant Escherichia coli colonies, after recovery of the plasmid DNA from the transfected cells and a second transfection into bacteria. The fSense and dsHES fragments were more effective than the conventional PCR fragment (pcrHES), as in our previous results on the correction of a substitution mutation. The frameshift correction efficiencies of fSense were comparable to those of dsHES. These results suggest that sense ss DNA and ds DNA fragments, prepared from phagemid and plasmid DNAs, respectively, have the potential to correct frameshift mutations.