Increased SFHR gene correction efficiency with sense single‐stranded DNA

Abstract

Background The correction of a mutated gene by the small fragment homologous replacement (SFHR) method is a highly attractive approach for gene therapy. However, the current SFHR method with a heat-denatured double-stranded PCR fragment yielded a low correction efficiency. Methods Single-stranded (ss) DNA fragments were prepared from ss phagemid DNA and tested in a gene correction assay with an inactivated Hyg-EGFP fusion gene, as a model target. Results A 606-nt sense, ss DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. Conclusions These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes. Copyright © 2004 John Wiley & Sons, Ltd.