Double targeting restriction endonucleases: towards correction of the acid-α-glucosidase gene in Pompe disease

Correction of a genetic mutation via homologous recombination (HR) is a promising approach to actually cure some genetic diseases. To increase HR effi ciency, DNA damage can be induced specifi cally to the target gene using DNA targeting agents such as triple helix forming oligonucleotides (TFOs), either alone or fused to DNA damaging agents. In this study, we report on sequence-specifi c TFOs conjugated to sequence-specifi c restriction endonucleases as double targeted nucleases. TFOs were designed for acid-α-glucosidase (GAA), the gene mutated in Pompe disease, and fused to camptothecin or restriction endonucleases. The agents were tested for target binding affi nity by BIAcore, for DNA damage by measuring γH2AX, and for HR induction using luciferase reporter plasmids. Co-transfecting TFO-MunI conjugates with a promoter-less reporter plasmid and a promoter-containing HR fragment resulted in restoration of luciferase activity to up to 24% of the promoter containing control. The data presented here demonstrate that TFOrestriction endonuclease conjugates are promising candidates for gene correction approaches for monogenetic disorders like Pompe disease.