Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break

Sequence-specific endonucleases with large recognition sites can cleave DNA in living cells, and, as a consequence, stimulate homologous recombination (HR) up to 10 000-fold. The recent development of artificial meganucleases with chosen specificities has provided the potential to target any chromosomal locus. Thus, they may represent a universal genome engineering tool and seem to be very promising for acute gene therapy. However, in toto applications depend on the ability to target somatic tissues as well as the proficiency of somatic cells to perform double-strand break (DSB)-induced HR.

In order to investigate DSB-induced HR in toto, we have designed transgenic mouse lines carrying a LagoZ gene interrupted by one I-SceI cleavage site surrounded by two direct repeats. The LagoZ gene can be rescued upon cleavage by I-SceI and HR between the two repeats in a process called single-strand annealing. beta-Galactosidase activity is monitored in liver after tail vein injection of adenovirus expressing the meganuclease I-SceI.

In toto staining revealed a strong dotted pattern in all animals injected with adenovirus expressing I-SceI. In contrast, no staining could be detected in the control. beta-Galactosidase activity in liver extract, tissue section staining, and PCR analysis confirmed the presence of the recombined LagoZ gene.

We demonstrate for the first time that meganucleases can be successfully delivered in animal and induce targeted genomic recombination in mice liver in toto. These results are an essential step towards the use of designed meganucleases and show the high potential of this technology in the field of gene therapy.