Targeted gene therapy using nicks to induce homologous recombination

Despite the early promise of gene therapy, monogenic diseases continue to be a significant source of morbidity and mortality. We have investigated the ability of the human retrovirus xenotropic murine leukemia virus-related virus (XMRV) to induce transformation in cell culture and found that it is not acutely oncogenic but can generate rare transformed foci, confirming the potential for transformation by untargeted integration of retroviral vectors and furthering our understanding of a possible human pathogen. Additionally, we investigated contamination of adeno-associated virus (AAV) vectors with cap DNA that can express capsid in vector-transduced cells, and found that capsid-expressing DNA is present in vectors, but it can be prevented through the use of an oversized cap gene in vector production. We next investigated gene targeting using homologous recombination (HR). HR can be induced by double strand breaks (DSBs), but these breaks can be toxic and mutagenic. Using the I-Anil homing endonuclease engineered to produce only nicks we found that nicks induce HR with both plasmid and AAV vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold for plasmid transfection and 4 to 6-fold for AAV vectors), but still represented a significant increase (240-fold and 30-fold, respectively). We observed severe toxicity with the I-Anil 'cleavase,' but no evidence of toxicity with the I-Anil 'nickase,' and we detected 100-fold fewer mutations at the I-AniI site with the nickase than with the cleavase. These results, and the observation that sequence surrounding the target site affects nick-induced but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs. Combining this with the oversized cap AAV production could allow for a targeted gene correction strategy without the toxicity of DSBs that could also have reduced contamination of the AAV vector.