Double-strand break-mediated homologous recombination as a tool for gene therapy.

摘要

An excellent strategy for gene therapy would be the site-specific integration of a therapeutic gene into a safe chromosomal location. Homologous recombination would be a good way to site-specifically insert a gene or correct a mutated gene. Unfortunately, the frequency of recombination in mammalian cells is too low for the strategy to be used successfully. However, induction of a double-strand break (DSB) in the targeted sequence can stimulate homologous recombination by 50-fold or more. This method warrants further examination as an approach for gene therapy.; Typical protocols for studying recombination in mammalian cells involve selection and are time-consuming and tedious. With the goal of studying recombination more efficiently, I developed an Epstein-Barr virus shuttle vector system. The shuttle vector system allows the recombination to occur in mammalian cells, while the screening and analysis of recombinants takes place in bacterial.; By using I-SceI to create a DSB in the EBV vector, I showed a significant increase in homologous recombination between the EBV and a donor plasmid. The lesion in the target sequence was repaired predominantly by insertion of the donor plasmid into the target. Circular donors were more effective than linearized donors.; Insertion of an entire therapeutic gene is a more relevant strategy for gene therapy. I showed a 175-fold stimulation in recombination of a homology-flanked 3.5 kb gene into a target EBV vector. The recombinants were the result of gene conversion events. Recombination of a donor plasmid arranged with the gene located outside the homologous sequence produced a low frequency of insertion events.; An important parameter in the construction of a donor plasmid is the amount of homology it shares with the target sequence. In targeting experiments where a DSB is not made in the target sequence, increasing the homology between the donor and the target exponentially increases the recombination frequency. When recombination is stimulated by a DSB, long homology tracts may not be as important. I examined the effect of decreasing the amount of homology between the donor and target. I found that the recombination frequency is considerably less dependent upon the length of homology when a DSB is present.