Establishment Of High Efficient Gene Targeting Modification Technique And Its Application

Genetic disease caused by genetic mutation is a human health issueand is difficult to be cured by current approach. Gene therapy is a new approach for the treatment of genetic disease. At present, the common used vector for gene therapy is lentivirus or retrovirus (Adeno-associated virus, AAV) which can be integrated into the genome, randomly. Due to the potential carcinoma risk of the random integration, its application in gene therapy is limited. AAV is suggested as the best virus vector used for treating genetic disease, but it still has some disadvantages in the aspects of the regulation of gene expression and lasting time of gene expression. As a new developed technique, zinc finger nuclease can cleave the target site on the genome, specifically, which will yield a double strand break. The reparation the double-stand break (DSB) will be carried out by through the homologous recombination (HR) or the non-homomogous end-joining (NHEJ) and the editing efficiency is increased by103-105fold At present, zinc finger nuclease has been successfully used in the study of gene regulation and gene therapy. High efficient homologous recombination based on the zinc finger nuclease technique with a high efficient homologous recombination is an ideal tool for the therapy of monogenetic diseases, but this technique still has some defects. The goal of our study is to improve ZFN technique as following:1) Improve the selection sensitivity of ZFPIt is still a challenge to generate the functional ZFNs for the specific targeting sits in mammalian genome. The first step of assembling ZFP is to generate the ZFP, which is able to bind on the target genome. However, the binding ability based ZFP selection is not sensitive enough and ZFP with low binding abilities is easily to be declined. Moreover, some recent studies suggested that a ZFP with low binding activity might be able to form an active ZFN complex with another binding active half-ZFP. To increase the concentration of ZFN, it is important to select these ZFP with lower binding abilities during the ZFP assembling process. In order to unveil the potential ZFP candidates among those with low binding activities, we established a highly sensitive mammalian cell-based transcriptional reporter system to assess the DNA binding activities of ZFPs by inserting multiple copies of ZFN target sequence fragment of an interested gene (e. g., hPGRN or hVEGF). Our results showed that this system increased the screening sensitivity up to50-fold and markedly amplified the differences in the binding activities between different ZFPs. We also found that the targeted chromosomal gene repair efficiency of each hPGRN or hVEGF ZFN pair was in proportion with the combination of the binding activities of the ZFL (Left zinc finger) and ZFR (Right zinc finger). A hPGRN ZFR with low binding ability was able to form a biological active ZFN if combined with a hPGRN ZFL with relatively high binding ability. Finally, the sequencing indicated that generated hPGRN ZFNsis suitable for the genome editing of specific sites., and compared with the wild type, the proliferation of hPGRN knock-out cell lineis significantly decreased.2) Improve the HR efficiency using a single adenovirus simultaneously ZFN and donor DNASite-specific genetic editing has been achieved in vitro by using plasmids or viral vectors that deliver ZFNs and DNA donor separately or simultaneously. However, ZFN-mediated targeted genome correction in vivo still faces a major challenge, which is the difficulty of delivering both ZFNs and the donor template into target cells with high efficiency. In this study, by using an inducible Tet-on promoter to tightly control ZFN expression, we successfully produced a single adenoviral vector with high titer that carried a ZFN expression cassette and donor template simultaneously in a single vector. We then demonstrated that site-specific genome correction could be mediated efficiently in vitro and in vivo in this system.3) Improve the HR efficiency by constructing a vector carrying multiple copies of linear donor DNAThe efficiency of HR can be increased by creating a targeted double-strand break (DSB) via zinc-finger nucleases (ZFNs) and/or by introducing linear donor DNA intracellular. Some studies have suggested that the increased copy numbers of linear donor DNA may further improve HR efficiency. However, the introduction of multiple copies of a linear donor fragment still remains a challenge. In particular, the transfection efficiency is very low in vitro, In this study, we developed a vector that can carry tandem repeats of a donor fragment, with each repeat flanked by ZFN target sequence fragments (TSFs). Cleave of the flanking TSF sequence will lead to the release of multiple linear fragments, which could improve ZFN-mediated HR efficiency. We found that linearization of the donor fragment will enhance the HR efficiency up to10times, and vector carrying4copies of linearizable donor fragments will increase the HR efficiency up to30times. To apply these techniques to gene therapy, we then introduced this system into an adenoviral vector, which also revealed improved ZFN-mediated HR efficiency.Recently, TALENs (Transcription activator-like effector nucleases) and RGNs (RNA-guided nucleases, RGNs) have been developed as new approaches for genome-editing. In order to apply the nucleases techniques to genetic diseases, we designed a single virus vector carried ZFN and donor DNA fragments. To confirm our findings, we carried out the GUS repair in mouse embryonic fibroblast (MEF) by using our approach. The results indicated that TALENs and Cas9-sgRNA efficiently repaired targeting mutated GUS gene. In summary, to optimize the current genome-editing approaches, we focus on the screen of zinc finger nuclease, the delivery of zinc finger nuclease and donor from a single adenoviral vector and the delivery of linear donor fragments. The goal is to improve the method of screening of zinc finger nuclease, and develop a high efficient gene target modification method for gene therapy. The data suggest that our developed method is a powerful tool for in-situ repair and could be further utilized in the therapy for monogenetic disease.