动物实验,遗传突变 z-bo 2020-09-01 484

　　The Juan Carlos Espis Abermont Institute of the Salk Institute in the United States, the Li Yingrui research group of the BGI Institute and the Liu Guanghui research group of the Institute of Biophysics of the Chinese Academy of Sciences used whole-genome sequencing (WGS ) To identify the existing disease genome. We worked together for the first time to clarify the safety and reliability of target modification tools. We have created a new type of human gene mutation repair tool, telHDAdV, whose efficiency is much higher than the current genome editing technology, which provides an important theoretical basis for the development of stem cell-based gene therapy. The emergence of human induced pluripotent stem cell technology (iPSC) has promoted the rapid development of correction technology for human disease genomes. The current methods that can be used to target and correct the human disease genome include ribozyme-mediated DNA homologous recombination technology (ZFN, TALEN, CRISPR/CAS9, etc.) and ribozyme-independent large-segment DNA homologous recombination technology (see typical It is a third-generation adenovirus vector HDAdV. Since gene repaired autologous stem cells have the potential to cure their own diseases, they have broad application prospects in individual and regenerative medicine.

　　Liu Guanghui’s research team used HDAdV-mediated genome-targeted editing technology to achieve the targeted repair of the disease-causing gene LMNA in the iPSC of patients with progeria, so that the concept of the feasibility of in-situ correction can be confirmed. Next, we corrected the pathogenic mutations in stem cells of patients with Parkinson's disease and Fanconi anemia, laying the foundation for the study of the mechanism of these genetic diseases, drug evaluation, and personalized stem cell and gene therapy.

　　In this study, the researchers used three different methods to target the mutant hemoglobin gene (HBB) in the iPSC of patients with sickle cell anemia for the first time: HDAdV, TALEN and CRISPR.. It can be seen that these three genetic modifications The method has similar targeting efficiency for HBB gene. At the same time, the whole genome deep sequencing results show that TALEN and HDAdV can maximize the patient's genome integrity during the genetic modification process, which proves the safety and reliability of these methods. In addition, the researchers have fully combined the TALEN and HDAdV With unique advantages, it can be used as a correction tool to target the genome to develop a new and efficient disease gene correction vector telHDAdV. telHDAdV has TALEN specific genome cutting and high HDAdV transfer efficiency, as well as accurate homologous recombination efficiency of large fragments. The same telHDAdV can effectively cover all possible gene mutations at the HBB locus, so it can be widely used to repair genes for different types of hemoglobin diseases (such as sickle cell anemia and thalassemia). Experimental results show that telHDAdV-mediated gene repair is dozens of times more efficient than using TALEN and CPRISPR alone, and it can correct gene mutations that cause different types of human diseases. Apply to

　　This research eliminates concerns about the safety of gene-targeted repair of diseases in the field of stem cells and regenerative medicine. At the same time, the emergence of new gene correction vectors also helps to accelerate the pace of clinical transformation of stem cells. CellStemCell released a preview comment entitled "What has changed in genome editing" during the same period: "These findings will undoubtedly promote the further application of targeted genome editing technology in disease research and clinical treatment."