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Objectives: This study explored whether TALENs-mediated non-homologous end joining (NHEJ) targeting the mutation site can correct the aberrant β-globin RNA splicing, and ameliorate the β-thalassaemia phenotype in β 654 mice., Material and Methods: TALENs vectors targeted to the human β-globin gene (HBB) IVS2-654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALENs + /β 654 mice. The gene editing and off-target effects were analysed by sequencing analysis. β-globin expression was identified by RT-PCR and Western blot analysis. Various clinical indices including haematologic parameters and tissue pathology were examined to determine the therapeutic effect in these TALENs + /β 654 mice., Results: Sequencing analysis revealed that the HBB IVS2-654C >T point mutation was deleted in over 50% of the TALENs + /β 654 mice tested, and off-target effects were not detected. RT-PCR and Western blot analysis confirmed the expression of normal β-globin in TALENs + /β 654 mice. The haematologic parameters were significantly improved as compared with their affected littermates. The proportion of nucleated cells in bone marrow was considerably decreased, splenomegaly with extramedullary haematopoiesis was reduced, and significant decreases in iron deposition were seen in spleen and liver of the TALENs + /β 654 mice., Conclusion: These results suggest effective treatment of the anaemia phenotype in TALENs + /β 654 mice following deletion of the mutation site by TALENs, demonstrating a simple and straightforward strategy for gene therapy of β 654 -thalassaemia in the future., (© 2018 John Wiley & Sons Ltd.)

Although β-thalassemia is one of the most common human genetic diseases, there is still no effective treatment other than bone marrow transplantation. Induced pluripotent stem cells have been considered good candidates for the future repair or replacement of malfunctioning organs. As a basis for developing transgenic induced pluripotent stem cell therapies for thalassemia, β(654) induced pluripotent stem cells from a β(654) -thalassemia mouse transduced with the normal human β-globin gene, and the induced pluripotent stem cells with an erythroid-expressing reporter GFP were used to produce chimeric mice. Using these chimera models, we investigated changes in various pathological indices including hematologic parameters and tissue pathology. Our data showed that when the chimerism of β(654) induced pluripotent stem cells with the normal human β-globin gene in β(654) mice is over 30%, the pathology of anemia appeared to be reversed, while chimerism ranging from 8% to 16% provided little improvement in the typical β-thalassemia phenotype. Effective alleviation of thalassemia-related phenotypes was observed when chimerism with the induced pluripotent stem cells owning the erythroid-expressing reporter GFP in β(654) mouse was greater than 10%. Thus, 10% or more expression of the exogenous normal β-globin gene reduces the degree of anemia in our β-thalassemia mouse model, whereas treatment with β(654) induced pluripotent stem cells which had the normal human β-globin gene had stable therapeutic effects but in a more dose-dependent manner., (Copyright© Ferrata Storti Foundation.)