Treatment of β 654 -thalassaemia by TALENs in a mouse model.

Objectives This study explored whether TALENs-mediated non-homologous end joining (NHEJ) targeting the mutation site can correct the aberrant beta-globin RNA splicing, and ameliorate the beta-thalassaemia phenotype in beta(654) mice. Material and methods TALENs vectors targeted to the human beta-globin gene (HBB) IVS2-654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALENs(+)/beta(654) mice. The gene editing and off-target effects were analysed by sequencing analysis. beta-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(+)/beta(654) mice. Results Sequencing analysis revealed that the HBB IVS2-654C >T point mutation was deleted in over 50% of the TALENs(+)/beta(654) mice tested, and off-target effects were not detected. RT-PCR and Western blot analysis confirmed the expression of normal beta-globin in TALENs(+)/beta(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(+)/beta(654) mice. Conclusion These results suggest effective treatment of the anaemia phenotype in TALENs(+)/beta(654) mice following deletion of the mutation site by TALENs, demonstrating a simple and straightforward strategy for gene therapy of beta(654)-thalassaemia in the future.