Treatment of β 654 ‐thalassaemia by TALEN s in a mouse model

Objectives This study explored whether TALEN s‐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 TALEN s vectors targeted to the human β‐globin gene ( HBB ) I VS 2‐654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALEN s + /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 TALEN s + /β 654 mice. Results Sequencing analysis revealed that the HBB IVS 2‐654C >T point mutation was deleted in over 50% of the TALEN s + /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 TALEN s + /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 TALEN s + /β 654 mice. Conclusion These results suggest effective treatment of the anaemia phenotype in TALEN s + /β 654 mice following deletion of the mutation site by TALEN s, demonstrating a simple and straightforward strategy for gene therapy of β 654 ‐thalassaemia in the future.