Treatment of β654-thalassaemia by TALENs in a mouse model
Treatment of β654-thalassaemia by TALENs in a mouse model
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TALENs 在小鼠模型中治疗β654-地中海贫血
DOI：
10.1111/cpr.12491
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发表时间：
2018-12-01
影响因子：
8.5
通讯作者：
Zeng, Fanyi
中科院分区：
文献类型：
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作者：
Fang, Yudan;Cheng, Yan;Zeng, Fanyi
目的探讨靶向突变位点的talens介导的非同源末端连接（NHEJ）是否能纠正β -珠蛋白RNA剪接异常，改善β(654)小鼠的β -地中海贫血表型。材料与方法构建针对人β -珠蛋白基因（HBB） IVS2-654C >T突变小鼠模型的TALENs载体，并选择其生成双杂合TALENs(+)/ β(654)小鼠。通过测序分析分析基因编辑和脱靶效应。RT-PCR和Western blot检测β -珠蛋白的表达。通过对TALENs(+)/ β(654)小鼠的血液学参数和组织病理学等临床指标的检测来确定其治疗效果。结果测序分析显示，在超过50%的TALENs(+)/beta（654）小鼠中，HBB IVS2-654C >T点突变被删除，未检测到脱靶效应。RT-PCR和Western blot分析证实TALENs(+)/beta（654）小鼠中正常β -珠蛋白的表达。血液学参数与受影响的幼崽相比有显著改善。TALENs(+)/ β(654)小鼠骨髓有核细胞比例明显降低，脾肿大伴髓外造血减少，脾脏和肝脏铁沉积明显减少。这些结果表明，在TALENs基因删除突变位点后，TALENs(+)/ β(654)小鼠的贫血表型得到了有效的治疗，为未来基因治疗β(654)-地中海贫血提供了一种简单直接的策略。
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.