BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector
BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector
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DOI：
10.1038/gt.2015.91
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发表时间：
2016-02-01
期刊：
影响因子：
5.1
通讯作者：
Hara, T.
中科院分区：
文献类型：
--
作者：
Yamamoto, H.;Ishimura, M.;Hara, T.
X连锁无丙种球蛋白血症（XLA）是一种常见的体液免疫缺陷病，由布鲁顿酪氨酸激酶（BTK）基因突变引起。为了研究XLA基因治疗的可能性，我们构建了辅助依赖性腺病毒/腺相关病毒BTK靶向载体（HD-Ad. AAV BTK载体），其由含有BTK外显子6-19的基因组序列和由巨细胞病毒启动子驱动的绿色荧光蛋白-潮霉素盒组成。我们首先使用NALM-6，一种人类男性前B急性淋巴细胞白血病细胞系，作为受体来测量通过同源重组进行基因靶向的效率。我们确定了10个克隆与同源重组的BTK基因之间的107潮霉素耐药稳定克隆分离两个独立的实验。我们接下来使用脐带血CD 34（+）细胞作为基因靶向的受体细胞。我们分离出在含有细胞因子和潮霉素的培养基中生长的菌落。我们发现755个潮霉素耐药菌落中有4个发生了BTK基因的靶向作用。重要的是，在选择培养基中生长期间从同源重组CD 34（+）细胞分化的CD 19（+）淋巴祖细胞中也观察到基因靶向。我们的研究显示了使用HD-Ad进行BTK基因治疗的潜力。通过造血干细胞中的同源重组的AAV BTK载体。
X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad. AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34(+) cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19(+) lymphoid progenitor cells that were differentiated from the homologous recombinant CD34(+) cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad. AAV BTK vector via homologous recombination in hematopoietic stem cells.