Severe congenital neutropenia (SCN) is a primary immunodeficiency disease in which a number of underlying gene defects are responsible for abnormalities in neutrophil development. The HCLS1-associated protein X1 (HAX1) mutation is associated with an autosomal-recessive form of SCN. Considering the potential of gene therapy approaches for the treatment of monogenic disorders, in this study we aimed to develop retroviral vectors expressing coding sequences (CDS) to be used for the removal of the genetic blockade in deficient hematopoietic cells. Following amplification of CDS with primers containing appropriate restriction sites, HAX1 CDS was cloned into an intermediate vector using TA-cloning. The sequence was transferred into a retroviral vector, followed by retroviral packaging in Plat-A cells. To show HAX1 protein expression, HEK293T cells were exposed to 10 multiplicity of infection (MOI) of retroviral particles and HAX1 expression was confirmed in these cells, using indirect intracellular flow cytometry. This vector was applied for in vitro transduction of hematopoietic stem cell with HAX1 mutation; after 11 days, cultured cells were analyzed for CD66acde and CD177 (neutrophil surface markers) expression. Increased neutrophil production in HAX1 viral vector-expressing hematopoietic cells was observed as compared to control vector transduced cells. Hence, according to the results, this type of therapy could be considered a potential treatment protocol for the disease.

中文翻译：

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逆转录病毒转导作为严重先天性白细胞减少症治疗靶点的HAX1基因治疗的体外研究。

严重的先天性中性粒细胞减少症（SCN）是一种主要的免疫缺陷疾病，其中许多潜在的基因缺陷导致嗜中性粒细胞发育异常。HCLS1相关蛋白X1 （HAX1）突变与SCN的常染色体隐性形式有关。考虑到基因治疗方法在治疗单基因疾病中的潜力，在这项研究中，我们旨在开发表达编码序列（CDS）的逆转录病毒载体，以去除缺乏的造血细胞中的遗传阻滞。用含有适当限制位点的引物扩增CDS之后，使用TA克隆将HAX1 CDS克隆到中间载体中。将该序列转移到逆转录病毒载体中，然后在Plat-A细胞中包装逆转录病毒。为了显示HAX1蛋白的表达，将HEK293T细胞暴露于10种逆转录病毒颗粒的感染（MOI）中，并使用间接细胞内流式细胞术确认了这些细胞中的HAX1表达。该载体已申请HAX1突变的造血干细胞的体外转导；11天后，分析培养的细胞的CD66acde和CD177（嗜中性粒细胞表面标记）表达。与对照载体转导的细胞相比，观察到表达HAX1病毒载体的造血细胞中嗜中性粒细胞产生的增加。因此，根据结果，可以认为这种治疗方法是该疾病的潜在治疗方案。