Phage φC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines
Phage φC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines
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DOI：
10.1002/jgm.891
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发表时间：
2006-05-01
影响因子：
3.5
通讯作者：
Sugamura, Kazuo
中科院分区：
文献类型：
--
作者：
Ishikawa, Yoshinori;Tanaka, Nobuyuki;Sugamura, Kazuo
背景资料：X连锁严重联合免疫缺陷病（X-linked severe combined immunodeficiency，SCID-X1，X-SCID）是由常见细胞因子受体γ链（gamma c）基因突变引起的一种危及生命的疾病。虽然离体基因治疗，即，将γ c基因转导到自体CD 34+细胞中，已经成功地用于治疗SCID-X1，逆转录病毒载体介导的转移允许失调的整合，引起白血病。在此，为了探索一种可提供较少插入诱变风险的替代基因转移方法，我们使用人T细胞系，包括γ c缺陷型ED 40515（-），采用基于phi C31整合酶的整合系统。方法：通过电穿孔将phi C31整合酶和含有phi C31 attB序列的neo（r）基因表达质粒共递送到Jurkat细胞中。在6418选择后，使用线性扩增介导的聚合酶链反应（LAM-PCR）进行整合位点分析。还用含有attB的γ c表达质粒转染ED 40515（-）细胞，并测定整合位点。IL-2刺激用于评估转导的γ c在ED 40515（-）衍生的clones.Results中的功能性：在共同引入phi C31整合酶表达质粒和携带attB的质粒后，评估整合到未修饰的人基因组中的效率。几个整合位点的特点，包括新的整合位点在染色体13和18上的基因间区域可能是首选的造血细胞。一个ED 40515（-）线轴承的整合γ c基因表现出稳定的γ c蛋白的表达，与正常的IL-2信号，评估STAT 5 activation.Conclusions：这项研究支持未来可能使用的OC 31整合酶介导的基因组整合策略作为一种替代的基因治疗方法治疗SCID-X1。版权所有（c）2006约翰威利父子有限公司。
Background: X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gamma c) gene. Although ex vivo gene therapy, i.e., transduction of the gamma c gene into autologous CD34+ cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phi C31 integrase-based integration system using human T-cell lines, including the gamma c-deficient ED40515(-).Methods: A phi C31 integrase and a neo(r) gene expression plasmid containing the phi C31 attB sequence were co-delivered by electroporation into Jurkat cells. After 6418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gamma c expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gamma c in an ED40515(-)-derived clone.Results: Following co-introduction of the phi C31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515 (-) line bearing the integrated gamma c gene exhibited stable expression of the gamma c protein, with normal IL-2 signaling, as assessed by STAT5 activation.Conclusions: This study supports the possible future use of this OC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1. Copyright (c) 2006 John Wiley & Sons, Ltd.