The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells.
The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells.
复制标题
β-珠蛋白复制剂大大增强了S/MAR基源载体在基因转移到人造血祖细胞中的潜力。
DOI：
10.1038/srep40673
复制
发表时间：
2017-01-20
影响因子：
4.6
通讯作者：
Athanassiadou A
中科院分区：
文献类型：
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作者：
Stavrou EF;Lazaris VM;Giannakopoulos A;Papapetrou E;Spyridonidis A;Zoumbos NC;Gkountis A;Athanassiadou A
特定的人类染色体元件增强附加型基因转移载体的性能。基于S/MAR的附加型载体pEPI-eGFP转染CD 34+造血细胞，但仅是瞬时的。为了解决这个问题，我们（1）通过用EF 1/HTLV或SFFV启动子替换驱动eGFP的CMV启动子来增强转基因转录，以分别产生载体pEPI-EF 1/HTLV和pEPI-SFFV;和（2）通过将来自β-珠蛋白基因座的复制起始区（IR）插入载体pEPI-SFFV中来增强质粒复制，以产生载体pEP-IR。来自健康供体外周血的CD 34+细胞的转染效率达到30%。在集落形成细胞（CFC）测定中评价CD 34 +/eGFP+细胞后，通过荧光显微镜观察，载体pEP-IR在14天后显示出上级的性能：100%eGFP +-集落，pEPI-eGFP为0%，pEPI-SFFV为56.9%，pEPI-EF 1/HTLV为49.8%;分别通过定量（q）PCR和RT-qPCR，与后两种构建体相比，每个细胞多50%的质粒拷贝和3倍的eGFP表达。重要的是，CFC测定中pEP-IR的建立率为15%，pEPI-SFFV为5.5%，pEPI-EF 1/HTLV为5%。载体pEP-IR显示极低的递送速率，但支持eGFP在地中海贫血小鼠造血祖细胞中的表达。IR是一种新的人类控制元件，用于改善向祖细胞中的附加型基因转移。
Specific human chromosomal elements enhance the performance of episomal gene-transfer vectors. S/MAR-based episomal vector pEPI-eGFP transfects CD34+ haematopoietic cells, but only transiently. To address this issue we reinforced (1) transgene transcription by replacing the CMV promoter driving eGFP with the EF1/HTLV or SFFV promoters to produce vectors pEPI-EF1/HTLV and pEPI-SFFV, respectively; and (2) plasmid replication by inserting the replication-Initiation Region (IR) from the β-globin locus into vector pEPI-SFFV to produce vector pEP-IR. All vectors supported stable transfections in K562 cells. Transfections of CD34+ cells from peripheral blood of healthy donors reached 30% efficiency. Upon evaluation of CD34+/eGFP+ cells in colony-forming cell (CFC) assays, vector pEP-IR showed superior performance after 14 days, by fluorescent microscopy: 100% eGFP+-colonies against 0% for pEPI-eGFP, 56.9% for pEPI-SFFV and 49.8% for pEPI-EF1/HTLV; 50% more plasmid copies per cell and 3-fold eGFP expression compared to the latter two constructs, by quantitative (q)PCR and RT-qPCR, respectively. Importantly, the establishment rate in CFC assays was 15% for pEP-IR against 5.5% for pEPI-SFFV and 5% for pEPI-EF1/HTLV. Vector pEP-IR shows extremely low delivery rate but supports eGFP expression in thalassaemic mouse haematopoietic progenitor cells. The IR is a novel human control element for improved episomal gene transfer into progenitor cells.