Non-viral ex vivo transduction of human hepatocyte cells to express factor VIII using a human ribosomal DNA-targeting vector
Non-viral ex vivo transduction of human hepatocyte cells to express factor VIII using a human ribosomal DNA-targeting vector
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DOI：
10.1111/j.1538-7836.2007.02355.x
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发表时间：
2007-02-01
影响因子：
10.4
通讯作者：
Xia, J.
中科院分区：
文献类型：
--
作者：
Liu, X.;Liu, M.;Xia, J.
背景：在基因治疗中，最重要的问题之一是载体的选择。pHrneo是本课题组前期构建的人源性载体，可将外源基因靶向导入人核糖体DNA（hrDNA）位点。方法与结果：在本研究中，我们将重组hFVIII（hFVIII-BDDAK 39）的表达盒插入pHrneo中，构建靶向载体：pHrneo-BDDAK 39。将pHrneo-BDDAK 39电穿孔至人肝细胞HL 7702中，PCR鉴定同源重组子，并检测位于hrDNA位点的hFVIII-BDDAK 39的表达。pHrneo-BDDAK 39成功地将hFVIII-BDDAK 39靶向到HL 7702的hrDNA位点上，位点特异性整合效率为1.1 × 10 ~（-5）。发现在HL 7702的hrDNA基因座处的hFVIII-BDDAK 39能够有效表达（4.3 +/- 0.9 ng 10（-6）细胞24 h（-1））。结论：pHrneo-BDDAK 39可用于血友病A的基因治疗。
Background: In gene therapy, one of the most important issues is the choice of the vectors. pHrneo is a human-derived vector previously constructed by our group, which can target a foreign gene into a human ribosomal DNA (hrDNA) locus. Methods and results: In this study, we inserted an expression cassette of reconstructive hFVIII (hFVIII-BDDAK39) to pHrneo to construct a targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HL7702 cells (human hepatocyte), we identified the homologous recombinants using polymerase chain reaction, and tested the expression of hFVIII-BDDAK39 located at the hrDNA locus. The hFVIII-BDDAK39 was successfully targeted into the hrDNA locus of HL7702 by pHrneo-BDDAK39, and the efficiency of site-specific integration was 1.1 x 10(-5). The hFVIII-BDDAK39 at the hrDNA locus of HL7702 was found to be able to express efficiently (4.3 +/- 0.9 ng 10(-6) cells 24 h(-1)). Conclusion: It has been indicated that the targeting vector pHrneo-BDDAK39 can be used in gene therapy for hemophilia A.