Zhao-Emonet J C, Marodon G, Pioche-Durieu C, Cosset F L, Klatzmann D
Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires UPMC-CNRS ESA 7087-CERVI-H pital de la Pitié, Paris, France.
J Gene Med. 2000 Nov-Dec;2(6):416-25. doi: 10.1002/1521-2254(200011/12)2:6<416::AID-JGM142>3.0.CO;2-Y.
Gene therapy of various immunological disorders will greatly benefit from improved retroviral vectors (RVs) with T cell specificity. Such vectors can be designed by placing a gene of therapeutic interest under the control of tissue-specific transcriptional elements. However, low titers and loss of specificity are frequently encountered with tissue-specific vectors. The aim of the present study was to develop a T cell-specific RV.
We constructed a series of Moloney murine leukemia virus (Mo-MLV)-based RVs expressing enhanced green fluorescent protein (EGFP) under the control of a mini-promoter/enhancer cassette derived from the CD4 gene (CD4pmE) and tested them in cell lines and peripheral blood lymphocytes. Expression of EGFP was monitored by fluorescence microscopy and analyzed by flow cytometry.
The CD4pmE cassette was inserted between the viral long terminal repeats (LTRs) in self-inactivating vectors (SIN vectors) or was substituted to the 3' U3 viral promoter/enhancer (hybrid vectors). High vector titers but poor specific expression of EGFP were achieved when CD4pmE was inserted in sense orientation in SIN vectors. Low titers but high specificity were observed when the CD4pmE cassette was in anti-sense orientation. In contrast, high titers and good T cell specificity were obtained with hybrid vectors.
An efficient T cell-specific retroviral vector was obtained.
各种免疫性疾病的基因治疗将极大地受益于具有T细胞特异性的改良逆转录病毒载体（RVs）。此类载体可通过将具有治疗意义的基因置于组织特异性转录元件的控制之下来设计。然而，组织特异性载体经常出现滴度低和特异性丧失的问题。本研究的目的是开发一种T细胞特异性RV。
我们构建了一系列基于莫洛尼鼠白血病病毒（Mo-MLV）的RVs，其在源自CD4基因的微型启动子/增强子盒（CD4pmE）的控制下表达增强型绿色荧光蛋白（EGFP），并在细胞系和外周血淋巴细胞中对其进行测试。通过荧光显微镜监测EGFP的表达，并通过流式细胞术进行分析。
CD4pmE盒被插入到自失活载体（SIN载体）的病毒长末端重复序列（LTRs）之间，或者被替换到3' U3病毒启动子/增强子（杂交载体）。当CD4pmE以正义方向插入SIN载体时，可获得高载体滴度，但EGFP的特异性表达较差。当CD4pmE盒以反义方向存在时，观察到滴度低但特异性高。相比之下，杂交载体获得了高滴度和良好的T细胞特异性。
获得了一种高效的T细胞特异性逆转录病毒载体。
