Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells
Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells
复制标题
DOI：
10.1038/sj.gt.3301675
复制
发表时间：
2002-04
期刊：
影响因子：
5.1
通讯作者：
N. Eliopoulos;A. Al-khaldi;C. Beauséjour;R. Momparler;L. Momparler;J. Galipeau
中科院分区：
文献类型：
--
作者：
N. Eliopoulos;A. Al-khaldi;C. Beauséjour;R. Momparler;L. Momparler;J. Galipeau
人类起源的自然产生的耐药基因可以用于选择共同表达所需治疗性转基因的基因工程细胞。它们在临床应用中的非免疫原性将是一大优势。人胞苷脱氨酶（hCD）是一种化学耐药基因，可使细胞毒性胞嘧啶核苷类似物失活，如胞嘧啶阿拉伯糖苷（Ara-C）。本研究的目的是确定hCD基因是否可以作为工程骨髓基质细胞（MSCs）的离体显性选择标记。构建了包含hCD cDNA和绿色荧光蛋白（GFP）报告基因的双链逆转录载体，并用于A549细胞和原代小鼠间充质干细胞的转导。对转导细胞的分析表明，前病毒DNA稳定整合，CD酶活性增加1000倍以上，并且对胞嘧啶核苷类似物具有耐药性。在转导和未转导的MSCs混合物中，通过1 μM Ara-C的体外药物选择，逆转录载体表达细胞的百分比可以增加到虚拟纯度（> 99.5%）。增加2.5 μM Ara-C的选择压力，与未处理的工程MSCs相比，可以富集表达约6倍高水平GFP和CD活性的混合MSCs。此外，这些体外选择和富集的基因修饰骨髓基质细胞的植入和内皮分化通过体内Matrigel试验得到证实。总之，这些发现概述了人类CD作为基因工程MSCs的离体选择和富集标记物用于转基因细胞治疗的潜力。
Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (> 99.5%) through in vitro drug selection with 1 μM Ara-C. Increased selective pressure with 2.5 μM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications.