Schwarzenberger P, Spence S E, Gooya J M, Michiel D, Curiel D T, Ruscetti F W, Keller J R
Laboratory of Leukocyte Biology, SAIC-Frederick, MD, USA.
Blood. 1996 Jan 15;87(2):472-8.
In this report, we describe a novel gene therapy approach for hematopoietic stem/progenitor cells using a specific receptor-mediated gene transfection procedure to target c-kit+ cell lines. The vector consists of plasmid DNA containing a luciferase reporter gene that is condensed by electrostatic forces with polylysine (PL) covalently linked to streptavidin (binds biotinylated ligand) and PL covalently linked to adenovirus (AD; to achieve endosomal lysis) with the final addition of biotinylated steel factor (SLF-biotin). Targeted transfection of growth factor-dependent hematopoietic progenitor cell lines that express c-kit showed specific luciferase gene expression over cell lines that did not express c-kit. This effect was dependent on the dose of SLF-biotin and was competed by excess SLF or with monoclonal antibodies that recognize c-kit and block the binding of SLF to its receptor. Maximum transfection efficiency (> 90%) requires a 2-hour incubation period of the vector with the cells, and maximum gene expression occurred 30 hours later. Removal of the endosomalytic agent, AD, from the vector resulted in the loss of gene expression. Vector targeting was versatile and could be changed by the addition of other biotinylated ligands. In principle, this vector should be broadly applicable to deliver genes to hematopoietic stem/progenitor cells in vitro and in vivo.
在本报告中，我们描述了一种针对造血干细胞/祖细胞的新型基因治疗方法，该方法使用特定的受体介导基因转染程序靶向c-kit+细胞系。载体由含有荧光素酶报告基因的质粒DNA组成，该质粒DNA通过静电力与共价连接到链霉亲和素（结合生物素化配体）的聚赖氨酸（PL）以及共价连接到腺病毒（AD；用于实现内体裂解）的PL凝聚，最后添加生物素化的钢因子（SLF-生物素）。对表达c-kit的生长因子依赖性造血祖细胞系进行靶向转染，与不表达c-kit的细胞系相比，显示出特异性的荧光素酶基因表达。这种效应取决于SLF-生物素的剂量，并可被过量的SLF或识别c-kit并阻断SLF与其受体结合的单克隆抗体所竞争。最大转染效率（>90%）需要载体与细胞孵育2小时，最大基因表达在30小时后出现。从载体中去除内体裂解剂AD会导致基因表达丧失。载体靶向具有通用性，可通过添加其他生物素化配体来改变。原则上，该载体应广泛适用于在体外和体内将基因递送至造血干细胞/祖细胞。
