Amara J F, Courage N L, Gilman M
ARIAD Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
Hum Gene Ther. 1999 Nov 1;10(16):2651-5. doi: 10.1089/10430349950016681.
Many therapeutic uses of gene-modified cells could benefit from inclusion of a surface marker for immunoselecting transduced cells. Another desired feature is a failsafe mechanism to ablate engineered cells if required. We describe here a system that combines a cell surface tag and an inducible apoptosis mechanism in a single protein. Spencer et al. (Curr. Biol. 1996;6:839-847) described an inducible cell suicide gene containing a myristoylation sequence, the human protein FKBP12, and the intracellular domain of Fas. Cells expressing this protein apoptose on treatment with a cell-permeable chemical dimerizing agent that binds two FKBP domains and cross-links the chimeric Fas proteins. We modified this system by anchoring a Fas-FKBP construct to the membrane with the extracellular and transmembrane domains of the low-affinity nerve growth factor receptor (LNGFR), thereby uniting cell surface tagging with the inducible apoptosis mechanism. Cells retrovirally transduced with this construct apoptosed on exposure to a chemical dimerizer, AP1903 (Clackson et al., Proc. Natl. Acad. Sci. U.S.A. 1998;95:10437-10442). The LNGFR-tagged construct showed an unpredicted clear advantage over the myristoylation-anchored construct in its efficiency of signaling in HT1080 cells. This linked marker and failsafe mechanism may have particularly attractive safety properties for gene therapy. The use of gene-modified cells in basic research and clinical studies is enhanced by the use of a selectable surface marker for immunoselection of transduced cells. Another desired feature for gene and cell therapies is an inducible suicide system to eliminate transduced cells when necessary. Spencer et al. (Curr. Biol. 1996;6:839-847) described a potential failsafe mechanism whereby exposure of cells to a chemical dimerizing agent activates the Fas-mediated apoptotic pathway. In this system, the intracellular signaling domain of Fas is linked to one or more copies of the human protein FKBP12. Treatment of engineered cells with a cell-permeable chemical dimerizing agent that simultaneously binds to two FKBP domains cross-links the chimeric Fas protein and induces apoptosis. Here, we modify the system by anchoring a Fas-FKBP construct to the membrane with the extracellular domain of the low-affinity nerve growth factor receptor (LNGFR), to unite cell surface tagging of transduced cells with the inducible apoptosis mechanism. Cells retrovirally transduced with this construct undergo apoptosis on exposure to a chemical dimerizer, AP1903. A linked marker and failsafe mechanism may have particularly attractive safety properties for gene therapy.
基因修饰细胞的许多治疗用途都可能受益于纳入一种用于免疫选择转导细胞的表面标志物。另一个理想的特性是一种故障安全机制，以便在需要时消除工程细胞。我们在此描述一种系统，该系统在单一蛋白质中结合了细胞表面标签和诱导性凋亡机制。斯宾塞等人（《当代生物学》，1996年；6：839 - 847）描述了一种诱导性细胞自杀基因，其包含一个肉豆蔻酰化序列、人FKBP12蛋白以及Fas的细胞内结构域。表达这种蛋白质的细胞在用一种可穿透细胞的化学二聚剂处理时会凋亡，该二聚剂结合两个FKBP结构域并使嵌合Fas蛋白交联。我们通过将Fas - FKBP构建体与低亲和力神经生长因子受体（LNGFR）的细胞外和跨膜结构域锚定到膜上对该系统进行了修饰，从而将细胞表面标记与诱导性凋亡机制结合起来。用该构建体进行逆转录病毒转导的细胞在暴露于化学二聚剂AP1903时会凋亡（克拉克森等人，《美国国家科学院院刊》，1998年；95：10437 - 10442）。在HT1080细胞中，带有LNGFR标签的构建体在信号传导效率方面比肉豆蔻酰化锚定的构建体具有意想不到的明显优势。这种相连的标志物和故障安全机制对于基因治疗可能具有特别有吸引力的安全特性。在基础研究和临床研究中使用基因修饰细胞时，通过使用可选择的表面标志物进行转导细胞的免疫选择得以增强。基因和细胞治疗的另一个理想特性是一种诱导性自杀系统，以便在必要时消除转导细胞。斯宾塞等人（《当代生物学》，1996年；6：839 - 847）描述了一种潜在的故障安全机制，即细胞暴露于化学二聚剂会激活Fas介导的凋亡途径。在该系统中，Fas的细胞内信号传导结构域与一个或多个拷贝的人FKBP12蛋白相连。用一种可穿透细胞的化学二聚剂处理工程细胞，该二聚剂同时结合两个FKBP结构域，使嵌合Fas蛋白交联并诱导凋亡。在此，我们通过将Fas - FKBP构建体与低亲和力神经生长因子受体（LNGFR）的细胞外结构域锚定到膜上对该系统进行修饰，以将转导细胞的细胞表面标记与诱导性凋亡机制结合起来。用该构建体进行逆转录病毒转导的细胞在暴露于化学二聚剂AP1903时会发生凋亡。一种相连的标志物和故障安全机制对于基因治疗可能具有特别有吸引力的安全特性。
