Fielding A K, Chapel-Fernandes S, Chadwick M P, Bullough F J, Cosset F L, Russell S J
Hematology and Molecular Medicine, Mayo Clinic, Rochester, MN 55902, USA.
Hum Gene Ther. 2000 Apr 10;11(6):817-26. doi: 10.1089/10430340050015437.
An important goal in cancer gene therapy is the development of novel targeted cytotoxic genes. The observation that transfection of a GaLV envelope glycoprotein lacking an R peptide into human cells results in considerable cell-cell fusion and subsequent cell death prompted us to explore the potential for using this fusogenic membrane glycoprotein (FMG) as a targeted cytotoxic gene. As proof of principle, we therefore displayed epidermal growth factor (EGF) on the N terminus of GaLV envelope glycoproteins both with and without an R peptide (GaLV R+ and GaLV R-). Transfection of the GaLVR+ envelope expression plasmids did not cause cell-cell fusion. The GaLV R+ envelopes were incorporated into retroviral vectors whose infectivity was investigated on EGF receptor-positive and -negative cells. The vector incorporating an N-terminally unmodified envelope was able to infect all human cell lines tested. Infectivity of the vector incorporating an envelope on which EGF was displayed was restricted on EGF receptor-positive cells (but not on EGF receptor-negative cells) and could be restored by protease cleavage of the displayed domain or competition with exogenous ligand. The cell-cell fusion capacity of the GaLV R- envelope glycoproteins (N-terminally unmodified and with N-terminal display of both EGF and insulin-like growth factor I [IGF-I]) was investigated by plasmid DNA transfection. While the N-terminally unmodified GaLV R- fused all human cell types tested, fusogenicity of GaLV R- on which EGF or IGF-I was displayed was considerably restricted on receptor-positive cells. "Reciprocal" competition experiments showed that fusogenicity could be restored by competition only with the relevant exogenous ligand. Thus the specificity of cell-cell fusion by a hyperfusogenic GaLV envelope glycoprotein can be regulated by N-terminal display of growth factor ligands. There is therefore significant potential for further development of the targeting of the cell-killing capability of this fusogenic viral glycoprotein by using strategies similar to those we have developed for the targeting of retroviral vectors.
癌症基因治疗的一个重要目标是开发新型靶向细胞毒性基因。将缺乏R肽的GaLV包膜糖蛋白转染到人类细胞中会导致大量细胞间融合并随后导致细胞死亡，这一观察结果促使我们探索将这种融合性膜糖蛋白（FMG）用作靶向细胞毒性基因的潜力。因此，作为原理验证，我们在有和没有R肽的GaLV包膜糖蛋白（GaLV R+和GaLV R-）的N末端展示了表皮生长因子（EGF）。转染GaLVR+包膜表达质粒不会引起细胞间融合。GaLV R+包膜被整合到逆转录病毒载体中，在EGF受体阳性和阴性细胞上研究其感染性。整合了N末端未修饰包膜的载体能够感染所有测试的人类细胞系。整合了展示EGF的包膜的载体的感染性在EGF受体阳性细胞上受到限制（但在EGF受体阴性细胞上不受限制），并且可以通过蛋白酶切割展示结构域或与外源性配体竞争来恢复。通过质粒DNA转染研究了GaLV R-包膜糖蛋白（N末端未修饰以及N末端同时展示EGF和胰岛素样生长因子I [IGF-I]）的细胞间融合能力。虽然N末端未修饰的GaLV R-与所有测试的人类细胞类型融合，但展示EGF或IGF-I的GaLV R-在受体阳性细胞上的融合性受到很大限制。“相互”竞争实验表明，只有与相关外源性配体竞争才能恢复融合性。因此，通过生长因子配体的N末端展示可以调节高融合性GaLV包膜糖蛋白的细胞间融合特异性。因此，通过使用与我们开发的逆转录病毒载体靶向策略类似的策略，进一步开发这种融合性病毒糖蛋白的细胞杀伤能力的靶向具有巨大潜力。
