In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate
In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate
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DOI：
10.1096/fj.99-1052com
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发表时间：
2000-10-01
期刊：
影响因子：
4.8
通讯作者：
Niitsu, Y
中科院分区：
文献类型：
--
作者：
Sato, Y;Yamauchi, N;Niitsu, Y
为了在体内靶向播散性肿瘤，转基因[β-半乳糖苷酶基因、绿色荧光蛋白（GFP）基因、单纯疱疹病毒胸苷激酶（HSV-TK）]通过化学计量可控的生物素-链霉亲和素桥接和Tf受体（Tf-R）亲和层析与转铁蛋白（Tf）缀合，Tf受体亲和层析选择具有完整受体结合位点的Tf缀合物与接头反应。将如此构建的Tf-β-半乳糖苷酶质粒偶联物通过Tf-R特异性转染至人红白血病细胞（K562），而无需任何促溶酶体剂的帮助。偶联物的转染效率上级高于脂质体转染（1%染色）和逆转录病毒载体（5%），略低于腺病毒（70%）。使用其他肿瘤细胞（M7609，TMK-1）证实了我们的缀合物的高水平表达，而在表达低水平Tf-R的正常二倍体细胞（HEL）中，表达可忽略不计。当GFP基因偶联物通过尾静脉全身给药皮下接种肿瘤的裸鼠时，发现GFP mRNA的表达几乎只在肿瘤中，在肌肉中的程度要小得多，而荧光显微镜显示的GFP仅在前者中检测到。为了利用该方法的治疗适用性，使用Tf-HSV-TK基因缀合物对严重联合免疫缺陷小鼠中的大规模转移的k562肿瘤进行自杀基因治疗，并且证实了存活期的显著延长和肿瘤负荷的显著降低。因此，该方法也可用于播散性肿瘤的基因治疗。
To target disseminated tumors in vivo, transgenes [beta-galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV-TK)] were conjugated to transferrin (Tf) by a biotin-streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf-R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf-beta-galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf-R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK-1) whereas in normal diploid cells (HEL), which express low levels of Tf-R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf-HSV-TK gene conjugate for massively metastasized k562 tumors in severe combined immune-deficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.