Development of the Methods for Regulated Expression of Transduced Gene in Transkaryotic Gene Therapy

阐述了在跨核或体细胞基因治疗中控制基因表达的原理。本研究的实际方法如下：首先将构建在pBMG-Neo质粒中的重组人胰岛素cDNA转染到培养的小鼠l细胞成纤维细胞中。选择胰岛素原分泌量最高的克隆。然后将含有小鼠基因组CD8.2基因的重组质粒进一步转染到该胰岛素原产生细胞系中。这种双重转染的细胞以3.4 × 10^<-6> ng/hr/细胞的速度产生胰岛素原。将2 × 10^6的该细胞系腹腔移植至链脲佐菌素诱导的糖尿病C3H小鼠。移植后第30天，血糖浓度由治疗前的430 mg/dl显著下降至80 mg/dl。不出所料，由于胰岛素原分泌过多，动物死于低血糖。为清除移植细胞，采用抗cd8.2单克隆抗体免疫安全系统进行检测。在移植后的第14天，每天1次，连续14天使用该抗体，完全体现了移植的降糖作用，证明了移植细胞的完全去除。因此，我们开发了体细胞基因治疗糖尿病的动物模型，免疫安全系统可以完全去除移植细胞。

The principle of controlling gene expression in transkaryotic or somatic cell gene therapy was developped. The practical methods used in this study were as follows : At first recombinant human insulin cDNA constucted in the plasmid of pBMG-Neo was transfected to mouse cultured fibroblasts of L-cells. Among the clones, the one with the highest proinsulin secretion was selected. Then the second recombinant plasmid containing mouse genomic CD8.2 gene in pHEBo was further transfected to this proinsulin producing cell line. This doubly transfected cells produced proinsulin at 3.4x10^<-6> ng/hr/cell. 2x10^6 of this cell line were intraperitoneally transplanted to streptozocininduced diabetic C3H mice. The blood glucose concentrations were remarkably decreased from 430 mg/dl of the pretreatment level to 80 mg/dl at the 30th day after the transplantation. As expected, animals died of hypoglycemia due to the excessive production of proinsulin. To remove the transplanted cells, an immunological safety system using anti-CD8.2 monoclonal antibodywas tested. The administation of this antibody on the 14th day after the transplantation, once a day for 14 days, completely revered the hypoglycemic effects of transplantation, proving the complete removal of transplated cells. Thus we have developed the animal model of somatic cell gene therapy against diabetes, with the immunological safety system eligible to completely remove the transplanted cells.