Gene therapy using promoters of glycosyltransferase genes which code tumor specific carbohydrate antigens.

1)GM2/GD2合成酶(β1，4-N-乙酰半乳糖胺转移酶)基因5‘侧翼区启动子/增强子活性。为了分析GM2/GD2合成酶基因表达的调控机制，我们确定了β1，4GalNAc-T基因的基因组结构，并定义了3个转录起始点和3个外显子1a、1b和1c的交替使用。氯霉素乙酰基转移酶分析表明，单个起始点的5‘-侧翼区具有启动子活性。当内含子1区域被添加到外显子1a和1b的5‘侧翼区时，检测到高水平的CAT活性。这些CAT活性与GM2/GD2合酶基因的表达水平相对应。2)细胞毒基因和GM2/GD2合酶基因启动子构建的表达载体的细胞毒作用。然后，我们建立了该基因高表达的AS(星形细胞瘤)细胞系的稳定表达株。当加入更昔洛韦(GCV)对细胞增殖的抑制作用时，稳定的转染体在GCV的5×10^-6&gt；M处表现出70%的增殖抑制，而亲本细胞甚至在GCV的5*10^-4&gt；M处也没有表现出抑制作用。此外，以TK基因为探针，通过Southern blotts筛选出与GCV基因拷贝数相同的AS和MeWo转基因株，并对其对GCV的敏感性进行了分析。因此，导入TK基因的AS表现出对GCV的生长抑制作用，而携带TK基因的MeWo则没有。

1) Promoter/enhancer activity of the 5'-flanking region of GM2/GD2 synthase (beta1,4-N-acetylgalactosaminyltransferase) gene.To analyze the regulatory mechanisms of gene expression of GM2/GD2 synthase, we determined the genomic organization of the beta1,4GalNAc-T gene and defined three transcription initiation sites and the alternative usage of three exons, exon 1a, 1b and 1c. 5'-flanking region of individual initiation sites showed promoter activity when analyzed by chloramphenicolacetyltransferase assay. The high level of CAT activity was detected when the intron 1 region was added to the 5'-flanking region of exon 1a and 1b. These CAT activities corresponded to the expression levels of GM2/GD2 synthase gene.2) Cytotoxic effects of an expression vector constructed by the combination of a cytotoxic gene and a promoter of GM2/GD2 synthase gene.We constructed a retroviral expression vector using a cytotoxic gene (herpes simplexthymidine kinase gene) and a promoter/enhancer region (from upsteram of exon 1b to intron 1) of GM2/GD2 synthase gene. Then, we established stable transfectants of AS (astrocytoma) cell line that was a high expressant of the gene. When suppressive effects of cell proliferation was examined by adding Gancyclovir (GCV) to the culture medium, the stable transfectants showed inhibition of proliferation (>70%) at 5*10^<-6>M of GCV,while the parent cell did not even at 5*10^<-4>M of GCV.As for MeWo transfectants (a low expressant of the gene) of the retrovival vector, no inhibition of proliferation was observed. Furthermore, AS and MeWo transfectant containing the same copy number of the transfected gene were selected based on Southern blots with TK gene as a probe, then sensitivity of them to GCV was analyzed. Consequently, AS introduced with TK gene showed a growth inhibition with GCV,though MeWo with TK gene did not.