Experimental Model for Gene Therapy of Malignant Glioma

一些恶性胶质瘤显示EGFR基因扩增，外显子2至外显子7缺失突变。EGFR异常基因的过度表达参与恶性胶质瘤的发展或发病。异常EGFR mRNA在外显子1和外显子8之间的连接处含有-GUA-序列。制备了靶向-GUA-位点的锤头状核酶。核酶（abEGFR-rib）在体外切割合成的异常EGFR-RNA底物。abEGFR-肋也在反映生理镁浓度（<2.5mM）和温度（37 ° C）的条件下切割底物。在锤头结构处具有点突变的禁用的核酶在相同条件下不切割异常的EGFR-RNA底物。abEGFR-rib不切割正常EGFR-RNA底物（外显子1-外显子2）。这些发现表明，abEGFR-肋可以特异性地切割异常的EGFR-RNA在体外。异常EGFR mRNA在有限数量的恶性胶质瘤细胞上表达。该分子在正常脑组织中不表达。核酶介导的EGFRmRNA异常切割有望用于恶性胶质瘤的特异性抗癌基因治疗。

Several malignant gliomas revealed EGFR gene amplification with a deletional mutation from exon 2 to exon 7. The overexpression of the aberrant EGFR gene is involved in the progression or pathogenesis of malignant glioma. The aberrant EGFR mRNA contains the -GUA- sequence at the junction between exon 1 and exon 8. A hammerhead ribozyme targeting the -GUA- site was prepared. The ribozyme (abEGFR-rib) cleaved the synthetic aberrant EGFR-RNA substrate in vitro. The abEGFR-rib also cleaved the substrate at the condition reflecting the physiological magnesium concentration (<2.5mM) and temperature (37ﾟC). The disabled ribozyme with a point mutation at the hammer head structure did not cleave the aberrant EGFR-RNA substrate at the same condition. The abEGFR-rib did not cleave the normal EGFR-RNA substrate (exon 1-exon 2). These findings suggest that the abEGFR-rib can specifically cleave the aberrant EGFR-RNA in vitro. The aberrant EGFR mRNA was expressed on a limited number of malignant glioma cells. The molecule was not expressed in the normal brain tissue. The ribozyme mediated aberrant EGFR mRNA cleavage should be useful for the malignant-glioma-specific anti-oncogene therapy.