Otomo T, Yamamoto S, Morishita R, Kaneda Y
Division of Gene Therapy Science, Osaka University School of Medicine, Suita, Japan.
J Gene Med. 2001 Jul-Aug;3(4):345-52. doi: 10.1002/jgm.199.
A major limitation of current cancer gene therapies is low and transient expression of the therapeutic gene. For long-term expression of transgenes in vivo, an Epstein-Barr virus (EBV) replicon vector has been developed. The present study examines the effect of the EBV replicon vector system and its application to a suicide gene therapy for melanoma in mice.
An EBV replicon vector system, pEBc, consisting of EBV nuclear antigen-1 (EBNA-1) and the origin of latent viral DNA replication, oriP, was used to express either the luciferase gene or the herpes simplex virus (HSV) thymidine kinase (TK) gene. The expression vector was introduced in vivo into melanoma tumor masses in mice by means of HVJ-cationic liposomes. The time-course of gene expression and the anticancer effect of the EBV replicon vector were investigated in comparison with pcLuc, which lacks the EBV components.
Luciferase expression was sustained in both cultured cells and melanoma masses by pEBc but not by pcLuc. The luciferase expression level in melanoma masses was higher by pEBcLuc than by pcLuc, although Southern blot analysis showed the number of copies of pEBcLuc retained in the melanoma masses to be fewer than that of pcLuc. The effectiveness of EBV replicon vector on suicide gene therapy of melanoma in mice was also demonstrated.
The EBV replicon vector appears useful for cancer gene therapy. Analysis of the transgene in tumors suggests that the EBV replicon system may be responsible for efficient transcription but not retention of the transgene.
当前癌症基因治疗的一个主要局限性是治疗基因的低表达和瞬时表达。为了在体内长期表达转基因，已开发出一种爱泼斯坦-巴尔病毒（EBV）复制子载体。本研究检测了EBV复制子载体系统的效果及其在小鼠黑色素瘤自杀基因治疗中的应用。
使用由EBV核抗原-1（EBNA-1）和潜伏病毒DNA复制起点oriP组成的EBV复制子载体系统pEBc来表达荧光素酶基因或单纯疱疹病毒（HSV）胸苷激酶（TK）基因。通过HVJ-阳离子脂质体将表达载体体内导入小鼠黑色素瘤肿瘤块中。与缺乏EBV成分的pcLuc相比，研究了EBV复制子载体的基因表达时间进程和抗癌效果。
pEBc可使荧光素酶在培养细胞和黑色素瘤块中持续表达，而pcLuc则不能。尽管Southern印迹分析显示黑色素瘤块中保留的pEBcLuc拷贝数少于pcLuc，但pEBcLuc在黑色素瘤块中的荧光素酶表达水平高于pcLuc。还证明了EBV复制子载体对小鼠黑色素瘤自杀基因治疗的有效性。
EBV复制子载体似乎对癌症基因治疗有用。对肿瘤中转基因的分析表明，EBV复制子系统可能负责转基因的高效转录，但不负责其保留。
