Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy
Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy
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DOI：
10.1038/gt.2011.129
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发表时间：
2011-09
期刊：
影响因子：
5.1
通讯作者：
Wen-Yi Luo;Yung-Shen Shih;Chie Hung;K. Lo;C. Chiang;Wen-Hsin Lo;S. Huang;S-C Wang;Chenghui Yu;C-H Chien;Y.-C. Hu
中科院分区：
文献类型：
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作者：
Wen-Yi Luo;Yung-Shen Shih;Chie Hung;K. Lo;C. Chiang;Wen-Hsin Lo;S. Huang;S-C Wang;Chenghui Yu;C-H Chien;Y.-C. Hu
抗血管生成是一种有吸引力的抗癌方法，但需要抗血管生成药物的持续存在，这可以通过基因治疗来补救。杆状病毒是一种新兴的基因递送载体，但仅能瞬时表达(7天)，因此，本研究的主要目的是开发一种可持续表达抗血管生成基因和癌症治疗的杂交杆状病毒。我们首次构建了以腺相关病毒反向末端重复序列(AAV ITRs)、ORip/Epstein-Barr病毒表达的核抗原1(EBNA1)或睡美人(SB)转座子为特征的载体，并比较了它们在持续表达方面的效果。在人胚胎肾(HEK293)细胞中，AAV ITR不能延长其表达，而ORIP/EBNA1适度延长其表达至35天。相比之下，SB系统即使在没有抗生素选择的情况下也能在77天后稳定表达。鉴于这一发现，我们构建了一个表达SB转座酶的混合SB杆状病毒，并携带有SB可识别的反向重复/直接重复(IR/DR)元件的转基因盒。由于转基因的持久性和整合性，杂交SB杆状病毒可以有效地转导哺乳动物细胞，并比传统杆状病毒介导更长的表达持续时间。表达含有内皮抑素和血管抑素(HEA)的抗血管生成融合蛋白的SB杆状病毒(Bac-SB-T2hEA/w)也能延长HEA的表达。持续表达HEA的Bac-Sb-T2hEA/w可抑制体内血管生成，抑制小鼠前列腺癌和人卵巢肿瘤移植瘤的生长，延长动物寿命。这些数据共同暗示了混合SB杆状病毒载体在延长HEA表达和治疗多种类型的血管生成依赖型肿瘤方面的潜力。
Antiangiogenesis is an appealing anticancer approach but requires continued presence of the antiangiogenic agents, which can be remedied by gene therapy. Baculovirus is an emerging gene delivery vector but only mediates transient expression (< 7 days); thus, this study primarily aimed to develop a hybrid baculovirus for sustained antiangiogenic gene expression and cancer therapy. We first constructed plasmids featuring adeno-associated virus inverted terminal repeats (AAV ITRs), oriP/Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1) or Sleeping Beauty (SB) transposon and compared their efficacies in terms of persistent expression. In human embryonic kidney (HEK293) cells, AAV ITR failed to prolong the expression while oriP/EBNA1 moderately extended the expression to 35 days. In contrast, the SB system led to stable expression beyond 77 days even without antibiotic selection. Given this finding, we constructed a hybrid SB baculovirus expressing the SB transposase and harboring the transgene cassette flanked by inverted repeat/direct-repeat (IR/DR) elements recognizable by SB. The hybrid SB baculovirus efficiently transduced mammalian cells and mediated an expression duration longer than that by conventional baculoviruses, thanks to the transgene persistence and integration. The SB baculovirus (Bac-SB-T2hEA/w) expressing the antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) also enabled prolonged hEA expression. With sustained hEA expression, Bac-SB-T2hEA/w repressed the angiogenesis in vivo, hindered the growth of two different tumors (prostate tumor allografts and human ovarian tumor xenografts) in mice and extended the life span of animals. These data altogether implicated the potential of the hybrid SB-baculovirus vector for prolonged hEA expression and for the treatment of multiple types of angiogenesis-dependent tumors.