携带GM-CSF和IL24双基因的重组溶瘤痘苗病毒天坛株靶向治疗肿瘤的研究

Oncolytic viruses are being developed as a novel strategy in cancer therapy. Vaccinia virus has many obvious advantages, including extensive safety, quick replication, large transgene-encoding capacity and stability. The recombinant oncolytic vaccinia viruses constructed by genetic engineering can selectively replicate in tumor cells, making them to become a great potential for the study. At present, several strains of vaccinia virus widely used in the global smallpox eradication campaign have been proven its oncolytic effect, while Chinese vaccinia virus Tian Tan strain lacks of research reports as oncolytic virus. In our previous study, we have explored the potential as oncolytic agent of the attenuated strain VG9 of Tian Tan strain as well as constructed the recombinant oncolytic vaccinia viruse VG9-GMCSF. Studies in vitro and in vivo have shown significant antitumor efficacy. To further explore the application of VG9 as an oncolytic backbone as well as to further improve the antitumor efficacy, in this study, we will construct a novel oncolytic vaccinia viruse carrying two promising immunostimulatory transgenes GM-CSF and IL-24 in TK locus and perform it in cancer therapy. We will further explore the mechanism and potential as oncolytic agent of VG9-GMCSF-IL24. The findings will provide the theoretical and experimental basis for the application of vaccinia virus Tian Tan strain as oncolytic virus in cancer therapy.

溶瘤病毒治疗肿瘤是当前肿瘤生物治疗的一个热点。痘苗病毒具有安全性高，复制快，基因组容量大，稳定性高等优点。利用基因工程对痘苗病毒基因组进行改造, 使其选择性地在肿瘤细胞中复制, 降低对正常细胞的毒性, 成为具有很大潜力的研究对象。目前，曾在消灭天花行动中广泛应用的几株痘苗病毒均已探明其溶瘤效果，而我国痘苗病毒天坛株缺少作为溶瘤病毒的研究报道。在前期的研究中，我们对天坛株减毒株VG9在溶瘤方面进行了初步探索，构建了重组溶瘤病毒VG9-GMCSF，体内外研究均显示了显著的抗肿瘤效果。为了进一步探索VG9作为肿瘤治疗载体的应用，进一步提高溶瘤病毒的肿瘤治疗效果，本项目将两种非常具有发展前景的免疫因子基因GM-CSF和IL24插入TK区，构建具有协同作用的靶向性双基因溶瘤痘苗病毒进行肿瘤治疗的研究，探索其溶瘤潜质和机制，为我国痘苗病毒天坛株作为溶瘤病毒在肿瘤治疗中的应用提供理论依据和实验基础。

溶瘤痘苗病毒复制快、可溶解感染的肿瘤细胞，还可作为治疗基因表达载体，从而成为肿瘤治疗有吸引力的候选方法。本课题以痘苗病毒中国天坛株减毒株广9（VG9）为骨架，构建了一种新型的同时携带粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素-24(IL-24)双基因的重组溶瘤痘苗病毒（VG9-GMCSF-IL24），评价其在不同肿瘤细胞中的复制能力，对不同肿瘤细胞及正常细胞的体外杀伤能力，以及引起细胞病变和细胞凋亡的机制。构建不同肿瘤模型（包括小鼠黑色素瘤B16模型，小鼠结肠癌CT26.WT模型，小鼠乳腺癌4T1模型），在体研究VG9-GMCSF-IL24体内分布及抗肿瘤疗效，评价其引起的肿瘤特异性免疫，检测血清中相关免疫因子水平。成功构建得到重组溶瘤痘苗病毒VG9-GMCSF-IL24，Western blotting和ELISA结果表明VG9-GMCSF-IL24可同时表达GM-CSF 和IL-24蛋白；体外细胞毒性实验显示VG9-GMCSF-IL24能强烈杀伤肿瘤细胞，而对正常细胞没有显著的杀伤作用；此外，VG9-IL24可通过多种细胞凋亡信号通路诱导细胞凋亡。不同肿瘤模型的体内实验显示，VG9-GMCSF-IL24可显著抑制肿瘤生长，延长生存期；还可以诱导机体产生较高水平的干扰素-γ（IFN-γ）, 肿瘤坏死因子-α（TNF-α）, IL-4 和 IL-6，介导细胞免疫和体液免疫反应；特异性毒性T淋巴细胞（CTL）杀伤实验和特异性抗肿瘤免疫保护实验结果显示 VG9-GMCSF-IL24可诱导产生肿瘤特异性免疫。本课题研究结果表明痘苗病毒VG9介导的GM-CSF和IL-24共表达可以产生协同抗肿瘤效应。溶瘤病毒携带一种或多种免疫因子基因是一种新颖而有效的肿瘤治疗方法，既有溶瘤作用又可进行免疫治疗，具有令人满意的临床应用前景。