Wu Qinjie, He Shasha, Wei Xiawei, Shao Bin, Luo Shuntao, Guo Fuchun, Zhang Hailong, Wang Yongsheng, Gong Changyang, Yang Li
1 State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University , Chengdu 610041, China .
Hum Gene Ther. 2014 Sep;25(9):811-23. doi: 10.1089/hum.2013.150. Epub 2014 Aug 21.
Adeno-associated virus (AAV) is an ideal choice for gene delivery; however, its further development has been limited owing to its low transduction efficiency. DNA-damaging agents can improve AAV-mediated transgene expression. Hyperthermia, as one of the oldest documented tumor treatment modalities, can cause DNA damage as well. However, combined treatment consisting of hyperthermia and AAV-mediated gene therapy has not been reported yet. In this work we investigated whether therapy consisting of AAV-mediated pigment epithelium-derived factor (PEDF) delivery combined with hyperthermia has synergistic antitumor effect on established solid tumors. We produced the recombinant AAV encoding PEDF (rAAV-PEDF). The therapeutic effect of rAAV-PEDF plus hyperthermia was evaluated in a subcutaneous fibrosarcoma mouse model, and the possible mechanism of antitumor effect was investigated. We found that rAAV-PEDF could infect a murine fibrosarcoma cell line (Meth-A) and express PEDF protein with bioactivity in vitro. In addition, in vivo experiments suggested that the combination of rAAV-PEDF with hyperthermia could significantly suppress tumor growth and prolong survival time of treated mice. Immunofluorescence studies indicated that the combination therapy could inhibit angiogenesis and induce apoptosis in tumor tissues. An immunohistochemistry assay of tumor tissue showed that PEDF expression in the combined treatment group was significantly higher than in the rAAV-PEDF group, which implied that hyperthermia could improve the expression of PEDF protein in vivo. No significant differences were observed in each group by hematoxylin-eosin staining of major organs, serum chemistry test, and complete blood assay. These results indicate that the combination of rAAV-PEDF with hyperthermia has synergistic therapeutic effects on established solid tumors, with no side effects. In addition, hyperthermia could improve AAV-mediated transgene expression, which suggests that hyperthermia could serve as a promising adjunctive therapy for AAV-mediated gene therapy.
腺相关病毒（AAV）是基因递送的理想选择；然而，由于其转导效率低，其进一步发展受到限制。DNA损伤剂可提高AAV介导的转基因表达。热疗作为有记载的最古老的肿瘤治疗方式之一，也可导致DNA损伤。然而，尚未见热疗与AAV介导的基因治疗联合应用的报道。在本研究中，我们探讨了AAV介导的色素上皮衍生因子（PEDF）递送联合热疗对已形成的实体瘤是否具有协同抗肿瘤作用。我们制备了编码PEDF的重组AAV（rAAV-PEDF）。在皮下纤维肉瘤小鼠模型中评估了rAAV-PEDF联合热疗的治疗效果，并研究了其抗肿瘤作用的可能机制。我们发现rAAV-PEDF可感染小鼠纤维肉瘤细胞系（Meth-A）并在体外表达具有生物活性的PEDF蛋白。此外，体内实验表明，rAAV-PEDF与热疗联合应用可显著抑制肿瘤生长并延长治疗小鼠的生存时间。免疫荧光研究表明，联合治疗可抑制肿瘤组织中的血管生成并诱导凋亡。肿瘤组织的免疫组化分析显示，联合治疗组中PEDF的表达明显高于rAAV-PEDF组，这表明热疗可提高体内PEDF蛋白的表达。主要器官苏木精-伊红染色、血清生化检测和全血细胞分析在各组中均未观察到显著差异。这些结果表明，rAAV-PEDF与热疗联合应用对已形成的实体瘤具有协同治疗作用，且无副作用。此外，热疗可提高AAV介导的转基因表达，这表明热疗有望成为AAV介导的基因治疗的辅助治疗方法。
