Development of AAV (adeno-associated virus) vectors and their application to cancer therapy

A)腺相关病毒（adeno-associated virus， AAV）介导癌症基因治疗的研究进展：1)利用杆状病毒表达载体构建AAV载体制备体系：在昆虫细胞中高效制备AAV载体。我们建立了一种从空衣壳中分离AAV载体颗粒的柱层析方法，该方法适用于AAV载体的大规模生产。在转基因序列中加入AAV p5启动子可提高AAV载体的生产效率。2) AAV载体修饰的基础研究：与2型VP1嵌合的5型AAV载体衣壳中VP1含量较高，其转导靶细胞的方式与亲本5型AAV载体相似。3) AAV载体介导基因转移及转基因表达调控的基础研究：建立了肌肉、肝脏、脂肪组织和腹膜基因转移的标准方法。一种组蛋白去乙酰化酶抑制剂增强了AAV载体介导的肿瘤细胞中的转基因表达。4)利用AAV载体进行肿瘤基因治疗策略的探讨：我们对难治性癌症（如血液和/或淋巴转移、腹膜播散）进行了基因治疗实验，并在荷瘤动物中显示了治疗效果。B)癌症基因治疗新策略的研究：对于难治性恶性淋巴瘤（B细胞非霍奇金淋巴瘤）的治疗，我们进行了实验，开发了一种新的增强过继免疫基因疗法，使用表达CAR（嵌合抗原受体）靶向cd19的t细胞。我们证明了基因工程t细胞在体外有效地溶解cd19阳性b细胞淋巴瘤细胞。

A) Development of AAV (adeno-associated virus)-mediated cancer gene therapy :1) Establishment of AAV vector production system using baculovirus expression vectors : AAV vectors were efficiently produced in insect cells. We developed a column chromatographic method to isolate AAV vector particles from empty capsids, which can be adaptable to large-scale production of AAV vectors. Incorporation of the AAV p5 promoter into a transgene sequence increased the efficiency of AAV vector production.2) Basic studies of the modification of AAV vectors : Chimeric type 5 AAV vectors with type 2 VP1 had a larger amount of VP 1 in their capsids and transduced target cells in a similar manner with parent type 5 AAV vectors.3) Basic studies of AAV vector-mediated gene transfer and the regulation of transgene expression : We established standard methods of gene transfer into muscle, liver, adipose tissue and peritoneum. A histone deacetylase inhibitor enhanced AAV vector-mediated transgene expression in tumor cells.4) Examination of strategies for cancer gene therapy using AAV vectors : We conducted gene therapy experiments for refractory cancers (e.g. hematogenous and/or lymphogenous metastasis, and peritoneal dissemination) and showed therapeutic efficacy in tumor-bearing animals.B) Research on novel strategies for cancer gene therapy : For the treatment of refractory malignant lymphoma (B-cell non-Hodgkin lymphoma), we conducted experiments to develop a novel reinforced adoptive immuno-gene therapy using T-cells expressing a CAR (chimeric antigen receptor) targeting CD 19. We demonstrated that genetically engineered T-cells efficiently lyzed CD 19positive B-cell lymphoma cells in vitro.