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

A）AAV（腺相关病毒）介导的癌症基因治疗的开发：1）使用杆状病毒表达载体的AAV载体生产系统的建立：我们开发了一种从空衣壳中分离AAV载体颗粒的柱层析方法，该方法可适用于AAV载体的大规模生产。将AAV p5启动子并入转基因序列中增加了AAV载体生产的效率。2）AAV载体修饰的基础研究：具有2型VP 1的嵌合5型AAV载体在其衣壳中具有更大量的VP 1，并且以与亲本5型AAV载体类似的方式转导靶细胞。介导的基因转移和转基因表达的调节：我们建立了将基因转移到肌肉、肝脏、脂肪组织和腹膜中的标准方法。组蛋白去乙酰化酶抑制剂增强了AAV载体介导的转基因在肿瘤细胞中的表达。4）使用AAV载体的癌症基因治疗策略的检查：我们对难治性癌症进行了基因治疗实验（例如，造血和/或淋巴转移，和腹膜播散），并在荷瘤动物中显示治疗功效。B）癌症基因治疗新策略的研究：为了治疗难治性恶性淋巴瘤（B细胞非霍奇金淋巴瘤），我们进行了实验以开发一种新的增强过继免疫基因疗法，其使用表达靶向CD 19的CAR（嵌合抗原受体）的T细胞。我们证明了基因工程T细胞在体外有效地溶解CD 19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.