基于可逆光控蛋白互作的细胞膜微囊包裹溶瘤腺病毒构建及其靶向治疗肝癌的研究

Targeted gene-virus therapy for hepatocellular carcinoma showed good results in preclinical experiments, but the immunoreaction and toxicity caused by this therapy restricted its application. We previously found that HEK293 cells, infected with oncolytic adenovirus ZD55, were treated with cytochalasin B to produce some cell membrane microcapsules (CMCs) that encapsulated ZD55, suggesting that CMCs can be a good carrier for ZD55-target transport, but the encapsulation efficiency was still to be further improved. Based on the development of CMCs vector and targeting gene-virus therapy in hepatocellular carcinoma, we constructed HEK293 cell line membrane-anchored CIBN protein and the capsid of ZD55 express the light-sensitive protein CRY2. Based on the rapid blue-light-mediated induction of protein interactions (CRY2-CIBN protein), ZD55 /IXCRY2@CMCs were prepared by adjusting the irradiation time and intensity. Subsequently, the ZD55/IXCRY2@CMCs was loaded the nucleolin targeting aptamer AS1411 on the surface of CMCs and produced ZD55/IXCRY2@CMCs-AS1411. The dynamic distribution and the proliferation of ZD55/IXCRY2@CMCs-AS1411 were evaluated and the biosafety of ZD55/IXCRY2@CMCs-AS1411 in the tumor-bearing mice was also assessed. The oncolytic adenovirus ZD55/IXCRY2-TIS@CMCs-AS1411 was constructed and its effect on hepatocellular carcinoma was studied. We demonstrate the utility of this system of light control protein interaction technology into the field of oncolytic virus targeting transportation.This research will effectively solve the problems of low targeting gene virus therapy effect and poor safety via intravenous administration, which can provide anovel approach for the targeting gene virus therapy of hepatocellular carcinoma.

肝癌靶向基因-病毒治疗在临床前研究中表现出良好效果，但静脉注射引起的免疫反应、毒副作限制了其临床应用。我们前期发现感染溶瘤病毒ZD55的HEK293细胞经Cytochalasin B处理能产生包裹ZD55的细胞膜微囊（CMCs），但CMCs病毒包裹效率仍需提高。本项目将构建表面展示CRY2的ZD55/IXCRY2病毒，感染HEK293/CIBN-CAAX细胞后利用CRY2-CIBN光控蛋白互作，通过调整光照时间、强度优化制备富含病毒的CMCs；再经AS1411修饰提高其肝癌靶向性，分析其在荷瘤鼠体内的动态分布、病毒复制扩散情况、生物安全性；最终静脉注射包裹溶瘤病毒的ZD55/IXCRY2-TIS@CMCs-AS1411研究其靶向治疗肝癌的效果及作用机制。本研究首次将光遗传学引入病毒靶向运输领域，开发基于光控蛋白互作的CMCs病毒包裹体系能进一步提高肿瘤靶向基因-病毒治疗的效果。

肝癌是一种常见的消化道恶性肿瘤，近年来发病率在我国逐渐增高，其临床表现隐匿，发展迅速，具有高度侵袭性，预后极差。近年来随着溶瘤腺病毒及细胞膜微囊载体的发展，溶瘤腺病毒已经成为肿瘤综合治疗模式的重要组成部分，关于肝癌的溶瘤腺病毒治疗研究也受到越来越多的重视。本研究结果显示，人类胚胎肾细胞 HEK293来源的CMCs可以作为溶瘤腺病毒的生物载体；负载溶瘤腺病毒额CMCs在生物体内外可以很好地保护溶瘤腺病毒，避免免疫系统的识别；CMCs-Ad对肝癌有很好的治疗效果。本研究为以CMCs为运输载体携带溶瘤病毒进行肝癌个体化治疗的新途径提供了实验依据，奠定理论基础。