基于生物矿化策略的新型溶瘤腺病毒的构建及其靶向治疗肝癌的研究

In recent years, the oncolytic virus therapy has been evolved to generate advanced therapeutic efficacy. In previous study, we generated a novel E1B-55K deleted oncolytic adenovirus(ZD55) which harbors TRAIL-IETD-Smac gene and showed significant anti-hepatoma effects. However, the systemic injection of oncolytic adenovirus in clinical applications is restricted due to significant liver toxicity and immunogenicity. Based on the systematic study of oncolytic adenovirus and virus mineralization, we suggest a combined, rational design approach to improve the tumor-targeting and stability of oncolytic adenovirus by self-biomineralization. The biomimetic nucleating peptide W6p are rationally integrated onto the capsid of oncolytic adenovirus ZD55 by reverse genetics so that calcium phosphate mineralization can be biologically induced onto virus surfaces under physiological conditions, generating a mineral exterior. First, we are aim to verify the self-biomineralization effect and the resistant capacity to host immune rejection of this engineered self-biomineralized virus. Then, the evaluation of in vivo biological behavior and safety for self-biomineralized virus is performed. Finally, the anti-hepatoma effects and mechanism of the self-biomineralized oncolytic adenovirus armed TRAIL-IETD-Smac gene are evaluated in vitro and in vivo. This study perform functional biomineralization to oncolytic virus for the first time and will provide a new approach and experimental basis for oncolytic adenovirus mediated gene therapy of hepatocellular carcinoma.

近年来，溶瘤病毒治疗已取得突破性成果。申请人前期利用溶瘤腺病毒载体携带治疗基因TRAIL-IETD-Smac表现出显著的抗肝癌效果，但发现溶瘤腺病毒存在静脉注射抗癌效果差、机体对腺病毒的免疫排斥强等局限性。本课题拟在前期溶瘤腺病毒研究及病毒矿化的工作基础上，进一步通过基因工程技术在腺病毒衣壳蛋白上克隆磷酸钙成核多肽W6p，诱导溶瘤腺病毒自发矿化形成纳米磷酸钙外壳，构建生物矿化溶瘤腺病毒载体。首先验证W6p多肽诱导的溶瘤腺病毒磷酸钙生物矿化效果及抵抗机体内免疫系统排斥作用，再分析矿化病毒体内趋向肿瘤的动态过程及评价矿化病毒的生物安全性，最后研究矿化病毒靶向治疗肝癌效果及作用机制。本研究首次对溶瘤病毒进行功能性生物矿化，以期为溶瘤腺病毒介导的肝癌基因治疗提供新的途径和有力实验依据。

为了利用生物矿化策略有效克服溶瘤腺病毒静脉给药治疗肿瘤的障碍，本课题在前期研究基础上，采取基因工程手段在溶瘤腺病毒衣壳蛋白上引入磷酸钙成核多肽，为病毒的磷酸钙矿化提供了有效的成核位点，对溶瘤腺病毒进行磷酸钙生物矿化修饰，成功制备具有核壳结构的磷酸钙矿化溶瘤腺病毒载体。采用靶向基因-病毒治疗策略，携带TRAIL-IETD-Smac融合基因，成功构建矿化病毒CaP@W6p-ZD55-TRAIL-IETD-Smac。静脉注射矿化病毒，利用实体瘤的EPR效应而靶向到肿瘤部位，发挥双基因溶瘤治疗作用，并对其作用机制进行探讨，进一步我们研究了矿化病毒重塑抑制性肿瘤免疫微环境的功能，并联合免疫检查点抑制剂PD-1抗体，验证了矿化病毒促进PD-1抗体抗肿瘤作用。本研究是首次对溶瘤病毒进行功能性生物矿化，所开发的磷酸钙生物矿化溶瘤腺病毒载体能够提高准确到达肿瘤部位的病毒数量、减少病毒用量，增强抗瘤效果，降低毒副作用，促进免疫治疗效果，为溶瘤腺病毒介导的基因治疗和免疫治疗提供新的途径和有力实验依据，具有重要的理论意义和应用价值。