腺病毒介导的RNA干扰策略抗流感病毒感染

Influenza virus infection is a major public health problem, causing millions of cases of severe illness and as many as 500,000 deaths each year worldwide. Currently，the licensed inactivated vaccines are 60-80% effective against matched influenza strains. Once infected with influenza virus, there is no specific and effective drug against it. The widespread use of antiviral drugs is limited by concerns about serious side effects and the possible emergence of drug-resistant variants. Given the limitations of current anti-influenza virus strategies, the need for novel strategies for prevention and treatment of influenza virus is urgent. RNA interference (RNAi) is an emerging technology that specifically inhibits gene expression. More and more studies have shown that RNAi could inhibit viral gene expression and protect cells from viral infection. In this regard, RNAi technology holds great promise to inhibit the replication of influenza virus. In this project, we aim to develop a new method against influenza infection based on RNAi and adenovirus delivery system. We designed and synthesized dozens of artificial mricroRNAs (amiRNAs) that target to conserved regions of influenza virus genes such as NP, M1 and M2 respectively, then cloned these amiRNAs into a commercially available pcDNATM6.2-GW/miR vector, respectively. We transfected these plasmids into cells and then infected the cells with influenza virus, screening for the amiRNAs that inhibit the replication of influenza virus efficiently in cells. For efficient delivery and expression, we cloned these amiRNAs into a chimpanzee adenoviral vector, termed AdC68, respectively, which is non-prevalent in humans so it can evade preexisting immunity to human adenovirus. Consequently, we are looking forward to developing a new method for the prevention and therapy of influenza virus infection.

流感对人类的公共健康构成严重威胁，每年在全球范围内造成数百万严重病例和50多万人死亡。目前尚无特效的治疗药物，常用的疫苗也只有60-80%的保护率。RNAi由于可以特异性地抑制基因表达, 近年来越来越多地应用于抗病毒治疗或预防的研究。在本项目中，我们设计分别针对流感病毒保守基因NP，M1，M2的microRNA，合成后克隆至pCDNA6.2TM-GW/miR载体，转染细胞，在细胞水平筛选出可有效抑制流感病毒复制的microRNA。然后，将筛选获得的microRNA克隆至黑猩猩型腺病毒载体AdC68，包装出表达microRNA的重组腺病毒，进一步研究其在体内和体外对多种流感病毒的抑制效果。由于腺病毒载体的高表达与高转导特性,通过本研究的开展，有望获得一种新型广谱型抗流感制剂，为流感的预防和治疗提供新策略。

流感对人类的公共健康构成了严重的威胁，每年会在全球范围内造成数百万的严重病例和50万人的死亡。RNAi由于可以特异性地抑制基因表达, 近年来越来越多的用于抗病毒治疗及预防的研究。在本研究中设计针对流感病毒的保守基因NP，M1，M2的microRNA，合成后插入到载体pCDNA6.2TM-GW/miR，转染MDCK细胞，在细胞水平上初步筛选出可有效抑制流感病毒复制的microRNA。本研究中选用黑猩猩型腺病毒（AdC68）作为将microRNA安全高效的运输到机体内的载体，以避免人群中广泛存在的腺病毒抗体的拮抗作用，进一步研究重组腺病毒在体内和体外对多种流感病毒的抑制效果。由于本研究中使用的是黑猩猩型腺病毒载体可避免人群中广泛存在的腺病毒抗体的拮抗作用，因此可显著提高载体的运输效率。通过本研究的开展，以期获得一种新型广谱型抗流感制剂，为研究流感预防和治疗的新策略提供理论依据。在本研究中，我们筛选具有抑制流感病毒复制的microRNA并以腺病毒作为转运载体，以此作为一种新的抗流感策略。研究结果表明：我们筛选获得的靶向流感病毒（PR8）M1、M2、NP等基因的人工合成microRNA可以在MDCK和HEK 293细胞中高效抑制流感病毒的复制；以复制缺陷的黑猩猩型腺病毒AdC68作为microRNA的转运载体，在小鼠体内可对致死剂量的同源流感病毒（PR8）的攻击感染提供完全保护，对异源的H9N2和H5N1的攻击感染也可提供部分保护。因此，以腺病毒为载体转运并表达靶向流感病毒保守基因的microRNA有望成为一种预防人群和动物中流感病毒感染的新策略。