一种具有肝靶向、抗吞噬和低免疫原性的抗乙肝病毒外泌体的构建及临床前初步研究

Chronic hepatitis B virus (HBV) infection is a major health problem in our country and around the world. Interferon alpha is the first-line treatment for hepatitis B but with a low response rate and a strong side-effect. Our previous data showed that exosomes could mediate cell-to-cell transfer of anti-HBV effect by packaging anti-viral molecules, e.g. APOBEC3G(A3G) from interferon alpha stimulated cells. However, these exosomes are not hepatocyte-specific, and have relative shorter half-life, stronger immunogenicity and lower anti-viral activity. To solve the problem, we first overexpress a hepatocyte-specific peptide (HSP) that binds to the low-density lipoprotein receptor-related protein (LRP) of the hepatocytes on the surface of exosome, which endows exosomes with hepatocyte-targeting capacity. Furthermore, we overexpress CD47 on the surface of exosome, which increase the half-life and reduce the immunogenicity of exosomes by inhibiting phagocytosis of macrophage and uptake by dendritic cells (DCs) via CD47-signal regulatory protein alpha (SIRP alpha) signaling pathway. Moreover, we overexpress A3G in the exosome producing cells, which confers the anti-HBV activity to exosomes. Finally, we study anti-viral ability of the recombinant exosomes in HBV-replicating cell and mouse models as well as preclinically evaluate their comprehensive toxicity and immunogenicity. In summary, we attempt to construct a type of hepatocyte-targeting, anti-phagocytic, low-immunogenic and anti-HBV exosomes by over-expressing a hepatocyte-specific peptide (HSP) and CD47 on the surface of and encapsulating the anti-HBV A3G molecules within exosome for hepatitis B treatment. Hopefully, this research may provide insights for new HBV therapy strategies.

乙肝病毒（HBV）持续感染是我国乃至世界的重要健康问题。干扰素是临床治疗乙型肝炎的一线药物，但是存在应答率低、副作用大等缺点。课题组前期研究表明，干扰素通过外泌体在细胞间传递APOBEC3G蛋白等抗病毒分子抑制HBV复制。但干扰素刺激细胞分泌的外泌体存在肝靶向性弱、半衰期短、免疫原性较强、抗病毒活性较弱等缺点。为此，本研究首先把一段能与肝细胞表面低密度脂蛋白受体相关蛋白结合的多肽序列表达在外泌体表面，使外泌体具有肝靶向性；然后把CD47分子表达于外泌体表面，使其通过与信号调节蛋白SIRPα结合抑制巨噬细胞的吞噬和树突状细胞的摄取，进而增加外泌体在体内的半衰期并减少其免疫原性；再通过过表达A3G，使其在外泌体中富集，从而使外泌体具有抗HBV活性；最后检测这种改造后的外泌体在体内的肝靶向性、抗HBV活性、稳定性、并对其综合毒性进行临床前评价。本研究可望为乙肝的治疗提供新的思路。

乙肝病毒（HBV）持续感染是我国乃至世界的重要健康问题。干扰素是临床治疗乙型肝炎的一线药物，但是存在应答率低、副作用大等缺点。课题组前期研究表明，干扰素通过外泌体在细胞间传递APOBEC3G蛋白等抗病毒分子抑制HBV复制。但干扰素刺激细胞分泌的外泌体存在肝靶向性弱、半衰期短、免疫原性较强、抗病毒活性较弱等缺点。为此，本研究首先把一段能与肝细胞表面低密度脂蛋白受体相关蛋白结合的多肽序列表达在外泌体表面，使外泌体具有肝靶向性；然后把CD47分子表达于外泌体表面，使其通过与信号调节蛋白SIRPα结合抑制巨噬细胞的吞噬和树突状细胞的摄取，进而增加外泌体在体内的半衰期并减少其免疫原性；再通过过表达A3G，使其在外泌体中富集，从而使外泌体具有抗HBV活性；最后检测这种改造后的外泌体在体内的肝靶向性、抗HBV活性、稳定性。我们的结果显示重组的外泌体具有肝细胞特异的肝靶向性，在小鼠体内能够抑制相关巨噬细胞吞噬进而有较强的稳定性和较低的免疫原性。在对重组外泌体进行抗乙肝病毒功能评价过程中，在体外乙肝病毒稳定复制细胞系或瞬时转染体系中，重组外泌体不能够有效地抑制HBV复制。在小鼠AAV8-HBV慢性感染体系中，重组外泌体在肝组织免疫组化水平能够降低HBV相关抗原水平但对小鼠乙肝病毒S抗原和DNA拷贝数没有明显影响。因此，本研究在方法学上成功地构建出了肝靶向、抗吞噬的重组外泌体，为肝靶向性药物的递送提供了新的思路。如果能结合有效的抗病毒分子，本研究可望应用于乙肝的治疗。