具逃逸人体预存免疫潜力的蝙蝠腺相关病毒载体的构建和鉴定

Adeno-associated virus (AAV) has been used for gene therapy in clinical trials. However, the pre-existing immunity against AAV remains a huge challenge against its effective application. Employment of alternative AAV serotypes has been a solution to the pre-exiting anti-AAV humoral immunity in human body. However, neutralizing antibodies against most of the primate AAV serotypes are detected in the human populations. Thus isolation of AAV from animals other than primates might help to circumvent this problem. This proposal intends to isolate multiple variable AAV capsid genes from the bat populations. They will be utilized to produce recombinant bat AAV containing reporter genes for investigation of their tissue tropism on human cell lines or in the C57B6 mice. Besides, bat AAV will also be studied on their cross-reactivity with mouse sera against the primate AAV or neutralization by human plasma containing primate AAV antibodies in the SCID mice to characterize their potentiality to evade humoral immunity against primate AAV. Furthermore, bioinformatic analysis will be done on the bat AAV capsid genes to detect positive selection during their evolution. Positively selected sites will be mutated to examine their effects on transduction properties of bat AAV. Through these studies, bat AAV vectors capable to evade pre-existing immunity to primate AAV and efficiently transduce specific organs will be developed for their application to human gene therapy.

腺相关病毒（adeno-associated virus，AAV）是重要的人类基因治疗载体，然而人体中预存的AAV免疫已成为其应用的一大障碍。采用备用的AAV血清型能在一定程度上避免针对AAV的体液免疫，然而多数灵长类AAV在人群中具有流行的中和抗体，因此从其它物种中分离的AAV将具有潜在的应用价值。本项目拟从蝙蝠中分离多个具遗传变异的AAV衣壳蛋白基因，用它们包装携带报告基因的重组蝙蝠AAV，在人源细胞系上或在C57B6小鼠体内研究蝙蝠AAV的组织趋向性，通过与灵长类AAV抗血清的交叉反应或在SCID小鼠体内与人源AAV抗体的中和实验研究蝙蝠AAV逃逸灵长类AAV体液免疫的能力。此外还采用生物信息学分析检测蝙蝠AAV衣壳蛋白受到的阳性选择，并对部分选择位点进行突变以研究对蝙蝠AAV转导性质的影响。最终获得能逃逸灵长类AAV预存免疫并有效转导特定器官的蝙蝠AAV载体以用于人类基因治疗。

在从前的研究中，我们的合作者报道了蝙蝠AAV在中国不同蝙蝠种群中的流行情况，展示出丰富的遗传多样性，然而对它们的生物学性质没有做详细研究。在本项目中，我们采用PCR方法从蝙蝠样本中分离出4个AAV衣壳蛋白基因的全长编码序列，它们与人AAV2的衣壳蛋白序列有约61%的氨基酸同源性。这些衣壳蛋白基因可以与AAV2的复制蛋白和末端重复序列合作生产野生型和重组型AAV载体，尽管效率较低。与此同时，蝙蝠AAV用于静脉注射基本不感染小鼠肝脏，却对肌肉有一定的转导能力，展示出较高的肌肉/肝脏转导比值。在直接肌肉注射中，蝙蝠AAV 10HB载体展示出和AAV2相近的肌肉转导效率。用人源AAV抗体处理免疫缺陷小鼠，然后用蝙蝠AAV进行肌肉注射，蝙蝠AAV 10HB载体相对于AAV2能够比较有效地逃逸人源AAV抗体的中和。进而采用生物信息学方法对包括蝙蝠AAV在内的32个依赖病毒属衣壳蛋白编码序列进行了分析，识别出在蝙蝠AAV衣壳蛋白中受到阳性选择的位点。其中对应于10HB衣壳蛋白V259和N691的两个位点分布于病毒衣壳的外面，对它们的突变改变了蝙蝠AAV对小鼠肌肉的转导能力，暗示这些位点的进化受到宿主感染选择压力的驱动。总之，蝙蝠AAV 10HB载体由于其中等的肌肉转导能力，并能在一定程度上逃逸人源AAV抗体的中和，在肌肉基因治疗中具有一定的应用潜力。