Coevolution of Adeno-associated Virus Capsid Antigenicity and Tropism through a Structure-Guided Approach
Coevolution of Adeno-associated Virus Capsid Antigenicity and Tropism through a Structure-Guided Approach
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DOI：
10.1128/jvi.00976-20
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发表时间：
2020-10-01
影响因子：
5.4
通讯作者：
Asokan, Aravind
中科院分区：
文献类型：
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作者：
Havlik, L. Patrick;Simon, Katherine E.;Asokan, Aravind
腺相关病毒(AAV)是由无包膜的二十面体蛋白质壳组成的，可以包装和运送重组治疗性DNA。用于基因治疗应用的工程重组衣壳的方法主要集中在合理设计或基于文库的方法，这些方法可以解决一到两个期望的属性；然而，在全面改善AAV载体特性方面仍有一个未得到满足的需求。这不能仅通过利用序列数据来实现，而需要利用AAV衣壳的三维(3D)结构属性。在这里，我们使用冷冻电子显微镜来解析与抗体复合的天然AAV分离株的结构，并利用这些结构信息通过饱和诱变不同的抗原足迹来设计AAV衣壳文库。每个表面环都是在辅助腺病毒存在的情况下通过感染性循环来进化的，以产生新的AAV变体，然后作为进化下一个表面环的模板。这个逐步的过程产生了人源化的AAV8衣壳(AAVhum.8)，它显示了非自然的表面环，同时显示了对人类肝细胞的趋向性，提高了基因转移效率，并中和了抗体逃避。具体地说，AAVhum.8比AAV8能更好地躲避多个物种的中和抗血清。此外，AAVhum.8在人肝移植小鼠模型中显示出强大的转导能力，对小鼠和人肝细胞都具有扩大的趋向性。这项工作支持了这样一种假设，即通过合理设计和基于文库的进化，AAV衣壳抗体的逃避和趋向性等关键特性可以在临床基因治疗中协同进化。在全面改善AAV的组织趋向性、转导效率和抗体逃逸方面，还有一个尚未得到满足的需求。这不能仅通过利用衣壳序列数据来实现，而需要利用AAV衣壳的3D结构属性。在这里，我们结合合理的设计和基于库的进化，通过利用3D结构信息将多种所需的属性共同进化到AAV上。
Adeno-associated viruses (AAV) are composed of nonenveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone but requires harnessing the three-dimensional (3D) structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying nonnatural surface loops that simultaneously display tropism for human hepatocytes, increased gene transfer efficiency, and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species than AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties, such as AAV capsid antibody evasion and tropism, can be coevolved by combining rational design and library-based evolution for clinical gene therapy.IMPORTANCE Clinical gene therapy with recombinant AAV vectors has largely relied on natural capsid isolates. There is an unmet need to comprehensively improve AAV tissue tropism, transduction efficiency, and antibody evasion. Such cannot be achieved by utilizing capsid sequence data alone but requires harnessing the 3D structural properties of AAV capsids. Here, we combine rational design and library-based evolution to coevolve multiple, desirable properties onto AAV by harnessing 3D structural information.