AAV capsid functions, immune evasion and neuronal targeting in mice and NHP

说明书(申请人提供)：腺相关病毒(AAV)是目前最有前景的体内病毒基因传递载体。然而，仍然有剩余的 需要解决的各种问题，包括先前存在的抗AAV中和抗体(NtAbbs)在人类中的高流行率，针对病毒蛋白的效力限制宿主免疫反应，以及混杂的病毒嗜性。此外，AAV介导的免疫反应和趋向性存在物种特异性差异，这往往使得从小动物研究中预测临床结果变得困难，并强调了非人类灵长类(NHP)研究的重要性。至于中枢神经系统(CNS)疾病的基因治疗，AAV无法有效地穿越血脑屏障(BBB)，这是一个需要克服的额外障碍。因此，为了确保基因治疗取得更大的成功，迫切需要彻底解决这些问题。该项目的最终目标是获得培养细胞、小鼠和NHP中AAV衣壳氨基酸序列-表型关系的大型数据集~利用这些数据来了解AAV衣壳多方面表型是如何在每种不同的环境中确定的~建立克服目前限制的方法~并创建NtAb逃逸AAV载体，通过静脉(IV)途径特异性靶向小鼠和NHP中的中枢神经系统神经元。为了实现这一目标，我们设计了一种新的基于测序的方法，称为AAV条形码-序列，它允许我们仅使用少量重复以高通量的方式研究数百种不同AAV物种的病毒衣壳表型。我们将充分利用我们的独特能力，利用这一当代技术进行AAV研究，以实现四个具体目标。在目标1中，我们将绘制肝脏、心脏、肌肉和嗜中枢神经系统的强健血清型衣壳(AAV8和9)的高分辨率功能图，并确定每个氨基酸在表现一系列表型(包括细胞表面结合、转导、趋向性和清除)中的功能作用。这一分析将使我们能够确定对CNS靶向重要的氨基酸。在目标2中，我们将使用一种新型的表达在病毒衣壳上的三维结构的多肽库来绘制抗AAV NtAbs的表位图，并建立一种创建NtAb逃逸突变体的方法。在目标3中，我们将使用体外和体内模型研究AAV如何跨越BBB，并建立一种创造具有更高BBB穿透性的AAV突变体的方法。在目标4中，通过结合AIMS 1-3的实验结果并利用一种新的基于知识的定向进化方法，我们将创建新的NtAb逃逸AAV衣壳，通过静脉注射特异性和有效地靶向小鼠和NHP大脑中的神经元。这个拟议项目的成功完成将1)产生丰富的关于病毒衣壳氨基酸序列与宿主相互作用的有洞察力的数据，从而进一步加深我们对AAV衣壳生物学的理解；2)产生新型神经元特异性NtAb逃逸AAV载体，能够有效且容易地用于临床相关的动物模型~3)提供有价值的工具 以及整个基因治疗社区的数据资源。

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is the most promising in vivo viral gene delivery vector currently available. However, there still remain various issues to be resolved, including the high prevalence of pre-existing anti- AAV neutralizing antibodies (NtAbs) in humans, efficacy-limiting host immune responses against viral proteins, and promiscuous viral tropism. In addition, there are species-specific differences in AAV-mediated immune responses and tropism, which often make it difficult to predict clinical outcomes from small animal studies and underscore the importance of nonhuman primate (NHP) studies. As for gene therapy for the central nervous system (CNS) diseases, the inability of AAV to efficiently cross the blood-brain barrier (BBB) poses an additional obstacle to be overcome. Therefore, to ensure a greater success for gene therapy, there is an urgent need to thoroughly address these issues. The ultimate goal of this project is to acquire a large dataset of AAV capsid amino acid sequence-phenotype relationships in cultured cells, mice and NHPs~ utilize the data to understand how the multifaceted AAV capsid phenotypes are determined in each different context~ establish means to overcome the current limitations~ and create NtAb escape AAV vectors that specifically target CNS neurons via the intravenous (IV) route in mice and NHPs. To achieve this goal, we have devised a novel next generation sequencing-based approach, termed AAV Barcode-Seq, which allows us to investigate an array of viral capsid phenotypes of hundreds of different AAV species in a high-throughput manner using only a small number of replicates. We will fully utilize our unique ability to conduct AAV research using this contemporary technology in order to achieve four specific aims. In Aim 1, we will draw high-resolution functional maps of the liver, heart, muscle and CNS-tropic robust serotype capsids (AAV8 and 9) and determine functional roles of each amino acid in manifesting a spectrum of phenotypes including cell surface binding, transduction, tropism and clearance. This analysis will allow us to identify amino acids important for CNS targeting. In Aim 2, we will map epitopes of anti-AAV NtAbs using a novel type of peptide libraries expressed on viral capsids in a native 3-D structure, and establish a means to create NtAb escape mutants. In Aim 3, we will investigate how AAV crosses the BBB using in vitro and in vivo models, and establish a means to create AAV mutants with increased BBB penetrability. In Aim 4, by combining the experimental outcomes of Aims 1-3 and utilizing a novel knowledge-based directed evolution approach, we will create novel NtAb escape AAV capsids that specifically and efficiently target neurons throughout the mouse and NHP brain by IV injection. Successful completion of this proposed project will 1) yield an abundance of insightful data on viral capsid amino acid sequence-host interactions that further our understanding of the AAV capsid biology~ 2) yield novel neuron- specific NtAb escape AAV vectors that can cross the BBB efficiently and readily be used in clinically relevant animal models~ and 3) provide valuable tools and data resources for the entire gene therapy community.