AAV capsids and their cellular interactions

描述（由申请人提供）：腺相关病毒重组AAV （rAAV）基因传递系统正进入一个关键和令人兴奋的阶段，有望进行20多年的深入研究，现已在许多成功的人体临床试验中实现。然而，为了完全实现，必须克服两个主要挑战：瞬时或低转基因表达和循环宿主抗体的有害影响。我们研究的主要目标是利用结构生物学方法，结合生物化学、分子生物学和动物体内研究来开发AAV病毒载体，克服这些挑战，提高临床治疗效果。成功的一个关键组成部分是确定衣壳中参与导致或改变感染的细胞相互作用的区域，特别是细胞受体附着和与宿主免疫的相互作用，特别是中和抗体。在这个项目中，我们将剖析组织转导的决定因素，并确定这些决定因素是否与衣壳的抗原区域重叠。我们将比较AAV1/6、AAV5、AAV8和AAV9，这些代表了AAV1/6、AAV5、AAV8和AAV9在AAV血清型中序列和结构多样性的范围，并显示出临床应用前景。我们将采取简化的方法来解决这个问题。我们将首先使用结构生物学，即x射线晶体学和冷冻电子显微镜和图像重建，来确定AAV衣壳最抗原活性的区域，然后通过分子和生化方法确定它们在感染中的作用。目标将是鉴定和突变不影响感染的残基，表征所产生的载体对人血清的抗原反应性，包括来自AAV临床基因治疗后患者的血清，并使用所产生的数据作为一般抗体逃逸AAV载体设计的指导原则。这些将在体内进行测试，包括使用临床相关的血友病B小鼠模型。该项目的总体影响将是了解成功感染所需的AAV衣壳决定因素以及宿主抗体中和这一过程，这是病毒学的基本目标。这一信息对于基因治疗界来说是至关重要的，以便在宿主免疫反应的存在下设计重组AAV载体以成功地传递基因。宿主免疫反应是一种无法阻止的自然宿主防御机制，但在基因传递应用中必须绕过它，因为它会显著减少或消除基因表达。

DESCRIPTION (provided by applicant): The Adeno-associated virus recombinant AAV (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, two major challenges that must be overcome for full realization are transient or low transgene expression and the detrimental effect of circulating host antibodies. The primary goal of our research is to use structural biology approaches, combined with biochemistry, molecular biology, and in vivo animal studies to develop AAV viral vectors that overcome these challenges and have improved clinical therapeutic efficacy. A key component to success is to identify regions of the capsid that participate in cellular interactions leading to or altering infection, i particular cell receptor attachment and interaction with host immunity, especially neutralizing antibodies. In this project, we will dissect the determinants of tissue transduction and also determine if these overlap with antigenic regions of the capsid. To get a general understanding we will compare AAV1/6, AAV5, AAV8, and AAV9, which represent the range of sequence and structure diversity among the AAV serotypes and have shown promise for clinical applications. We will take a reductionist approach to tackle the problem. We will first use structural biology, namely X-ray crystallography and cryo-electron microscopy and image reconstruction, to define the most antigenically reactive regions of the AAV capsids and then determine their role in infection through molecular and biochemical methods. The goal will then be to identify and mutate the residues that do not affect infection, characterize resulting vectors with respect to their antigenic reactivity against human serum, including those from post-AAV clinical gene therapy patients, and use the data arising as the guiding principle for general antibody escape AAV vector design. These will be tested in vivo, including the use of a clinically relevant hemophilia B mouse model. The overall impact of this project will be an understanding of the AAV capsid determinants necessary for successful infection and the neutralization of this process by host antibodies, a fundamental goal of virology. This information is critical for the gene therapy community in order to engineer recombinant AAV vectors for successful gene delivery in the presence of a host immune response, a natural host defense mechanism that cannot be prevented but must be circumvented in gene delivery applications because it dramatically reduces or eliminates gene expression.