AAV capsids and their cellular interactions

腺相关病毒（Adeno-associated viruses，AAV）是属于依赖细小病毒属的ssDNA包装病毒 细小病毒科基于AAVs的基因递送系统最近进入了一个令人兴奋的阶段， 批准Luxturna，一种基于AAV血清型2（AAV 2）的基因疗法，用于治疗 眼睛然而，Luxturna的成功是由于眼睛是一个免疫特权器官， 直接施用避免了预先存在的宿主免疫。这仍然是对治疗方法的重大挑战。 AAV基因递送系统的功效。最近，研究人员发现了Bocaparvovirus属的成员， 细小病毒科也已被开发为病毒基因递送载体，也用于治疗单基因疾病。 然而，针对AAV和博卡病毒（BoV）的宿主抗体的高水平血清阳性率（≥ 70%）， 对两个系统的完全治疗实现来说是一个重大挑战。该项目的主要重点是 已经使用小鼠单克隆抗体（Mab）表征灵长类AAV的抗原结构，如 它们与受体附着的衣壳决定子、组织向性和转导效率（基因 表达式）。我们率先使用这些信息进行AAV载体的分子工程， 抗体识别，目前正在评估作为潜在的临床载体。然而，有必要 证实了通过对每种AAV血清型的几种小鼠Mab作图获得的“多克隆”信息 研究概括了多克隆人的反应。在这次更新申请中，我们将描述 中和或结合而不中和载体转导。这 将指导抗体逃逸和/或转导功效的工程化，从而指导治疗效用。我们扩大 我们的病毒模型包括非灵长类AAV和BoV载体，以努力扩大细小病毒库， 可供使用的载体。我们的三个具体目标将提出四个新问题：（1）“灵长类动物的抗体是否共享 表位与先前描述的鼠单克隆抗体？”(2)“中和和非中和的结合位点 中和结合抗体重叠”(3)“非灵长类动物的AAVs是否能自然地逃脱预先存在的中和作用？ 灵长类抗体并能够转导人类细胞？”和（4）“我们能不能设计抗原位点， BoV载体逃避抗体中和，同时保留或改善亲本转导 效率？”我们将使用冷冻电子显微镜和图像重建来确定高分辨率 非灵长类AAV和BoV衣壳的结构，单独和与聚糖受体复合，分辨率≤3 μ m 和AAV/BoV衣壳-人/NHP抗体的结构达到3至4 μ m分辨率。这是例行公事 在我们组里。我们将利用获得的信息来设计载体，保留其细胞结合特性， 逃避人类/NHP的识别。我们将在体外和体内评估这些载体， IVIG，分别。我们将创造新的临床生物制剂，就像在过去的一轮融资中所做的那样，以扩大 细小病毒病毒载体库，因此可治疗的疾病靶标的数量，以及可治疗的患者的队列。

The Adeno-associated viruses (AAVs) are ssDNA packaging viruses belonging to the Dependoparvovirus genus of the Parvoviridae. Gene delivery systems based on the AAVs recently entered an exciting phase with the FDA approval of Luxturna, an AAV serotype 2 (AAV2)-based gene therapy for treating a monogenetic defect in the eye. However, the success of Luxturna was ushered by the fact that the eye is an immune privileged organ and direct administration avoids pre-existing host immunity. This remains a significant challenge to the therapeutic efficacy of the AAV gene delivery system. More recently, members of the Bocaparvovirus genus of the Parvoviridae have also been developed as viral gene delivery vectors, also for treating monogenetic diseases. However, high level of seroprevalence of host antibodies against AAVs and bocaviruses (BoVs), at ≥70%, represents a major challenge to full therapeutic realization of both systems. The primary focus of this project has been to characterize the antigenic structures of primate AAVs, using mouse monoclonal antibodies (Mabs), as they relate to capsid determinants of receptor attachment, tissue tropism, and transduction efficiency (gene expression). We pioneered the use of this information for molecular engineering of AAV vectors able to escape antibody recognition and currently under evaluation as potential clinical vectors. However, there is need to confirm that the “polyclonal” information obtained by mapping several mouse Mabs for each AAV serotype studied recapitulates the polyclonal human response. In this renewal application, we will characterize the ability of human and non-human primate (NHP) sera to neutralize or bind and not neutralize vector transduction. This will guide the engineering of antibody escape and/or transduction efficacy and thus therapeutic utility. We expand our viral models to include non-primate AAVs and BoV vectors in an effort to expand the pool of parvoviral vectors available for use. Our three specific aims will ask four new questions: (1) “Do primate antibodies share epitopes with the previously described murine Mabs?” (2) “Do the binding sites of neutralizing and non- neutralizing binding antibodies overlap”? (3) “Do non-primate AAVs naturally escape pre-existing neutralizing primate antibodies and capable of transducing human cells?” And (4) “Can we engineer the antigenic sites on BoV vectors to evade neutralization by antibodies while retaining or improving the parental transduction efficiency?”. We will use cryo-electron microscopy and image reconstruction to determine high-resolution structures of non-primate AAVs and BoV capsids, alone and in complex with glycan receptors, to ≤3 Å resolution and the structures of AAV/BoV capsid – human/NHP antibodies to between 3 to 4 Å resolution. This is routine in our group. We will use the information obtained to engineer vectors that retain their cell binding properties but evade recognition by human/NHP. We will evaluate these vectors in vitro and in vivo in the presence of IgG and IVIG, respectively. We will create new clinical biologics, as was done in the past funding round, to expand the parvovirus viral vector repertoire, thus the number of treatable diseases targets, and cohort of treatable patients.