AAV capsids and their cellular interactions

腺相关病毒(AAVs)是DependoparvoVirus属的单链DNA包装病毒 属于细小病毒科。基于AAVs的基因递送系统最近与FDA进入了一个令人兴奋的阶段 批准Luxturna，一种基于AAV2(AAV2)的基因疗法，用于治疗先天性心脏病的单基因缺陷 眼睛。然而，卢克图纳的成功是因为眼睛是一个免疫特权器官， 直接管理避免了预先存在的宿主免疫。这仍然是一个巨大的挑战，治疗 AAV基因递送系统的有效性。最近，猪瘟病毒属的成员 细小病毒科也被开发为病毒基因传递载体，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，在≥70%， 对于这两个系统的完全治疗实现来说，这是一个重大挑战。这个项目的主要关注点是 用鼠单抗(MAb)鉴定灵长类AAVs的抗原结构。 它们与受体附着、组织趋向性和转导效率(基因)的衣壳决定因素有关 表达)。我们率先将这些信息用于能够逃逸的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 确认通过为每种AAV血清型映射几个小鼠单抗而获得的“多克隆”信息 这项研究概括了人类的多克隆反应。在此续订申请中，我们将描述以下能力 人和非人灵长类(NHP)血清中和或结合而不是中和载体转导。这 将指导抗体逃逸和/或转导效率的工程，从而指导治疗用途。我们进行扩张 我们的病毒模型将包括非灵长类AAVs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)灵长类抗体 与前面描述的鼠源单抗的表位？“(2)”中和和非- 中和结合抗体重叠？(3)非灵长类AAVs会自然逃避先前存在的中和作用吗？ 灵长类抗体和转导人类细胞的能力？“和(4)“我们能不能把抗原点设计成 在保留或改进亲本转导的同时避免抗体中和的BoV载体 效率？“。我们将使用低温电子显微镜和图像重建来确定高分辨率 非灵长类AAVs和Bov衣壳的结构，单独和与多糖受体的复合体，到≤3？分辨率 AAV/BoV衣壳-人/NHP抗体的结构分辨率在3-4？之间。这是例行程序 在我们的团队里。我们将使用获得的信息来设计保持其细胞结合属性的载体，但 躲避人类/NHP的识别。我们将在体外和体内对这些载体进行评估，包括免疫球蛋白和 IVIG。我们将创造新的临床生物制剂，就像在上一轮融资中所做的那样，以扩大 细小病毒病毒载体曲目，因此可治疗疾病的目标数量，以及可治疗患者的队列。

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.