Interactions of AAV5 with Sialic Acid and PDGF Receptors

虽然目前的治疗方法已经改善了囊性纤维化（CF）患者的生活，但没有一种是针对潜在的缺陷，CF仍然是一种致命的疾病。基因转移很有吸引力，因为它可以纠正由于CFTR丢失而导致的所有生理异常。腺相关病毒已被研究用于基因治疗。然而，AAV 2基因转移到人气道上皮的效率低。我们发现，重组AAV 5结合到顶端表面，介导基因转移的效率比AAV 2高50倍。我们的工作表明，AAV 5向气道上皮细胞的基因转移是受体介导的， 感染需要2，3 N-连接构型的唾液酸。Chiorini实验室最近的工作已经确定了血小板衍生生长因子受体（PDGF-R）作为AAV 5的受体。因此，2，3 N-连接构型的唾液酸和PDGF-R都是细胞的AAV 5转导所需的。在本申请中，我们提出研究AAV 5衣壳与其受体之间的分子相互作用。我们提出了3个具体的目标：PDGF-Rs是负责AAV 5在人气道上皮细胞中结合和内化的受体吗？我们假设PDGF-R的N-糖基化对于AAV 5结合和内化是必需的。或者，我们假设另一种膜蛋白中的2，3 N -连接的唾液酸 是AAV 5与PDGF-R结合所必需的，PDGF-R作为内化或其他后续感染步骤所需的共受体。AAV 5的结合是否导致PDGF-Rs二聚体形成和信号传导？我们假设AAV 5与PDGF-Rs的结合导致受体二聚化和磷酸化，并且PDGF-Rs的磷酸化在AAV 5感染细胞中起作用。AAV 5衣壳的哪些区域是结合唾液酸和PDGF所必需的？基于细小病毒家族其他成员的结构-功能分析和AAV 5的15 Angstrom cryo-EM结构，我们已经鉴定了AAV 5衣壳蛋白中的区域，该区域可接近表面并且可能在唾液酸介导的细胞结合中起作用。我们假设AAV 5衣壳的该区域负责AAV 5与细胞的结合和基因转移。此外，我们将研究与PDGFR结合的AAV 5区域。

Although current treatments have improved the lives of patients wit cystic fibrosis (CF), none are directed at the underlying defect and CF remains a lethal disease. Gene transfer is attractive because it could correct all of the physiologic abnormalities due to the loss of CFTR. Adeno-associated viruses have been studied for gene therapy. However, the efficiency of AAV2 gene transfer to human airway epithelia is low. We found that recombinant AAV5 binds to the apical surface and mediates gene transfer 50-fold more efficiently than AAV2. Our work indicates that gene transfer with AAV5 to the airway epithelia is receptor-mediated and that sialic acids in a 2,3 N-linked configuration are required for infection. Recent work by Chiorini's laboratory has identified the platelet derived growth factor receptors (PDGF-R), as receptors for AAV5. Thus, both sialic acids in a 2,3 N- linked configuration and PDGF-Rs are required for AAV5 transduction of cells. In this Grant application we propose to study the molecular interactions between AAV5 capsid and its receptors. We propose 3 specific aims: Are PDGF-Rs the receptors responsible for AAV5 binding and internalization in human airway epithelia? We hypothesize that N-glycosylation of PDGF-R is necessary for AAV5 binding and internalization. Alternatively, we hypothesize that 2,3 N -linked sialic acids in another membrane protein are required for AAV5 binding with the PDGF-Rs acting as a co-receptor required for internalization or other subsequent steps of infection. Does binding of AAV5 result in PDGF-Rs dimer formation and signaling? We hypothesize that AAV5 binding to PDGF-Rs results in receptor dimerization and phosphorylation and that phosphorylation of PDGF-Rs plays a role in AAV5 infection of cells. What regions of the capsid of AAV5 are required for binding to sialic acid and PDGF? Based on structure-function analysis of other members of the parvovirus family, and the 15 Angstroms cryo-EM structure of AAV5, we have identified a region in the capsid protein of AAV5 that is accessible to the surface and may play a role in sialic acid-mediated cell binding. We hypothesize that this region of AAV5 capsid is responsible for AAV5 binding to cells and gene transfer. Moreover we will investigate the region of AAV5 that binds to PDGFR.