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患者的生活，但没有一种是针对潜在的缺陷，而且囊性纤维化仍然是一种致命的疾病。基因转移是一种很有吸引力的方法，因为它可以纠正由于CFTR缺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，AAV2基因转移到人呼吸道上皮细胞的效率较低。我们发现，重组AAV5结合到根尖表面，并比AAV2有效地介导基因转移50倍。我们的工作表明，AAV5基因转移到呼吸道上皮细胞是由受体介导的，并且 感染需要2，3N连接构型的唾液酸。Chiorini的实验室最近的工作已经确定血小板衍生生长因子受体(PDGF-R)是AAV5的受体。因此，AAV5转导细胞既需要2，3 N-连接构型的唾液酸，也需要PDGF-R。在这项GRANT申请中，我们建议研究AAV5衣壳与其受体之间的分子相互作用。我们提出了三个特定的目标：PDGF-Rs是人呼吸道上皮细胞中负责AAV5结合和内化的受体吗？我们推测，PDGF-R的N-糖基化是AAV5结合和内化所必需的。或者，我们假设另一种膜蛋白中的2，3N-连接唾液酸 AAV5与作为内化或其他后续感染步骤所需的辅助受体的PDGF-Rs结合是必需的。AAV5结合是否导致PDGF-Rs二聚体的形成和信号转导？我们假设AAV5与PDGF-Rs结合导致受体二聚化和磷酸化，PDGF-Rs的磷酸化在AAV5感染细胞中起作用。AAV5衣壳的哪些区域需要与唾液酸和PDGF结合？基于对细小病毒家族其他成员的结构和功能的分析，以及AAV5的15埃冷冻-EM结构，我们在AAV5的衣壳蛋白中发现了一个表面可及的区域，可能在唾液酸介导的细胞结合中发挥作用。我们推测AAV5衣壳的这个区域负责AAV5与细胞的结合和基因转移。此外，我们还将研究AAV5与PDGFR结合的区域。

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.