The Progranulin C-Terminal Domain and AAV-Progranulin Gene Therapy for Frontotemporal Dementia

项目摘要 此应用程序是为F30支持Shreya Kashyap在她的MD/PhD培训。科学的焦点在于 一个提议是确定颗粒蛋白前体C末端结构域（CTD）对AAV衍生的颗粒蛋白前体水平的作用 在体内和AAV-颗粒蛋白前体基因治疗的有效性。基因组中的杂合性功能丧失突变 颗粒蛋白前体基因（GRN）是导致额颞叶痴呆（FTD）的原因之一，FTD是一种毁灭性的神经退行性疾病， 以社会行为和语言缺陷为特征的疾病。埃里克·罗伯森博士实验室的研究成果， 已经建立了AAV-颗粒蛋白前体基因治疗在颗粒蛋白前体不足的患者中的治疗潜力， FTD小鼠模型。用于这些实验的载体具有与CTD融合的标签，其排除了 颗粒蛋白前体与其典型运输受体分拣蛋白的相互作用。这些实验表明， AAV-颗粒蛋白前体的溶酶体定位和功效独立于分拣蛋白。然而，功能 暴露的颗粒蛋白前体CTD（其允许颗粒蛋白前体-分拣蛋白结合）的结果仍然未知。 值得注意的是，分拣蛋白介导颗粒蛋白前体的摄取和降解，分拣蛋白的消融或抑制增强了颗粒蛋白前体的摄取和降解。 野生型和颗粒蛋白前体不足小鼠模型中的脑和CSF颗粒蛋白前体水平。我的初步 实验表明，阻断颗粒蛋白前体C-末端结构域可以提高AAV衍生的腺病毒的实质水平。 本发明提供了一种AAV-颗粒蛋白前体，并且提高了AAV-颗粒蛋白前体在挽救溶酶体病理和神经炎症中的功效。 具有与颗粒蛋白前体CTD融合的标签的AAV-颗粒蛋白前体载体的临床翻译是不可能的。因此， 确定改善AAV-颗粒蛋白前体剂量和水平的CTD序列修饰是重要的。 免疫介导的AAV颗粒和AAV转导的细胞的清除降低了AAV-1的有效性。 基因疗法。颗粒蛋白前体缺陷小鼠对AAV-鼠颗粒蛋白前体产生免疫应答， 目前尚不清楚FTD-GRN患者（具有一个功能性颗粒蛋白前体等位基因）是否会产生免疫应答， AAV-人颗粒蛋白前体。然而，正如最近的AAV-胶质源性神经营养因子的临床试验所证明的， AAV-GDNF和AAV-AADC在帕金森病患者中的作用 在疾病中，患者甚至可以对具有非外源表位的AAV衍生蛋白产生免疫应答。因此，在本发明中， 重要的是鉴定减少分泌型AAV衍生的免疫细胞摄取的序列修饰， 颗粒蛋白前体由于分拣蛋白由脑中的各种抗原呈递细胞表达，因此阻断了细胞内的免疫应答。 前颗粒蛋白-分拣蛋白相互作用具有降低AAV-前颗粒蛋白载体的免疫原性的潜力。 Shreya Kashyap的拟议培训计划由她的项目导师Erik Roberson博士赞助。的 培训计划的总体目标是为PI作为一名医生的成功职业生涯提供坚实的基础 科学家一个基于转化方法的项目，同时关注疾病导向的发病机制， 是任何有抱负的医生科学家的理想培训环境。

Project Summary This application is for F30 support of Shreya Kashyap during her MD/PhD training. The scientific focus of this proposal is to determine the role of the progranulin C-terminal domain (CTD) on AAV-derived progranulin levels in vivo and the effectiveness of AAV-Progranulin gene therapy. Heterozygous loss of function mutations in the progranulin gene (GRN) are one cause of frontotemporal dementia (FTD), a devastating neurodegenerative disease characterized by social behavior and language deficits. Work from the lab of Dr. Erik Roberson, sponsor of the PI, has established the therapeutic potential of AAV-Progranulin gene therapy in progranulin-insufficient mouse models of FTD. The vector used for these experiments has a tag fused to the CTD that precludes progranulin's interaction with its canonical trafficking receptor, sortilin. These experiments show that the lysosomal localization and efficacy of AAV-Progranulin is independent of sortilin. However, the functional consequences of an exposed progranulin CTD, which allows for progranulin-sortilin binding, are still unknown. Notably, sortilin mediates the uptake and degradation of progranulin and sortilin ablation or inhibition boosts both brain and CSF progranulin levels in wild type and progranulin-insufficient mouse models. My preliminary experiments indicate that blocking the progranulin C-terminal domain boosts parenchymal levels of AAV-derived progranulin, and improves efficacy of AAV-Progranulin in rescuing lysosomal pathology and neuroinflammation. The clinical translation of AAV-Progranulin vectors with tags fused to the progranulin CTD is unlikely. Thereby, it is important to determine CTD sequence modifications that improve dosage and levels of AAV-Progranulin. Immune mediated clearance of AAV particles and AAV-transduced cells reduce the effectiveness of AAV- based gene therapies. Progranulin-deficient mice mount an immune response to AAV-murine progranulin, and it is unclear if FTD-GRN patients (who have one functioning progranulin allele) will mount an immune response to AAV-human progranulin. However, as evidenced by recent clinical testing of AAV-glial derived neurotrophic growth factor (AAV-GDNF) and AAV-amino acid decarboxylase (AAV-AADC) in patients with Parkinson's disease, patients can mount immune responses even to AAV-derived proteins with non-foreign epitopes. Thus, it is important to identify sequence modifications that reduce immune cell uptake of secreted AAV-derived progranulin. Because sortilin is expressed by various antigen presenting cells in the brain, blocking the progranulin-sortilin interaction has the potential to decrease the immunogenicity of AAV-Progranulin vectors. The proposed training plan for Shreya Kashyap is sponsored by her project mentor, Dr. Erik Roberson. The overall goal of the training plan is to provide the PI with a solid foundation for a successful career as a physician scientist. A project based both in translational approaches, while focused on a disease-oriented pathogenesis, is the ideal training environment for any aspiring physician scientist.