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

项目摘要 此申请是为Shreya Kashyap在医学博士/博士培训期间提供F30支持。其科学关注点在于 建议确定原颗粒蛋白C末端结构域(CTD)在AAV衍生的原颗粒蛋白水平上的作用 体内和AAV-原颗粒基因治疗的效果。功能突变的杂合性丢失 原颗粒蛋白基因(GRN)是额颞叶痴呆(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。发起人埃里克·罗伯逊博士的实验室工作 已经确立了AAV-原颗粒基因治疗前颗粒缺乏的治疗潜力 FTD小鼠模型。用于这些实验的载体有一个与CTD融合的标签，该标签排除了 原颗粒与其典型的运输受体山梨素的相互作用。这些实验表明， AAV-原颗粒的溶酶体定位和疗效不依赖于山梨素。然而，功能性的 暴露的原颗粒蛋白CTD允许原颗粒蛋白-山梨素结合，其后果尚不清楚。 值得注意的是，山梨素介导原颗粒的摄取和降解，而山梨素的消融或抑制可促进两者的摄取和降解 野生型和原颗粒缺乏小鼠模型的脑和脑脊液原颗粒水平。我的初选 实验表明，阻断原颗粒蛋白C末端结构域可提高AAV来源的实质水平 原颗粒，并提高AAV-原颗粒在挽救溶酶体病理和神经炎症方面的疗效。 带有融合到原颗粒CTD的标签的AAV-原颗粒载体的临床翻译是不可能的。从而， 重要的是要确定CTD序列的修改，以提高AAV-PROGROGIN的剂量和水平。 免疫介导的AAV颗粒和AAV转导细胞的清除降低了AAV- 以基因疗法为基础。原颗粒缺乏的小鼠对AAV-小鼠原颗粒产生免疫反应，并且 目前尚不清楚FTD-GRN患者(有一个功能前颗粒等位基因)是否会产生免疫反应 到AAV-人类前颗粒。然而，最近AAV-胶质源性神经营养的临床测试证明 帕金森病患者血清中生长因子(AAV-GDNF)和AAV-氨基酸脱羧酶(AAV-AADC)的变化 对于疾病，患者甚至可以对具有非外来表位的甲型肝炎病毒衍生蛋白产生免疫反应。因此， 确定减少免疫细胞摄取分泌型AAV来源的序列修饰是很重要的 普罗米林。因为山梨素是由大脑中的各种抗原提呈细胞表达的，所以阻断了 前颗粒蛋白-山梨素相互作用有可能降低AAV-前颗粒蛋白载体的免疫原性。 Shreya Kashyap拟议的培训计划是由她的项目导师Erik Roberson博士赞助的。这个 培训计划的总体目标是为PI作为医生的成功职业生涯提供坚实的基础 科学家。这是一个以翻译方法为基础的项目，同时专注于以疾病为导向的发病机制， 是任何有抱负的内科科学家的理想训练环境。

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.