Design and use of EpiEditors to mitigate alpha-synuclein overload in Parkinson’s disease and related synucleinopathies

帕金森病（PD）是最常见的神经退行性运动障碍。这种复杂疾病的标志性神经病理学特征是α-突触核蛋白（由SNCA编码）蛋白聚集体在PD脑中的路易体中的积累。罕见的SNCA点突变和基因组倍增与高突变率的家族性PD病例相关，因此构成PD的主要遗传危险因素。本研究方案的关键方法是通过SNCA位点的表观基因组编辑沉默SNCA表达。因此，PD脑病理应该被减弱，细胞和组织的稳态恢复。我们将应用基于Sun-Tag和DNMT 3A/3L的先进表观基因组编辑系统，这将允许在SNCA调控区的不同靶区域中非常精确、强大和稳定地递送DNA甲基化。之后，我们将通过成熟的细胞系和动物模型来研究SNCA沉默对PD症状的影响。设计和生成用于通过靶向DNA甲基化抑制SNCA表达的EpiEditors.优化表观编辑器以最大化位点特异性编辑、表观基因组编辑的长期稳定性和最小化脱靶效应。产生表达成功的EpiEditor的病毒载体。应用人类细胞培养模型评估EpiEditing在细胞培养水平缓解PD相关病理学的疗效. EpiEditors在BAC SNCA小鼠和大鼠模型中的应用，以评估SNCa启动子甲基化对SNCA基因表达、aSYN病理学和行为的影响。开发和应用先进的EpiEditor系统，用于精确时间控制的区域特异性神经表观编辑。该项目的成功工作取决于Jeltsch和Schulze-Hentrich研究小组的密切合作，利用他们在表观基因组编辑和PD调查方面的互补和成熟的专业知识。

Parkinson’s Disease (PD) represents the most common neurodegenerative movement disorder. The hallmark neuropathologic feature of this complex disease is the accumulation of α-synuclein (encoded by SNCA) protein aggregates in Lewy bodies in the PD brain. Rare SNCA point mutations and genomic multiplications are linked to familial PD cases with high penetrance and therefore constitute major genetic risk factors for PD.The key approach of this research proposal is to silence SNCA expression by epigenome editing at the SNCA locus. Thereby, PD brain pathology should be attenuated and cell and tissue homeostasis restored. We will apply advanced epigenome editing systems based on a Sun-Tag and DNMT3A/3L, which will allow very precise, strong, and stable delivery of DNA methylation in different target region in the SNCA regulatory regions. Afterwards, sophisticated cell line and animal models will enable us to investigate the effect of the SNCA silencing on PD symptoms.The specific objectives of this project are to:1. Design and generation of EpiEditors for repression of SNCA expression by targeted DNA methylation.2. Optimization of EpiEditors to maximize locus specific editing, long-term stability of epigenome editing and minimize off-target effects.3. Generation of viral vectors expressing successful EpiEditors.4. Application of human cell culture models to assess the efficacy of EpiEditing in the alleviation of PD-associated pathology at cell culture level.5. Application of EpiEditors in a BAC SNCA mouse and rat model to assess effects of the SNCa promoter methylation on SNCA gene expression, aSYN pathology and behavior.6. Development and application of advanced EpiEditor systems for region specific neuroepiediting with precise temporal control.Successful work in the project depends on the close cooperation of the Jeltsch and Schulze-Hentrich research groups exploiting their complementary and well established expertise in Epigenome editing and investigation of PD.