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PD脑路易小体中α-突触核蛋白（SNCA编码）蛋白聚集体的积累。罕见的SNCA点突变和基因组复制与家族性PD病例有关，具有高外显率，因此构成PD的主要遗传危险因素。本研究方案的关键途径是通过在SNCA位点进行表观基因组编辑来沉默SNCA的表达。因此，PD脑病理应减弱和细胞和组织的稳态恢复。我们将应用基于Sun-Tag和DNMT3A/3L的先进表观基因组编辑系统，这将允许在SNCA调控区域的不同目标区域进行非常精确、强大和稳定的DNA甲基化递送。随后，复杂的细胞系和动物模型将使我们能够研究SNCA沉默对PD症状的影响。该项目的具体目标是：1。靶向DNA甲基化抑制SNCA表达的EpiEditors的设计和生成。优化EpiEditors，最大限度地实现位点特异性编辑、表观基因组编辑的长期稳定性和最大限度地减少脱靶效应。成功表达epieditors的病毒载体的生成。应用人类细胞培养模型在细胞培养水平上评估EpiEditing在减轻pd相关病理方面的功效。应用EpiEditors在BAC SNCA小鼠和大鼠模型中评估SNCA启动子甲基化对SNCA基因表达、aSYN病理和行为的影响。先进的EpiEditor系统的开发和应用，具有精确的时间控制区域特异性神经上皮编辑。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研究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.