Sastre Danuta, Zafar Faria, Torres C Alejandra Morato, Piper Desiree, Kirik Deniz, Sanders Laurie H, Qi Stanley, Schüle Birgitt
Stanford University School of Medicine, Department of Pathology, Stanford, CA 94305, U.S.A.
San Jose State University, Department of Biological Sciences, San Jose, 95192 CA, U.S.A.
bioRxiv. 2023 Jan 24:2023.01.24.525105. doi: 10.1101/2023.01.24.525105.
Parkinson's disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept for precision medicine by targeting the gene promoter. The CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.
帕金森病（PD）是最常见的神经退行性疾病之一，但尚无疾病修饰疗法在临床转化中取得成功，这是一个重大的未满足医疗需求。一个有前景的靶点是α-突触核蛋白或其聚集形式，它在帕金森病患者大脑中以路易小体的形式积累。虽然尚不完全清楚哪种α-突触核蛋白物种与疾病相关，但遗传性形式中α-突触核蛋白的单纯过表达会导致神经退行性变。为了特异性地解决α-突触核蛋白的基因调控问题，我们开发了一种基于与转录抑制结构域Krüppel相关盒融合的核酸酶失活Cas9（dCas9）的CRISPR干扰（CRISPRi）系统，以在转录水平上可控地抑制α-突触核蛋白的表达。我们在启动子区域筛选了单向导（sg）RNA，并鉴定出了几种能在不同水平介导α-突触核蛋白下调的sgRNA。在一名基因组三倍体患者的诱导多能干细胞衍生的神经元培养物中，CRISPRi介导的α-突触核蛋白下调通过降低氧化应激和线粒体DNA损伤实现了功能恢复。我们的结果证明了通过靶向基因启动子实现精准医学的概念验证。CRISPRi方法为理解α-突触核蛋白下调的安全水平提供了一个新模型，并为帕金森病及相关α-突触核蛋白病提供了一种新的治疗策略。
