O'Donovan Bernadette, Rittiner Joseph, Upadhya Suraj, Hodgson Dellila, Kantor Boris, Chiba-Falek Ornit
Division of Translational Brain Sciences, Department of Neurology, Duke University School of Medicine; Durham, NC 27710, USA.
Center for Genomic and Computational Biology, Duke University School of Medicine; Durham, NC 27710, USA.
bioRxiv. 2025 Jun 17:2025.06.16.659948. doi: 10.1101/2025.06.16.659948.
Alpha-synuclein (SNCA) overexpression is implicated in Parkinson's disease (PD) pathogenesis, making SNCA downregulation a promising therapeutic strategy. We developed a -targeted epigenome therapy using an lentiviral vector (LV) carrying deactivated CRISPR/(d)Cas9, gRNA targeted at -intron1, and either the catalytic domain of DNA-methyltransferase3A (DNMT3A), or a synthetic repressor molecule of Krüppel-associated box (KRAB)/ methyl CpG binding protein 2 transcription repression domain (MeCp2-TRD). Therapeutic efficacy was evaluated in a new PD mouse model, generated with an adeno-associated viral vector carrying an engineered minigene comprised of the human (h)A53T- expressed via the human native regulatory region. Both therapeutic vectors reduced expression of α-synuclein in the substantia nigra (SN), with LV/dSaCas9- KRAB-MeCP2(TRD) demonstrating greater repression. LV/dSaCas9- KRAB-MeCP2(TRD) also significantly reduced pathological α-synuclein aggregation and phosphorylation (Ser129), and preserved tyrosine hydroxylase expression in the SN and the striatum. Behavioral analysis following LV/dSaCas9- KRAB-MeCP2(TRD) injection, showed significant improvement in motor deficits characteristic of our PD-mouse model. Safety assessments found normal blood counts, serum chemistry, and weights. Collectively, we provide proof-of-concept for our -targeted epigenome therapy in a PD-mouse model. Our results support the system's therapeutic potential for PD and related synucleinopathies and establish the foundation for further preclinical studies toward investigational new drug enablement.
α-突触核蛋白（SNCA）的过表达与帕金森病（PD）的发病机制有关，因此下调SNCA成为一种有前景的治疗策略。我们开发了一种靶向表观基因组疗法，使用携带失活CRISPR/Cas9（dCas9）的慢病毒载体（LV）、靶向SNCA内含子1的引导RNA（gRNA），以及DNA甲基转移酶3A（DNMT3A）的催化结构域或Krüppel相关盒（KRAB）/甲基化CpG结合蛋白2转录抑制结构域（MeCp2-TRD）的合成阻遏分子。在一种新的PD小鼠模型中评估了治疗效果，该模型由携带经工程改造的小基因的腺相关病毒载体构建而成，该小基因由人源天然调控区表达的人（h）A53T组成。两种治疗载体均降低了黑质（SN）中α-突触核蛋白的表达，其中LV/dSaCas9-KRAB-MeCP2（TRD）表现出更强的抑制作用。LV/dSaCas9-KRAB-MeCP2（TRD）还显著减少了病理性α-突触核蛋白的聚集和磷酸化（Ser129），并保留了SN和纹状体中酪氨酸羟化酶的表达。注射LV/dSaCas9-KRAB-MeCP2（TRD）后的行为分析表明，我们的PD小鼠模型的运动缺陷有显著改善。安全性评估发现血细胞计数、血清化学指标和体重均正常。总的来说，我们为靶向SNCA的表观基因组疗法在PD小鼠模型中提供了概念验证。我们的结果支持了该系统对PD及相关突触核蛋白病的治疗潜力，并为进一步开展研究性新药临床前研究奠定了基础。
