Wolter Justin M, James Lucas M, Boeshore Samantha L, Mao Hanqian, McCoy Eric S, Ryan Daniel F, Fragola Giulia, Taylor-Blake Bonnie, Stein Jason L, Zylka Mark J
UNC Neuroscience Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Department of Cell Biology and Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Commun Biol. 2025 Sep 2;8(1):1332. doi: 10.1038/s42003-025-08794-2.
Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of maternally-inherited UBE3A. In neurons, paternally-inherited UBE3A is silenced in cis by a long non-coding RNA called Ube3a-ATS. Here, we found that Neisseria meningitidis Cas9 with two mutations (D15A and H587A) in the nuclease domains (dNmCas9) can unsilence the dormant paternal Ube3a allele in mouse and human neurons when targeted to Snord115 snoRNA genes located in introns of Ube3a-ATS. Importantly, dNmCas9 disrupted Ube3a-ATS with a non-template bias and in the absence of a chromatin modifying domain, supporting a transcriptional interference mechanism. When packaged into an adeno-associated virus (AAV) vector, dNmCas9 exhibited dose-dependent Ube3a-ATS knock-down and paternal Ube3a unsilencing in vitro and in vivo. This vector also partially rescued the hind limb clasp phenotype when delivered to neonatal AS model mice. Collectively, our study underscores the potential of dCas9-based therapeutics without chromatin repression domains to mediate transcriptional downregulation.
天使综合征（AS）是一种由母系遗传的UBE3A缺失引起的使人衰弱的神经发育障碍。在神经元中，父系遗传的UBE3A通过一种名为Ube3a-ATS的长链非编码RNA在顺式作用下被沉默。在此，我们发现核酸酶结构域有两个突变（D15A和H587A）的脑膜炎奈瑟菌Cas9（dNmCas9），当靶向位于Ube3a-ATS内含子中的Snord115 snoRNA基因时，可使小鼠和人类神经元中处于休眠状态的父系Ube3a等位基因去沉默。重要的是，dNmCas9以非模板偏向性方式破坏Ube3a-ATS，且在没有染色质修饰结构域的情况下，这支持了一种转录干扰机制。当被包装到腺相关病毒（AAV）载体中时，dNmCas9在体外和体内均表现出剂量依赖性的Ube3a-ATS敲低以及父系Ube3a去沉默。当将该载体递送至新生AS模型小鼠时，还部分挽救了后肢紧握表型。总的来说，我们的研究强调了不含染色质抑制结构域的基于dCas9的疗法介导转录下调的潜力。
