Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA.
Department of Urology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, PR China; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA.
CRISPR J. 2020 Dec;3(6):562-572. doi: 10.1089/crispr.2020.0077.
Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the experiment, single-guide RNAs targeting myostatin () were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the experiment, a total of 14 female lean ZUC-Lepr 186 and 14 fatty ZUC-Lepr 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis . , the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis and It also improved urethral continence in the OA-SUI rat model.
超重女性易患肥胖相关压力性尿失禁（OA-SUI），而对于这种常见的泌尿科疾病尚无明确的医学治疗方法。本研究旨在验证以下假设，即恢复尿道横纹肌（stM）和盆底肌的再生疗法可能代表一种有价值的治疗方法。为此实验，使用靶向肌生成抑制素（myostatin，MSTN）的单指导 RNA 进行 CRISPRi/dCas9-Kruppel 相关盒（KRAB）介导的基因沉默。在实验中，将 14 只瘦 ZUC-Lepr 186 雌性大鼠和 14 只肥胖 ZUC-Lepr 185 大鼠分别作为对照和 CRISPRi-MSTN 处理组。结果表明，慢病毒介导的 MSTN CRISPRi/dCas9-KRAB 表达导致大鼠 L6 成肌细胞中 MSTN 的持续下调，并显著增强了成肌作用。此外，尿道括约肌内注射慢病毒-MSTN sgRNA 和慢病毒-dCas9-KRAB 显著增加了漏尿点压力、stM 层厚度、stM 与平滑肌的比例以及神经肌肉接头的数量。CRISPRi/dCas9-KRAB 介导的基因沉默下调 MSTN 显著增强了成肌作用，并改善了 OA-SUI 大鼠模型中的尿道控尿功能。
