Design, Synthesis and Efficacy of New Small Molecule Therapeutics to Impede Myotonic Dystrophy

项目摘要 强直性肌营养不良1型(DM1)是成人发作性肌营养不良症的主要原因，但没有有效的治疗方法 DM1患者可采用治疗策略。家长对这项补充计划的资助旨在 了解强直性肌营养不良1型(DM1)的发病机制并寻找可能的治疗方法 治疗这种多系统疾病。DM1是由3‘非翻译区(UTR)中的CTG重复序列引起的 营养不良症肌强直蛋白激酶(DMPK)基因。当转录时，扩展的重复序列隔离 肌盲样蛋白(MBNL)，一种选择性剪接调节蛋白家族，破坏其正常细胞 拼接功能。我们的团队之前已经发现了拯救分子的现有小分子 DM1的特征，如纠正异常剪接和核核灶减少，现在已经 设计了一系列新的改性多环化合物(MPC)进行评价。虽然我们目前 研究这些新的MPC的治疗潜力和作用机制，初步数据表明 它们通过减少有毒RNA的转录和/或增加表达来发挥作用 MBNL蛋白。本附录的项目侧重于互补性研究，寻求更好地 了解MBNL蛋白、CUG扩展RNA和全基因组剪接靶标之间的相互作用 通过各种人工合成的MBNL蛋白的产生、分析和评估。伯格伦德实验室已经 之前产生了几个合成的MBNL蛋白，并证明这些蛋白显示出改进或 改变了剪接活动。这些数据表明，人工合成的MBNL有可能被用作 研究剪接调控以及糖尿病的蛋白质疗法。Brianna的项目将延长并 通过产生更多的合成MBNL蛋白并探测其结构和功能来利用这些研究 通过体外生化分析和转录研究对DM1中RNA GOF的影响 细胞培养。这个为期一年的毕业后项目将提供以下方面的研究和职业发展技能 一个中心研究项目旨在：(1)在体外识别和表征靶结合特异性 强直性肌营养不良1型扩展RNA背景下的工程合成MBNL；以及(2)确定 基因工程合成MBNL对DM1患者体内转录的特异性和治疗潜力 衍生的细胞系。确定合成的MBNL蛋白的功能和整体转录影响将 加强我们对糖尿病疾病生物学的了解，同时提供关键信息以改善他们的 治疗DM1和其他可能与剪接相关的重复序列扩张性疾病的治疗潜力。

Project Abstract Myotonic dystrophy type 1 (DM1) is the leading cause of adult-onset muscular dystrophy, yet no effective treatment strategies are available to DM1 patients. The parental grant to this supplement project is aimed at understanding the disease mechanisms of myotonic dystrophy type 1 (DM1) and identifying potential therapeutics for this multisystemic disease. DM1 is caused by a CTG expansion repeat in the 3’ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. When transcribed, the expanded repeats sequester muscleblind-like (MBNL) proteins, a family of alternative splicing regulators, disrupting their normal cellular splicing function. Our group has previously identified existing small molecules that rescue the molecular hallmarks of DM1, such as correction of aberrant splicing and reduction of ribonuclear foci and have now designed a series of new modified polycyclic compounds (MPCs) for evaluation. While we are currently investigating the therapeutic potential and mechanism of action of these new MPCs, preliminary data suggests that they operate through either reduction of the transcription of the toxic RNA and/or increases the expression of MBNL proteins. The project for this supplement is focused on complementary studies that seek to better understand the interaction between MBNL proteins, CUG expansion RNAs, and genome-wide splicing targets through the generation, analysis, and evaluation of various synthetic MBNL proteins. The Berglund lab has previous generated several synthetic MBNL proteins and demonstrated that these proteins show improved or altered splicing activity. These data suggested that synthetic MBNL may have the potential to be used as tools for investigating splicing regulation as well as for protein therapeutics for DM. Brianna’s project will extend and leverage these studies by generating additional synthetic MBNL proteins and probing their structure and function through in vitro biochemical assays and their impact on the RNA GOF in DM1 through transcriptomic studies in cell culture. This one-year post-baccalaureate project will provide research and career development skills around a central research project aim at: (1) identifying and characterizing in vitro the target engagement specificity of engineered synthetic MBNL in the context of myotonic dystrophy type 1 expansion RNAs; and (2) determining the in vivo transcriptomic specificity and therapeutic potential of engineered synthetic MBNL in DM1 patient- derived cell lines. Determining the functional and global transcriptomic impacts of synthetic MBNL proteins will enhance our understanding of the disease biology of DM while provide critical information to improve their therapeutic potential for the treatment of DM1 and potentially other splicing-related repeat expansion diseases.