Smooth Muscle Cell-Based Assesment and Therapy for Myopathic forms of CIPO

项目摘要/摘要 先天性肠动力障碍，如慢性假性肠梗阻(CIPO)和 巨结肠肠道动力低下(MMIH)是一种罕见且研究不足的疾病，具有 巨大的管理成本和不利的终身后果。这些疾病的一个主要原因是异常 平滑肌细胞(SMC)的功能。最近的基因研究发现了一种肌动蛋白的突变。 作为CIPO/MMIH的原因，该基因仅在肠肌(ACTG2)中表达，在很大一部分 案子。肌动蛋白与分子马达肌球蛋白相互作用，对细胞和肠道施加收缩力量。 SMC这些力量转化为蠕动运动，在肠道内混合消化的食物并逐渐 通过肠道将其移动。人们对这些突变的功能后果知之甚少 尚未在实验室中对平滑肌细胞进行研究，这在很大程度上是因为难以维持 稳定培养的人肠平滑肌组织的收缩表型。 最近，三项技术发展使得开始纠正致病因素的工作成为可能 突变。已经有可能创建一个逼真的自然肠道平滑肌模型，它包括 通过特殊的细胞培养技术，将SMC、神经胶质细胞、肠神经元和起搏器细胞混合在一起 取自体外培养的病人。此外，CRISPR/Cas9的首次令人兴奋的发现使 突变基因的特定校正。此外，生物工程师已经开发出一种新的高通量 测量单个电池产生的力的动态变化的平台，以测试 基因修正。 作为一个长期目标，这些技术承诺提供一种手段，为患有癌症的患者开发基于细胞的疗法 使用转基因SMC的CIPO。目前的建议是进行一项试点研究，以了解更多关于 用遗传学方法研究ACTG2突变对人SMC基因表达和收缩功能的影响 方法对携带ACTG2基因突变的人血管内皮细胞进行挽救，并检测其增殖和存活情况。 通过在小鼠体内植入SMC和其他细胞成分。 在这项研究完成后，我们预计将深入了解一项重要的 改变内脏平滑肌功能的单基因疾病和我们的发现可能会推动对 更常见的运动障碍和新的治疗方法。

PROJECT SUMMARY/ABSTRACT Congenital intestinal dysmotility disorders, such as chronic intestinal pseudo-obstruction (CIPO) and megacystis microcolon intestinal hypoperistalsis (MMIH), are rare and under-researched diseases having enormous management costs and adverse life-long outcomes. A major cause of these diseases is abnormal function of smooth muscle cells (SMCs). Recent genetic findings have identified mutations in a form of actin that is exclusively expressed in enteric muscle (ACTG2) as the cause of CIPO/MMIH in a large portion of cases. Actin interacts with the molecular motor myosin to exert contractile forces on cells, and in intestinal SMCs these forces translate into peristaltic movement that mixes digesting food in the intestine and gradually moves it way through the intestine. Little is known about the functional consequences of these mutations and have not been studied in the laboratory in smooth muscle cells largely due to the difficulty of maintaining the contractile phenotype of human intestinal smooth muscle tissue in stable cultures. Three technological developments have recently made it feasible to begin work on correcting disease-causing mutations. It has become possible to create a realistic model natural intestinal smooth muscle, which consists of a mixture of SMCs, glial cells, enteric neurons, and pacemaker cells, by special techniques of growing cells taken from patients in vitro. In addition the exciting discovery of CRISPR/Cas9 for the first time enables the specific correction of mutant genes. Furthermore, bioengineers have developed a new a high-throughput platform to measure dynamic changes in force generated by single cells, in order to test the effectiveness of genetic corrections. As a long-term goal these technologies promise a means to develop cell-based therapies for patients with CIPO using genetically modified SMCs. The current proposal is for a pilot study to learn more about the functional effects of ACTG2 mutations on human SMC gene expression and contractility, to use genetic methods to rescue human SMCs with ACTG2 mutations, and to evaluate proliferation and survival of modified SMCs and the other cellular components in vivo by implantation in mice. At the completion of this study, we anticipate insight into basic physiological consequences of an important monogenetic disorder that alters visceral smooth muscle function and our findings may propel further study of more common dysmotility aberrations and open new therapeutic approaches.