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

基于平滑肌细胞的 CIPO 肌病评估和治疗

• 批准号: 9263710
• 负责人: James C Dunn
• 金额: $ 23.12万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9263710
• 关键词: Actins; Address; Agonist; Autologous; Biomedical Engineering; Birth; CRISPR/Cas technology; Cell Culture System; Cell Culture Techniques; Cell Line; Cell Therapy; Cell physiology; Cells; Characteristics; Child; Chronic; DNA Sequence Alteration; Data; Development; Disease; Dominant-Negative Mutation; Effectiveness; Enteral; Exhibits; Food; Foundations; Future; Gene Expression; Genes; Genetic; Genotype; Goals; Human; Implant; In Vitro; Injectable; Interstitial Cell of Cajal; Intestinal Pseudo-Obstruction; Intestines; Laboratories; Learning; Life; Literature; Measures; Methods; Modeling; Molecular Motors; Movement; Mus; Muscle; Muscle Cells; Muscle function; Mutate; Mutation; Myopathy; Myosin ATPase; Neuroglia; Neurons; Organoids; Outcome; Patients; Phenotype; Physiological; Pilot Projects; Protein Isoforms; Proteins; Publishing; Research; Sampling; Smooth Muscle; Smooth Muscle Actin Staining Method; Smooth Muscle Myocytes; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Translating; Variant; Visceral; Work; actin 2; base; cell preparation; cohort; cost; disease-causing mutation; dosage; exome sequencing; fetal; genetically modified cells; implantation; in vivo; induced pluripotent stem cell; innovation; insight; motility disorder; mutant; nanomaterials; nodal myocyte; novel; novel therapeutic intervention; overexpression; scaffold; tissue culture; tissue/cell culture

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.

项目概要/摘要 先天性肠道动力障碍，例如慢性假性肠梗阻 (CIPO) 和 巨囊虫小结肠肠蠕动减退 (MMIH) 是一种罕见且研究不足的疾病， 巨大的管理成本和不利的终生结果。导致这些疾病的一个重要原因是异常 平滑肌细胞（SMC）的功能。最近的遗传学发现发现了肌动蛋白形式的突变 专门在肠肌 (ACTG2) 中表达，是大部分患者中 CIPO/MMIH 的病因 案例。肌动蛋白与分子运动肌球蛋白相互作用，对细胞和肠道施加收缩力 SMC 将这些力转化为蠕动运动，在肠道中混合消化食物并逐渐 它通过肠道移动。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，主要是因为维持平滑肌细胞的困难 稳定培养物中人肠平滑肌组织的收缩表型。 最近的三项技术发展使得开始纠正致病因素成为可能 突变。创建真实的天然肠道平滑肌模型已成为可能，该模型由 通过特殊的细胞生长技术，由 SMC、神经胶质细胞、肠神经元和起搏细胞混合而成 取自体外患者。此外，令人兴奋的 CRISPR/Cas9 首次发现使得 突变基因的特异性校正。此外，生物工程师开发了一种新的高通量 平台测量单细胞产生的力的动态变化，以测试其有效性 基因修正。 作为长期目标，这些技术有望为患有以下疾病的患者开发基于细胞的疗法： CIPO 使用转基因 SMC。目前的建议是进行试点研究，以了解更多关于 ACTG2 突变对人类 SMC 基因表达和收缩性的功能影响，利用遗传 拯救具有 ACTG2 突变的人类 SMC 的方法，并评估修饰后的增殖和存活 通过植入小鼠体内的 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重要的生理后果 改变内脏平滑肌功能的单基因疾病，我们的发现可能会推动进一步的研究 更常见的运动障碍畸变并开辟新的治疗方法。