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.

项目总结/摘要 先天性肠动力障碍，如慢性假性肠梗阻（锡波）和 巨囊肿性小结肠肠下垂（MMIH）是一种罕见的研究不足的疾病， 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不正常的 平滑肌细胞（SMC）的功能。最近的遗传学研究发现， 它只在肠肌（ACTG 2）中表达，是大部分锡波/MMIH的原因。 例肌动蛋白与分子运动肌球蛋白相互作用，对细胞施加收缩力， SMC这些力量转化为蠕动运动，混合消化食物在肠道，并逐渐 让它穿过肠道人们对这些突变的功能性后果知之甚少， 尚未在实验室中在平滑肌细胞中进行研究，主要是由于难以维持 稳定培养的人肠平滑肌组织的收缩表型。 最近有三项技术发展使开始纠正致病因素的工作成为可能。 突变。它已成为可能，以创造一个现实的模型天然肠平滑肌，其中包括 平滑肌细胞，神经胶质细胞，肠神经元和起搏细胞的混合物，通过特殊的细胞生长技术， 从体外培养的病人身上提取。此外，令人兴奋的CRISPR/Cas9首次发现， 突变基因的特异性校正。此外，生物工程师已经开发出一种新的高通量 平台来测量单细胞产生的力的动态变化，以测试 基因矫正 作为一个长期目标，这些技术有望为患有癌症的患者开发基于细胞的疗法。 锡波使用转基因SMC。目前的建议是进行一项试验性研究， ACTG 2突变对人SMC基因表达和收缩性的功能影响，使用遗传学方法， 方法拯救具有ACTG 2突变的人SMC，并评估修饰的SMCs的增殖和存活。 SMC和其他细胞成分通过植入小鼠体内。 在这项研究完成后，我们预计深入了解一个重要的生理后果， 单基因疾病改变内脏平滑肌功能，我们的研究结果可能会推动进一步的研究， 更常见的运动障碍畸变和开放的新的治疗方法。