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

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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.