Development of Novel Tools for Gene Targeting in Smooth Muscle

描述（由申请人提供）：以异常平滑肌（SM）功能为特征的疾病，如膀胱过度活动、哮喘和肠道动力障碍，每年影响美国数千万人。治疗这种疾病的经济成本是巨大的，每年高达数百亿美元。尽管有如此巨大的医疗负担，SM生物学领域的研究，特别是内脏SM，已经落后于其他学科。这部分是由于缺乏作为现代生物医学研究支柱的遗传模型。 在这个修订后的申请中，我们提出了一个创新的策略，用于识别和验证内脏SM中Cre重组酶表达的候选驱动程序。我们相信，成功的示范选择性基因靶向SM，在器官特异性的方式，将代表SM生物学领域的重大突破，并将提供新的机会，SM的病理生理机制和翻译研究。 尽管已经报道了SM特异性靶向Cre，但这些模型在基本上所有含有平滑肌的组织中显示Cre介导的重组，并且不允许器官特异性基因靶向。此外，目前的策略通常依赖于编码SM收缩蛋白（如SM-MHC和SM 221）的启动子来实现Cre的SM特异性靶向。然而，这需要SM分化在这样的启动子活化之前已经发生。为了规避这些限制，我们提出了两种互补的方法。在目标1中，我们将利用我们小组最近未发表的数据，这些数据表明嘌呤能受体P2 rx 1的表达高度局限于膀胱SM，以产生P2 rx 1-Cre基因敲入和转基因小鼠系，并确定它们在器官选择性、SM特异性基因靶向中的实用性。在目标2中，我们将筛选新的候选启动子，从信息学分析预测将富集在内脏SM中，其细胞类型和器官特异性，以及其以器官特异性，SM特异性方式驱动Cre表达的能力。 在2年的项目期结束时，我们预计已经开发出一套全新的SM特异性基因靶向试剂，这将首次提供以器官特异性方式敲除（或敲入）涉及SM功能障碍的基因的机会。我们相信，这些基因“工具包”的增加将大大促进我们对SM生物学的理解。 公共卫生相关性：内脏中空器官（例如膀胱、肠道和气道）的机制和转化研究由于缺乏允许基因以组织特异性方式靶向平滑肌（SM）的遗传工具而受到严重限制。所提出的实验将产生一套全新的遗传修饰的小鼠品系，其中Cre重组酶在一种或多种SM特异性启动子的控制下以器官选择性方式在中空器官SM中表达。我们预计这些试剂将能够精确的空间和时间靶向涉及SM功能障碍的基因，从而提供更深入的了解SM发挥主导作用的疾病（膀胱肥大，肠梗阻，哮喘）的病理生理学。基于多年来已经有这种靶向方法的器官系统（例如大脑和心脏），我们相信我们提出开发的工具将为人类开发新的合理疗法铺平道路。

DESCRIPTION (provided by applicant): Diseases characterized by aberrant smooth muscle (SM) function, such as bladder over activity, asthma, and motility disorders of the gut, affect tens of millions of people in the US each year. The economic cost of treating such conditions is substantial and runs into the tens of billions of dollars annually. Despite this significant healthcare burden, research in the field of SM biology, especially visceral SM, has lagged behind that in other disciplines. This results in part from the paucity of genetic models that are the mainstay of modern biomedical research. In this revised application we propose an innovative strategy for the identification and verification of candidate drivers for Cre recombinase expression in visceral SM. We believe that successful demonstration of selective gene targeting in SM, in an organ-specific manner, would represent a major breakthrough in the field of SM biology and would provide new opportunities for mechanistic and translational investigation of SM pathophysiology. Although SM-specific targeting of Cre has been reported, such models display Cre-mediated recombination in essentially all smooth muscle-containing tissues, and do not allow for organ-specific gene targeting. Furthermore, current strategies typically rely on promoters encoding SM contractile proteins such as SM-MHC and SM221 to achieve SM-specific targeting of Cre. However, this requires SM differentiation to have occurred before such promoters are active. To circumvent these limitations, we propose two complementary approaches. In Aim 1, we will exploit recent unpublished data from our group showing that expression of the purinergic receptor P2rx1 is highly restricted to bladder SM, to generate P2rx1-Cre knock-in and transgenic mouse lines and determine their utility for organ-selective, SM-specific gene targeting. In Aim 2, we will screen novel, candidate promoters, predicted from informatics analysis to be enriched in visceral SM, for their cell type- and organ-specificity, and their ability to drive Cre expression in an organ-specific, SM-specific manner. At the end of the 2-year project period, we expect to have developed a completely novel suite of SM- specific gene-targeting reagents that will provide, for the first time, an opportunity to knock out (or knock in) genes implicated in SM dysfunction in an organ-specific manner. We believe these additions to the genetic 'tool-kit' will greatly facilitate advancements in our understanding of SM biology. PUBLIC HEALTH RELEVANCE: Mechanistic and translational studies of visceral hollow organs, such as the bladder, gut and airways have been severely limited by the lack of genetic tools that would allow genes to be targeted, in a tissue- specific manner, to smooth muscle (SM). The proposed experiments will create an entirely novel suite of genetically modified mouse lines in which Cre recombinase is expressed in hollow organ SM in an organ- selective manner under the control of one or more SM-specific promoters. We anticipate these reagents will enable precise spatial and temporal targeting of genes implicated in SM dysfunction, thereby providing greater insight into the pathophysiology of diseases where SM plays a dominant role (bladder hypertrophy, intestinal obstruction, asthma). Based on organ systems that have had such targeting approaches available for years (e.g. brain and heart), we believe the tools we proposed to develop will pave the way for development of novel, rational therapies in humans.