Development of Novel Tools for Gene Targeting in Smooth Muscle

描述(申请人提供)：以异常的平滑肌(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，每年在美国影响数千万人。治疗这种疾病的经济成本是巨大的，每年高达数百亿美元。尽管有这种巨大的医疗负担，但SM生物学领域的研究，特别是内脏SM，一直落后于其他学科。这在一定程度上是由于缺乏作为现代生物医学研究支柱的遗传模型。在这个修订的申请中，我们提出了一种创新的策略，用于识别和验证内脏SM中Cre重组酶表达的候选驱动因素。我们相信，在SM中成功地以器官特异性的方式进行选择性基因打靶，将代表着SM生物学领域的重大突破，并将为SM的机制和翻译研究提供新的机会。尽管已经报道了针对Cre的SM特异性靶向，但这些模型基本上在所有含有平滑肌的组织中都显示了Cre介导的重组，并且不允许器官特异性的基因靶向。此外，目前的策略通常依赖于编码SM-MHC和SM221等SM收缩蛋白的启动子来实现SM对Cre的特异性靶向。然而，这需要在这些启动子激活之前发生SM分化。为了绕过这些限制，我们提出了两种互补的方法。在目标1中，我们将利用我们团队最近未发表的数据，表明嘌呤能受体P2rx1在膀胱SM中的表达高度受限，建立P2rx1-Cre敲入和转基因小鼠系，并确定它们对器官选择性和SM特异性基因靶向的作用。在目标2中，我们将筛选从信息学分析中预测富含内脏SM的新的候选启动子，以了解它们的细胞类型和器官特异性，以及它们以器官特异性、SM特异性的方式驱动Cre表达的能力。在为期两年的项目期结束时，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)与SM功能障碍有关的基因的机会。我们相信，这些对遗传“工具包”的补充将极大地促进我们对SM生物学的理解。 与公共卫生相关：对内脏中空器官，如膀胱、肠道和呼吸道的机械学和翻译研究一直受到严重限制，因为缺乏基因工具，可以以组织特异性的方式将基因靶向到平滑肌(SM)。拟议的实验将创造一套全新的转基因小鼠品系，其中Cre重组酶在一个或多个SM特异性启动子的控制下，以器官选择性的方式在中空器官SM中表达。我们预计，这些试剂将能够对SM功能障碍所涉及的基因进行精确的时空靶向，从而更好地了解SM起主导作用的疾病(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)，我们相信我们提议开发的工具将为开发新的、合理的人类疗法铺平道路。

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.