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In Aim 1, we will exploit recent unpublished data from our group showing that expression of the purinergic receptor P2rx1 is highly restricted to bladder SM, to generate P2rx1-Cre knock-in and transgenic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.
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.