A forward genetic screen for PKD pathways in mice using the PiggyBac transposon

描述（由申请人提供）：常染色体显性多囊肾病（ADPKD）的特点是受累肾脏中大量囊肿进行性扩大，导致约一半的ADPKD患者肾功能衰竭。仅在美国，ADPKD的疾病负担估计就有60万人，而在全球范围内，这一数字超过了1200万。在美国，ADPKD患者肾脏替代治疗的费用每年超过20亿美元。在过去的15年里，我们对PKD发病机制的理解取得了实质性的进展，包括致病基因的鉴定，体细胞第二步突变引发囊肿生长的认识，以及纤毛/基底体复合体是肾囊性疾病发病机制的焦点，特别是ADPKD。尽管取得了这些重大进展，但我们对该疾病的分子发病机制的了解仍然存在很大差距，特别是当涉及到PC1/PC2受体通道复合物下游的直接信号时。这些空白削弱了我们全面了解ADPKD的能力，并直接限制了我们合理有效地针对ADPKD进行治疗的能力。促进对多囊肾病潜在因素的更深入的科学理解和促进对开发治疗方法的兴趣的关键一步是实现对ADPKD靶通路的令人信服和精确的定义。实现对ADPKD的全面理解的障碍是双重的。首先，PKD是一种影响肾脏和肝脏的发育，更重要的是影响三维实体器官结构维持的疾病。因此，基于二维或三维细胞培养的代理离体系统不足以发现PKD的核心途径。在这样的系统中，囊性表型的相关读数，如果存在的话，是未知的。其次，目前还没有成功的策略来发现PKD基因下游的未知途径。相反，途径发现是基于对已知途径的研究，例如与增殖和平面极性相关的途径，作为PKD失调的候选者。我们假设多囊毒素在一个尚未被发现的新途径中起作用，该途径在完整的器官中最明显地起作用，并且需要在整个哺乳动物生物体中进行无偏见的、表型驱动的前向遗传方法的发现。目前的建议提出了一套强大而新颖的研究来实现这一目标。我们建议使用基于独特修饰的PiggyBac （PB）转座子系统的转座子介导的体细胞插入突变来发现野生型背景下导致小鼠肾脏囊肿形成的激活突变。我们将使用ADPLD基因Sec63突变对囊肿形成敏感的背景对功能丧失突变做同样的研究。由于PB转位也可能在肾脏中产生微肿瘤，我们也将使用该系统来揭示肾脏的致癌途径。最后，我们将使用该系统来确定囊肿细胞中多囊蛋白途径选择性再激活所产生的治疗预期。

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of numerous cysts in affected kidneys that lead to renalfailure in about half of ADPKD patients. The disease burden from ADPKD in U.S. alone is estimated at 600,000 individuals and worldwide it exceeds 12 million. The costs of renal replacement therapy in ADPKD patients in the US exceeds $2 billion annually. The past 15 years have seen substantial progress in our understanding of the pathogenesis of PKD with notable discoveries including the identification of the causative genes, the recognition that somatic second step mutations initiate cyst growth and the discovery that the cilia/basal body complex is the focal point of the pathogenesis of renal cystic diseases in general, and of ADPKD specifically. Despite these major advances, there remain substantial gaps in our knowledge of the molecular pathogenesis of the disease, particular when it comes to the immediate signals downstream of the PC1/PC2 receptor channel complex. These gaps undermine our ability to achieve a comprehensive understanding of ADPKD and directly limit our ability to rationally and effectively target ADPKD for therapy. A critical step for promoting deeper scientific understanding of the factors underlying polycystic kidney diseases and for promoting interest in efforts to develop therapies, is to achieve a compelling and precise definition of the target pathway(s) in ADPKD. The impediments to achieving a comprehensive understanding of ADPKD are two-fold. First, PKD is a disease affecting the development and, more importantly, the maintenance of three dimensional solid organ structure in the kidney and liver. As such, surrogate ex vivo systems based in two or three dimensional cell culture are inadequate for discovery of pathways central to PKD. The relevant readout for the cystic phenotype in such systems, if it exists, is not known. Second, there has been no strategy that has been successful for unknown pathway discovery downstream of the PKD genes. Rather, pathway discovery has been based on examining known pathways, such as those associated with proliferation and planar polarity, as candidates for dysregulation in PKD. We hypothesize that the polycystins act in an as yet undiscovered novel pathway(s) that is most clearly functional in intact organs and the discovery of which requires unbiased, phenotypically-driven forward genetic approaches in whole mammalian organisms. The current proposal puts forth a powerful and novel set of studies to achieve this goal. We propose to use transposon mediated somatic insertional mutagenesis based on a uniquely modified PiggyBac (PB) transposon system to discover activating mutations on a wild type background that result in cyst formation in the mouse kidney. We will do the same for loss-of-function mutations using a background sensitized to cyst formation by mutations in the ADPLD gene, Sec63. Since PB transposition may also yield micro-tumors in the kidney, we will use this system to uncover oncogenic pathways in the kidney as well. Finally, we will use this system to define the therapeutic expectations resulting from selective reactivation of the polycystin pathways in cyst cells. PUBLIC HEALTH RELEVANCE: Autosomal dominant polycystic kidney disease (ADPKD) affects 600,000 individuals in the US adds over $2 billion to health care costs annually. Improved understanding of the molecular basis for this disease is the best means of moving toward treatment. This proposal will use a novel state-of-the-art genetic analysis in mouse kidneys to discover mechanisms underlying ADPKD that have not been previously known and which will improve the prospects for finding effective treatments for the disease.