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

描述(申请人提供)：常染色体显性遗传性多囊肾病(ADPKD)的特征是受累肾脏中大量的囊肿进行性增大，导致大约一半的ADPKD患者发生肾功能衰竭。仅在美国，ADPKD造成的疾病负担估计就达到60万人，全球范围内超过1200万人。在美国，ADPKD患者的肾脏替代治疗费用每年超过20亿美元。在过去的15年里，我们对PKD的发病机制的了解取得了实质性的进展，取得了显著的发现，包括确定了致病基因，认识到体细胞第二步突变启动了囊性生长，发现纤毛/基底体复合体是一般肾囊性疾病和ADPKD的发病机制的焦点。尽管取得了这些重大进展，但我们对该病分子发病机制的了解仍然存在很大差距，特别是涉及到PC1/PC2受体通道复合体下游的即时信号时。这些差距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。要促进对多囊肾病潜在因素的更深入的科学了解，并提高人们对开发治疗方法的兴趣，关键的一步是对ADPKD的靶途径(S)实现令人信服和准确的定义。全面了解ADPKD的障碍是双重的。首先，PKD是一种影响发育的疾病，更重要的是影响肾脏和肝脏三维实体器官结构的维持。因此，基于二维或三维细胞培养的替代体外系统不足以发现PKD的中心通路。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，对于PKD基因下游未知途径的发现，目前还没有成功的策略。相反，通路的发现是基于检查已知通路，例如那些与增殖和平面极性相关的通路，作为PKD失调的候选通路。我们假设，多囊蛋白以一种尚未发现的新途径(S)发挥作用，该途径在完整的器官中最明显地发挥功能，而要发现这种途径，需要在整个哺乳动物生物体中采用无偏见的、表型驱动的向前遗传方法。目前的提案提出了一套强大而新颖的研究来实现这一目标。我们建议使用转座子介导的体细胞插入突变，基于一个独特的改良的iggyBac(PB)转座子系统来发现在野生型背景下导致小鼠肾脏囊变形成的激活突变。我们将使用通过ADPLD基因突变而对囊形成敏感的背景，对功能丧失突变进行同样的处理，Sec63。由于PB转位也可能在肾脏中产生微小肿瘤，我们将使用该系统来揭示肾脏中的致癌途径。最后，我们将使用这个系统来确定通过选择性地重新激活囊细胞中的多囊蛋白通路而产生的治疗期望。 公共卫生相关性：常染色体显性遗传性多囊肾病(ADPKD)在美国影响60万人，每年增加超过20亿美元的医疗费用。提高对这种疾病的分子基础的了解是走向治疗的最好方法。这项提议将使用一种新的最先进的小鼠肾脏遗传分析来发现ADPKD的潜在机制，这些机制以前并不为人所知，这将改善找到有效治疗该疾病的前景。

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.