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

使用 PiggyBac 转座子对小鼠 PKD 通路进行正向遗传筛选

• 批准号: 7829572
• 负责人: STEFAN SOMLO
• 金额: $ 50万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7829572
• 关键词: Affect; Alleles; Arts; Autosomal Dominant Polycystic Kidney; Biochemical; Cell Count; Cell Culture Techniques; Cell Nucleus; Cells; Cilia; Complex; Coupled; Cyst; Cystic Kidney Diseases; Cystic kidney; DNA; Development; Disease; Dose; Embryonic Development; Event; Generations; Genes; Genetic; Genetic Screening; Goals; Growth; Health Care Costs; Individual; Insertional Mutagenesis; Kidney; Knowledge; Lead; Liver; Maintenance; Mediating; Membrane; Molecular; Mus; Mutagenesis; Mutation; Oncogenes; Oncogenic; Organ; Organism; Outcome; Paste substance; Pathogenesis; Pathway interactions; Patients; Phenotype; Physiological; Polycystic Kidney Diseases; Principal Investigator; Proteins; Renal Replacement Therapy; Renal carcinoma; Renal tubule structure; Screening procedure; Sensitivity and Specificity; Signal Transduction; Solid; Staging; Structure; System; Tamoxifen; Testing; Therapeutic; Time; Tissues; Transgenes; base; burden of illness; cost; effective therapy; expectation; gene discovery; genetic analysis; improved; in vivo; interest; kinetosome; loss of function; loss of function mutation; mouse model; mutant; novel; programs; public health relevance; receptor; response; therapy development; transcription factor; tumor

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.

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囊肿的进行性增大，导致约一半的ADPKD患者出现肾衰竭。仅在美国，ADPKD的疾病负担估计为60万人，全球超过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发病机制的理解取得了实质性进展，有了显著的发现，包括致病基因的鉴定，认识到体细胞第二步突变启动囊肿生长，以及发现纤毛/基体复合体是一般肾囊肿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认识仍存在很大差距，特别是当涉及到PC 1/PC 2受体通道复合物下游的直接信号时。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。促进对多囊肾疾病潜在因素的更深入科学理解和促进对开发治疗方法的兴趣的关键步骤是对ADPKD的靶途径进行令人信服和精确的定义。全面了解儿童多动症的障碍有两个方面。首先，PKD是一种影响肾脏和肝脏三维实体器官结构发育的疾病，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离体系统不足以发现PKD的核心途径。在这样的系统中的囊性表型的相关读数，如果它存在，是未知的。其次，对于PKD基因下游的未知途径发现，还没有成功的策略。相反，通路发现是基于检查已知的通路，例如与增殖和平面极性相关的通路，作为PKD中调节异常的候选者。我们假设多囊蛋白在一个尚未发现的新途径中起作用，该途径在完整器官中最明显地起作用，并且该途径的发现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的基础上，一个独特的修改PiggyBac（PB）转座子系统，发现激活突变的野生型背景下，导致囊肿形成的小鼠肾脏。我们将使用对ADPLD基因Sec 63突变引起的囊肿形成敏感的背景对功能丧失突变进行同样的研究。由于PB转座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定义治疗的期望，从选择性激活多囊蛋白途径在囊肿细胞。 公共卫生相关性：常染色体显性遗传性多囊肾病（ADPKD）影响美国60万人，每年增加超过20亿美元的医疗保健费用。提高对这种疾病的分子基础的理解是走向治疗的最佳手段。该提案将在小鼠肾脏中使用一种新的最先进的遗传分析，以发现以前未知的ADPKD潜在机制，这将改善寻找有效治疗该疾病的前景。