MicroRNA-Based Therapeutics for Rare Cystic Kidney Diseases

描述(由申请人提供)：这项为期两年的提案的目标是测试基于microRNA的疗法作为治疗罕见囊性肾脏疾病的新策略的可行性。常染色体隐性遗传性囊性肾病包括常染色体隐性遗传性多囊肾病(ARPKD)、肾囊肿伴糖尿病(RCAD)和肾小球肾炎(NPHP)。这些罕见的遗传性疾病的特点是在肾脏形成囊肿和进行性肾功能衰竭。肾外表现包括先天性肝纤维化(ARPKD)、糖尿病(RCAD)和失明(NPHP)。虽然这些疾病的发病率为1：20,000或更低，但它们具有重要的临床意义。ARPKD和NPHP是婴幼儿终末期肾病最常见的单基因致病因素。在人类ARPKD、RCAD和NPHP中突变的基因已经被确定。然而，目前还没有针对这些疾病的特效疗法，目前也没有药物处于临床试验阶段。因此，需要新的治疗方法。在过去的10年里，我们实验室对常染色体隐性遗传性囊性肾病的分子发病机制进行了研究。我们已经开发出携带相同基因突变的同源小鼠模型，这些突变基因在患有这些疾病的人类中发生突变。突变小鼠的表型与受影响人类的表型相似，表明它们代表了优秀的动物模型。对突变小鼠的分析有助于揭开人类疾病的发病机制，并揭示它们以一种共同的途径联系在一起。特别是，在RCAD中突变的转录因子HNF-1β调节在ARPKD中突变的PKHD1和在肾炎中突变的GLIS2和NPHP3的转录。因此，纠正这一通路的异常可能是治疗囊性肾脏疾病的有效方法。最近的研究发现，microRNAs(MiRNAs)是人类疾病中潜在的分子靶点。MiRNAs是一种短的、非编码的RNA，通过抑制翻译或促进互补信使RNA的切割来调节转录后基因的表达。在初步研究中，我们已经确定了几个在HNF-1突变细胞中异常过表达的miRNAs家族。MiRNAs的序列与已知囊性疾病基因编码的mRNAs互补，如PKD2和PKHD1。这些研究确定了miRNAs在囊性肾脏疾病发病机制中的新作用，并提示抑制miRNAs可能是减少囊性形成的有效策略。最近，已经发展了有效的方法来调节体内miRNAs的活性。反交配子是一种化学修饰的寡核苷酸，可以非肠道给药，并特异性和持久地灭活靶向miRNAs。在这个方案中，我们将使用miRNA微阵列分析来全面识别在ARPKD、RCAD和NPHP的同源小鼠模型中异常表达的miRNAs。接下来，我们将测试针对过表达的miRNAs的反义体的注射是否抑制了突变小鼠的包囊形成和改善了肾功能。这类研究将提供重要的临床前数据，证明这种方法治疗这些罕见但具有临床重要性的遗传病的可行性，并将对其他影响肾脏的罕见疾病的治疗产生广泛影响。 公共卫生相关性：常染色体隐性遗传性囊性肾病是一种罕见的遗传性疾病，可导致儿童肾功能衰竭。目前还不存在特定的治疗方法。这项提案将测试使用一种新技术--基于microRNA的疗法--治疗这些疾病的可行性。

DESCRIPTION (provided by applicant): The goal of this two-year proposal is to test the feasibility of microRNA-based therapeutics as a novel strategy for the treatment of rare cystic kidney diseases. Autosomal recessive cystic kidney diseases include autosomal recessive polycystic kidney disease (ARPKD), renal cysts and diabetes (RCAD), and nephronophthisis (NPHP). These rare genetic disorders are characterized by the formation of cysts in the kidneys and progressive kidney failure. Extrarenal manifestations include congenital hepatic fibrosis (ARPKD), diabetes (RCAD), and blindness (NPHP). Although these disorders are rare with incidences of 1:20,000 or less, they have significant clinical importance. ARPKD and NPHP are the most common monogenic causes of end-stage kidney disease in infants and children. The genes that are mutated in human ARPKD, RCAD, and NPHP have been identified. However, no specific and effective therapies exist for these diseases, and no drugs are currently in clinical trials. Therefore, new therapeutic approaches are needed. For the past 10 years, our laboratory has investigated the molecular pathogenesis of autosomal recessive cystic kidney diseases. We have developed orthologous mouse models carrying mutations in the same genes that are mutated in humans with these disorders. The phenotypes of the mutant mice resemble the phenotypes of affected humans indicating that they represent excellent animal models. Analysis of the mutant mice has helped unravel the pathogenesis of the human diseases and revealed that they are linked in a common pathway. In particular, the transcription factor HNF-1¿, which is mutated in RCAD, regulates the transcription of PKHD1, which is mutated in ARPKD, and GLIS2 and NPHP3, which are mutated in nephronophthisis. Thus, correction of abnormalities in this pathway may be an effective therapeutic approach for cystic kidney diseases. Recent studies have identified microRNAs (miRNAs) as potential molecular targets in human diseases. miRNAs are short, non-coding RNAs that regulate post-transcriptional gene expression by inhibiting translation or promoting cleavage of complementary messenger RNAs. In preliminary studies, we have identified several families of miRNAs that are abnormally overexpressed in HNF-1¿ mutant cells. The sequences of the miRNAs are complementary to mRNAs encoded by known cystic disease genes, such as PKD2 and PKHD1. These studies identify a novel role of miRNAs in the pathogenesis of cystic kidney diseases and suggest that inhibition of miRNAs may be an effective strategy for reducing cyst formation. Recently, effective methods for modulating the activity of miRNAs in vivo have been developed. Antagomirs are chemically modified oligonucleotides that can be administered parenterally and that specifically and durably inactivate target miRNAs. In this proposal, we will use miRNA microarray analysis to comprehensively identify miRNAs that are abnormally expressed in orthologous mouse models of ARPKD, RCAD, and NPHP. Next, we will test whether the administration of antagomirs targeting the overexpressed miRNAs inhibit cyst formation and improve renal function in mutant mice. Such studies would provide important pre-clinical data demonstrating the feasibility of this approach for the treatment of these rare but clinically important genetic disorders and would also have broad implications for the treatment of other rare disorders affecting the kidney. PUBLIC HEALTH RELEVANCE: Autosomal recessive cystic kidney diseases are rare genetic disorders that produce kidney failure in children. No specific treatment currently exists. This proposal will test the feasibility of treating these disorders using a new technology, microRNA-based therapeutics.