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的治疗方法作为治疗罕见囊性肾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多囊性肾病（ARPKD）、肾囊肿合并糖尿病（RCAD）和肾病（NPHP）。这些罕见的遗传性疾病的特征是肾脏囊肿的形成和进行性肾衰竭。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或更少，但它们具有重要的临床意义。ARPKD和NPHP是婴儿和儿童终末期肾病最常见的单基因原因。人类ARPKD、RCAD和NPHP的突变基因已被确定。然而，目前还没有针对这些疾病的特异性和有效的治疗方法，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，我们的实验室一直在研究常染色体隐性遗传性囊性肾病的分子发病机制。我们已经开发出携带与这些疾病患者相同基因突变的同源小鼠模型。突变小鼠的表型与受影响的人类的表型相似，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示人类疾病的发病机制，并揭示了它们在共同途径上的联系。特别是在RCAD中发生突变的转录因子HNF-1¿调节在ARPKD中发生突变的PKHD1的转录，以及在肾病中发生突变的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病的有效途径。最近的研究已经确定了microRNAs （miRNAs）作为人类疾病的潜在分子靶点。mirna是一种短的非编码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表达。在初步研究中，我们已经确定了几个mirna家族在HNF-1突变细胞中异常过表达。这些mirna序列与已知囊性疾病基因（如PKD2和PKHD1）编码的mrna是互补的。这些研究确定了miRNAs在囊性肾病发病机制中的新作用，并提示抑制miRNAs可能是减少囊肿形成的有效策略。近年来，研究人员已经开发出了有效的体内调节mirna活性的方法。安塔戈米是经化学修饰的寡核苷酸，可经肠外给药，并能特异性和持久地灭活靶mirna。在本提案中，我们将使用miRNA微阵列分析来全面鉴定在ARPKD， RCAD和NPHP同源小鼠模型中异常表达的miRNA。接下来，我们将测试靶向过表达miRNAs的安塔戈米是否能抑制突变小鼠的囊肿形成并改善肾功能。这些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临床上重要的遗传疾病的可行性，并将对其他影响肾脏的罕见疾病的治疗产生广泛的影响。

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.