Augmented Notch signaling as a therapeutic approach for Alagille Syndrome

项目摘要 Alagille综合征（ALGS）是一种常染色体显性遗传疾病，以新生儿和成人多效性为特征， 由单倍不足的JAGGED/NOTCH信号传导导致的病理（1）。具体来说，ALGS是由以下原因引起的： 主要在Notch配体基因JAGGED 1（JAG 1）中的杂合性功能丧失突变， 通常，在NOTCH 2（N2）（2 - 5）中。虽然这种疾病的特点是多器官系统的缺陷， 心血管和肝脏病变是最威胁生命的。然而，在体内遗传建模， 由于Jag1杂合子小鼠的轻度和可变的突变率，疾病一直具有挑战性（6）。此外，本发明还 目前还没有成熟的药物可以增强Notch信号传导，以潜在地治疗这种遗传性疾病。 disorder.为了生成一个表型上更稳健的ALGS脊椎动物模型，我们利用了斑马鱼模型， 控制锯齿状等位基因的遗传剂量，以产生持续强烈和渗透性的病理 与ALGS类似，包括肝胆管缺乏未能解决和自发性再狭窄。 此外，我们已经验证了一种小分子Notch激动剂，其可以直接增强Notch信号传导， 哺乳动物细胞和斑马鱼。这些重要的新工具将使我们能够第一次调查救援工作， Notch信号传导作为这种单倍不足的Jag/Notch信号传导遗传疾病的治疗方法。 在ALGS肝脏中，胆管细胞缺乏可导致胆汁淤积和肝功能衰竭。患病率 估计为1/40，000的出生率（7），对于那些没有肝移植的人，19岁时的死亡率为76% (8)ALGS迫切需要有效的治疗。我们的斑马鱼体内研究首次揭示， 恢复Jagged/Notch信号传导导致失去的肝导管细胞的再生。这一发现，与 Jagged 1杂合子小鼠肝管缺乏的出生后恢复（6，9）和 ALGS患者（10），表明ALGS病理可能是可逆的。可逆性，以及变量 ALGS患者的动态病理学变化及其JAG 1杂合性，使我们提出了一个假设， Jag/Notch信号可能在不足和充足之间摇摆不定。因此，这些ALGS 病理学可能潜在地可以用Notch信号传导的轻微增强来治疗。我们严格验证了 一种新的小分子Notch激动剂，可以在小鼠肝细胞和ALGS中强烈增强Notch信号传导 JAG1突变的患者成纤维细胞。我们建议在斑马鱼中测试这种新验证的Notch激动剂， 小鼠、人Jag突变ALGS模型。我们新的初步研究表明，这种Notch激动剂 确实刺激了JAG突变斑马鱼的肝导管细胞再生。我们的研究对于 使用Notch激动剂作为解决大多数生命周期问题的治疗策略的概念验证数据。 威胁ALGS病理学。这些研究是临床前研究的必要基础步骤。

Project Summary Alagille Syndrome (ALGS) is an autosomal dominant disorder characterized by pleiotropic neonatal and adult pathologies resulting from haploinsufficient JAGGED/NOTCH signaling (1). Specifically, ALGS is caused by heterozygous loss-of-function mutations predominantly in the Notch ligand gene, JAGGED1 (JAG1), and less frequently, in NOTCH2 (N2) (2-5). Although this disorder is characterized by defects in multiple organ systems, cardiovascular and hepatic pathologies are the most life-threatening. However, in vivo genetic modeling of this disease has been challenging due to the mild and variable penetrance of Jag1 heterozygous mice (6). Further, there is currently no well-established drug that can enhance Notch signaling to potentially treat this genetic disorder. To generate a more phenotypically robust vertebrate model of ALGS, we leveraged the zebrafish model to control the genetic dosage of jagged alleles to produce consistently strong and penetrant pathologies analogous to ALGS, including failure of liver bile duct paucity to resolve and spontaneous hemorrhaging. Moreover, we have validated a small molecule Notch agonist that can directly enhance Notch signaling in mammalian cells and in zebrafish. These critical new tools will allow us for the first time to investigate the rescuing of Notch signaling as a therapeutic approach for this haploinsufficient Jag/Notch signaling genetic disease. In the ALGS liver, bile duct cell paucity can lead to cholestasis and liver failure. With a prevalence estimated at 1/40,000 births (7) and a 76% mortality rate by the age of 19 years for those without a liver transplant (8), ALGS urgently requires an effective treatment. Our zebrafish in vivo studies reveal for the first time that restoring Jagged/Notch signaling leads to regeneration of the lost liver duct cells. This discovery, together with postnatal recovery of liver duct paucity in Jagged1 heterozygous mice (6, 9) and fluctuations in liver function in ALGS patients (10), suggest that ALGS pathologies may be reversible. The reversibility, as well as the variable and dynamic pathological penetrance in ALGS patients and their JAG1 heterozygosity, led us to the hypothesis that Jag/Notch signaling may be teetering between being insufficient and sufficient. Therefore, these ALGS pathologies may potentially be treatable with a slight augmentation of Notch signaling. We rigorously validated a new small molecule Notch agonist that can robustly enhance Notch signaling in mouse livers cells and ALGS patient fibroblasts with JAG1 mutations. We propose here to test this newly validated Notch agonist in zebrafish, mouse, human Jag mutant models of ALGS. Our emerging preliminary studies reveal that this Notch agonist does indeed stimulate liver duct cell regeneration in jag mutant zebrafish. Our studies will be critical to yield proof-of-concept data for the use of a Notch agonist as a therapeutic strategy for resolving the most life- threatening ALGS pathologies. These studies are necessary fundamental steps towards pre-clinical studies.