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性疾病，其特征是新生儿和成人具有多向性 单倍体不充分的锯齿/缺口信号引起的病理(1)。具体来说，ALGS是由以下原因引起的 杂合性功能丧失突变主要发生在Notch配体基因Jagged1(JAG1)和更少 通常，在NOTCH2(N2)(2-5)中。虽然这种疾病的特征是多个器官系统的缺陷， 心血管和肝脏的病理是最具生命威胁的。然而，这一点的体内遗传模型 由于Jag1杂合子小鼠温和且可变的外显性，疾病一直具有挑战性(6)。此外， 目前还没有成熟的药物可以增强Notch信号来潜在地治疗这种基因 无序。为了生成一个更具表型健壮的ALGS脊椎动物模型，我们利用斑马鱼模型 控制参差不齐的等位基因的遗传剂量，以产生一致的强而透彻的病理 类似于ALGS，包括肝胆管稀疏不能溶解和自发性出血。 此外，我们已经验证了一种小分子Notch激动剂，它可以直接增强Notch信号转导 哺乳动物细胞和斑马鱼体内。这些关键的新工具将使我们第一次能够调查救援 Notch信号作为治疗这种单倍体JAG/Notch信号缺陷遗传病的一种方法。 在ALGS肝脏中，胆管细胞缺乏可导致胆汁淤积和肝功能衰竭。与流行的 据估计，没有肝脏移植的人出生人数为1/40,000(7)，19岁时的死亡率为76% (8)ALGS急需有效的治疗方法。我们的斑马鱼活体研究首次显示 恢复锯齿状/Notch信号导致丢失的肝管细胞再生。这一发现，连同 Jagged1杂合子小鼠(6，9)肝管缺失的出生后恢复和肝功能的波动 ALGS患者(10例)，提示ALGS的病理可能是可逆的。可逆性，以及变量 ALGS患者的动态病理外显性及其JAG1杂合性，使我们得出了假设 JAG/Notch信令可能在不足和足够之间摇摇欲坠。因此，这些ALG 病理可能可以通过略微增强Notch信号来治疗。我们严格验证了 一种新的小分子Notch激动剂，可显著增强小鼠肝细胞和ALG中的Notch信号 具有JAG1突变的患者成纤维细胞。我们建议在斑马鱼身上测试这种新验证的Notch激动剂， ALGS的小鼠、人类JAG突变模型。我们新出现的初步研究表明，这种Notch激动剂 确实能刺激JAG突变斑马鱼的肝管细胞再生。我们的研究将是取得成果的关键 使用Notch激动剂作为治疗策略的概念验证数据，以解决大多数生命- 威胁着ALGS的病理。这些研究是迈向临床前研究的必要的基本步骤。

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.