Testing Substrate-Flux Therapies for Glycosylation Disorders using Zebrafish

描述（由申请人提供）：在两年的时间里，四名研究人员将各自雇用一名新的全职员工，以确定和验证一组罕见遗传性代谢疾病的新型治疗策略，即先天性糖基化疾病I型（CDG-I）。这些疾病导致蛋白质糖基化不足，并且由于绝大多数CDG-I患者无法治疗，因此相关的多系统病理、发病率和死亡率是常见的。目前，13种I型CDG在临床和遗传学上被定义。大多数缺乏合成N-连接糖蛋白前体葡萄糖3甘露糖9 GlcNAc 2-P-P-多萜醇（也称为“脂连接寡糖”或“LLO”）所需的转移酶和底物。这些缺陷导致蛋白质的糖基化不足和相关的多系统病理、发病率和死亡率。在许多CDG-I患者中，原发性缺陷损害甘露糖代谢。CDG-I患者都有亚型等位基因，我们建议通过一种新的治疗策略来利用剩余的酶活性，我们称之为“底物通量”。例如，在CDG-Ia中，（CDG最常见的形式），我们将使用各种化学试剂来“哄”出更多患者自身有限的代谢物库（在这种情况下，甘露糖-6-磷酸）向缺陷酶（磷酸甘露变位酶），使酶池远离竞争酶（甘露糖磷酸异构酶），和/或改变相关途径（提供多萜酚-P或调节蛋白质合成），从而更有效地利用有限的底物库。我们的研究团队手头有三种已知的药物和五种实验化合物，所有这些药物都在初步的细胞培养实验中获得了有希望的结果，并准备进行动物试验。然而，CDG研究的一个严重限制是缺乏代表性的动物模型。敲除小鼠是胚胎致死的，尽管CDG基因亚型小鼠刚刚出现，但它们还没有准备好作为评估治疗的模型。因此，为了立即测试底物通量疗法，我们正在利用斑马鱼遗传学的力量和速度，用吗啉代寡核苷酸产生新的脊椎动物CDG-I斑马鱼模型（本质上是一种“敲除”方法）。这些正在病理学和生物化学方面得到证实。最近的初步数据支持成功产生pmm 2变形斑马鱼作为CDG-Ia的模型;它们具有适当的LLO缺陷和再现CDG患者方面的表型。使用斑马鱼模型的阳性药物结果将快速确定通量改变剂是否影响CDG-I表型，然后将使用真实的CDG-I患者细胞培养物进行确认。与这些研究同时，将通过在正常和CDG-I人类细胞培养物和斑马鱼模型中的甘露糖通量实验直接测试所讨论的药剂的机制。有效化合物通过正常小鼠的通量研究得到证实。下一代通量增强分子的筛选正在进行中。在两年内，我们将确定通量调节剂，并证明CDG治疗的真实的潜力。在这一点上，已知的药物将适合于标签外临床评价，然后将在CDG-I的新兴小鼠模型中测试实验药物。 公共卫生相关性：很少有“先天性糖基化障碍”（CDG）患者能够成功治疗。这些患者遭受多系统病理学、发病率和死亡率，因为他们不能正常地糖基化蛋白质。我们的四名研究人员团队将聘请四名科学工作人员来测试一种名为“底物通量”疗法的新治疗策略。由于小鼠模型还不存在，我们将在我们创建的一系列新的斑马鱼CDG模型中评估治疗。

DESCRIPTION (provided by applicant): For two years, four investigators will each hire a new full-time employee to identify and validate a novel therapeutic strategy for a group of rare inherited metabolic disorders, the Congenital Disorders of Glycosylation-Type I (CDG-I). These disorders result in underglycosylation of proteins and associated multisystem pathologies, morbidity, and mortality are common since the vast majority of CDG-I patients cannot be treated. Currently, 13 Type-I CDGs are clinically and genetically defined. Most have deficiencies in transferases and substrates necessary to synthesize the precursor of N-linked glycoproteins, glucose3mannose9GlcNAc2-P-P-dolichol, also known as "lipid-linked oligosaccharide" or "LLO". These deficiencies result in underglycosylation of proteins and associated multi-system pathologies, morbidity, and mortality. In many CDG-I patients, the primary defect impairs mannose metabolism. CDG-I patients all have hypomorphic alleles, and we propose to take advantage of the residual enzyme activities by a novel therapeutic strategy we term "substrate-flux". For example, in CDG-Ia (the most common form of CDG), we will use various chemical agents to "coax" more of the patients' own limited metabolite pool (in this case mannose- 6-phosphate) toward the defective enzyme (phosphomannomutase), divert the pool away from competing enzymes (mannose phosphate isomerase), and/or alter associated pathways (providing dolichol-P or regulating protein synthesis) so that the limited substrate pool is used more effectively. Our research team has three known drugs and five experimental compounds in-hand, all of which have given promising results in preliminary cell culture experiments and are ready for animal testing. However, a severe limitation of CDG research is the absence of representative animal models. Knockout mice are embryonic lethal and although CDG-gene hypomorphic mice are just now emerging, they are not yet ready as a model to evaluate therapies. Thus, to test substrate-flux therapy immediately, we are harnessing the power and speed of zebrafish genetics to generate new vertebrate CDG-I zebrafish models with morpholino oligonucleotides (essentially a "knockdown" approach). These are being validated both pathologically and biochemically. Recent preliminary data support the successful generation of pmm2 morphant zebrafish as a model of CDG-Ia; they have an appropriate LLO defect and phenotypes recapitulating aspects of CDG patients. Positive drug results with zebrafish models will rapidly determine whether flux-altering agents affect CDG-I phenotypes, and will then be confirmed with authentic CDG-I patient cell cultures. Concurrent with these studies, the mechanisms of the agents in question will be tested directly by mannose flux experiments in both normal and CDG-I human cell cultures and zebrafish models. Efficacious compounds are confirmed by flux studies in normal mice. Screening of the next generation of flux-enhancing molecules is underway. Within two years, we will identify fluxmodifying agents and demonstrate real potential for CDG treatment. At this point the known drugs would be appropriate for off-label clinical evaluation, and the experimental drugs will then be tested in emerging mouse models of CDG-I. PUBLIC HEALTH RELEVANCE: Very few patients with "Congenital Disorders of Glycosylation" (CDG) can be successfully treated. These patients suffer multi-system pathology, morbidity, and mortality because they do not glycosylate proteins normally. Our four-investigator team will hire four scientific staff to test a new treatment strategy called "substrate-flux" therapy. Since mouse models do not yet exist we will evaluate therapy in a series of novel zebrafish CDG models that we've created.