Novel Therapy for a Human Glycosylation Disorder

描述(申请人提供)：先天性糖基化障碍(CDGs)是一种罕见的、可筛查的遗传性疾病，它会耗尽N-连接的多糖的合成。编码磷酸甘露聚糖酶2(PMM2，Man-6-P？Man-1-P)的PMM2突变导致最常见的形式CDG-Ia。患者有一系列的问题，包括低眼压、变异性精神运动迟缓、癫痫发作、周围神经病、心肌病和蛋白丢失肠病。目前还没有治疗这种疾病的方法。其他由磷酸甘露糖异构酶(PMI)突变引起的CDG-Ib患者的症状较轻，可以通过补充甘露糖来治疗。甘露糖被转运到细胞内，转化为Man-6-P，绕过了PMI缺乏症。请注意，PMM2和PMI都竞争它们的共同底物Man-6-P。在这里，研究人员提出了一种新的、非正统的治疗方法来治疗PMM2缺陷的细胞和小鼠。通过降低PMI活性和提供外源甘露糖，他们将试图推动更多的Man-6-P进入消瘦的糖基化途径，并挽救糖基化。通常，在生物合成途径中组合多个致病突变会加剧病理，但在这种情况下不是这样。初步结果表明，研究人员可以挽救Pmm2缺陷细胞的糖基化，并逆转Pmm2缺陷小鼠的胚胎死亡。他们将同时提供外源甘露糖，并使用遗传和药物方法来增加Man-6-P的可用性和进入糖基化途径的流量。他们开发了携带PMI亚型等位基因的可存活和可生育的小鼠品系，并在基于细胞的糖基化分析中确定了几种抑制PMI的化合物。因此，特异性目标1将通过敲除PMI或特异性PMI抑制剂来增加Pmm2缺陷细胞的甘露糖进入糖基化途径的流量；特异性目标2将确定在多大程度上，特异性PMI抑制剂在多大程度上增加正常小鼠甘露糖进入糖基化途径的流量；以及特异性目标3将向PMM缺陷小鼠引入亚型Pmi等位基因以挽救e18.5天的胚胎死亡。这一应用具有很高的影响，因为它的成功将验证CDGIa患者潜在的小分子疗法。它还可以基于从饮用水中添加或移除甘露糖来创建灵活的CDG-Ia小鼠模型。高风险在于，这种方法不能挽救胚胎的致死性，也不能为Pmm2缺陷细胞提供足够的前体。对于没有任何治疗选择的CDG-Ia患者来说，潜在的影响值得冒险。 项目简介：PMM2缺乏导致先天性Ia型糖基化(CDG)紊乱，目前尚无治疗方法。PMI缺乏导致密切相关的CDG-Ib，而饮食甘露糖疗法有效。这两种酶都竞争相同的底物甘露糖-6-P。研究人员预测，如果竞争性PMI活性降低，CDG-Ia患者也会对甘露糖补充剂产生反应。这个应用程序旨在验证这一假设。

DESCRIPTION (Provided by Applicant): Congenital disorders of glycosylation (CDGs) are rare, screenable genetic disorders that deplete the synthesis of N-linked glycans. Mutations in PMM2, encoding phosphomannomutase 2 (PMM2, Man-6-P?Man-1-P) cause the most common form, CDG-Ia. Patients have a host of problems including hypotonia, variable psychomotor retardation, seizures, peripheral neuropathy, cardiomyopathy, and protein losing enteropathy. There is no therapy for this disorder. Other patients with CDG-Ib caused by mutations in phosphomannose isomerase (PMI) (Man-6-P<?Fructose-6-P) have milder symptoms, and they can be treated with dietary mannose supplements. Mannose is transported into cells, converted to Man-6-P, and bypasses the PMI deficiency. Note that both PMM2 and PMI compete for their common substrate, Man-6-P. Here the investigators propose a novel and unorthodox therapeutic approach to treat Pmm2-deficient cells and mice. By reducing PMI activity and providing exogenous mannose, they will try to drive more Man-6-P into the emaciated glycosylation pathway and rescue glycosylation. Usually, combining multiple disease-causing mutations within a biosynthetic pathway exacerbates the pathology, but not in this case. Preliminary results suggest that the investigators can rescue glycosylation of Pmm2-deficient cells and reverse embryonic lethality in a Pmm2-deficient mouse. They will simultaneously provide exogenous mannose and use both genetic and pharmacological methods to increase the availability and flux of Man-6-P into the glycosylation pathway. They developed viable and fertile mouse lines carrying hypomorphic alleles for Pmi and also identified several compounds that inhibit Pmi in cell-based glycosylation assays. Thus Specific Aim 1 will increase the flux of mannose into the glycosylation pathway of Pmm2 deficient cells by knockdown of Pmi or a specific Pmi inhibitor; Specific Aim 2 will determine to what extent, the specific Pmi inhibitor increases the flux of mannose into glycosylation pathway in normal mice; and Specific Aim 3 will introduce a hypomorphic Pmi allele into Pmm-deficient mice to rescue embryonic lethality at e18.5 days. This application has high impact because its success would validate a potential small molecule therapy for CDGIa patients. It may also create a flexible mouse model of CDG-Ia based on the addition or withdrawal of mannose from the drinking water. The high risk is that this approach will fail to rescue embryonic lethality or provide sufficient precursors for Pmm2 deficient cells. For CDG-Ia patients without any therapeutic options, the potential impact is worth the risk. PROJECT NARRATIVE: PMM2 deficiency causes congenital disorder of glycosylation (CDG) Type Ia, and no therapy exists. PMI deficiency causes the closely related CDG-Ib where dietary mannose therapy works. Both enzymes compete for the same substrate, mannose-6-P. The investigators predict that CDG-Ia patients will also respond to mannose supplements if the competing PMI activity is reduced. This application aims to validate that hypothesis.