Novel Therapy for a Human Glycosylation Disorder

描述（由申请人提供）：先天性糖基化疾病（CDGs）是一种罕见的、可筛查的遗传性疾病，它会消耗n链聚糖的合成。编码磷酸腺苷转氨酶2 (PMM2, Man-6-P？Man-1-P)引起最常见的CDG-Ia。患者有许多问题，包括张力低下、可变精神运动迟缓、癫痫发作、周围神经病变、心肌病和蛋白质丢失性肠病。这种疾病没有治疗方法。其他由磷酸甘糖异构酶（PMI）突变引起的CDG-Ib患者(Man-6-P<？果糖-6- p)症状较轻，可通过膳食甘露糖补充剂治疗。甘露糖被运输到细胞中，转化为Man-6-P，并绕过PMI缺陷。请注意，PMM2和PMI都在争夺它们共同的基质Man-6-P。在这里，研究人员提出了一种新颖而非正统的治疗方法来治疗pmm2缺陷细胞和小鼠。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进入弱糖基化途径并挽救糖基化。通常，在生物合成途径中结合多个致病突变会加剧病理，但在这种情况下并非如此。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缺陷小鼠的胚胎致死。他们将同时提供外源性甘露糖，并使用遗传和药理学方法来增加Man-6-P进入糖基化途径的可用性和通量。他们开发了携带Pmi亚型等位基因的可育小鼠系，并在基于细胞的糖基化实验中发现了几种抑制Pmi的化合物。因此，特异性Aim 1通过敲低Pmi或特异性Pmi抑制剂，增加甘露糖进入Pmm2缺陷细胞糖基化途径的通量；特异性Aim 2将决定特异性Pmi抑制剂在多大程度上增加甘露糖进入正常小鼠糖基化途径的通量；特异性Aim 3将在pmm缺陷小鼠中引入一个半形的Pmi等位基因，以挽救胚胎18.5天的死亡。该应用具有很高的影响力，因为它的成功将验证CDGIa患者潜在的小分子治疗方法。它也可能基于从饮用水中添加或提取甘露糖来创建一个灵活的CDG-Ia小鼠模型。这种方法的高风险在于无法挽救胚胎的致命性，也无法为缺乏Pmm2的细胞提供足够的前体。对于没有任何治疗选择的CDG-Ia患者，潜在的影响是值得冒险的。

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.