Abstract：
Non-Ketotic Hyperglycinemia (NKH) is a rare inborn error of metabolism caused by impaired function of the glycine cleavage system (GCS) and characterised by accumulation of glycine in body fluids and tissues. NKH is an autosomal recessive condition and the majority of affected individuals carry mutations in GLDC (glycine decarboxylase). Current treatments for NKH have limited effect and are not curative. As a monogenic condition with known genetic causation, NKH is potentially amenable to gene therapy. An AAV9-based expression vector was designed to target sites of GCS activity. Using a ubiquitous promoter to drive expression of a GFP reporter, transduction of liver and brain was confirmed following intra-venous and/or intra-cerebroventricular administration to neonatal mice. Using the same capsid and promoter with transgenes to express mouse or human GLDC, vectors were then tested in GLDC-deficient mice that provide a model of NKH. GLDC-deficient mice exhibited elevated plasma glycine concentration and accumulation of glycine in liver and brain tissues as previously observed. Moreover, the folate profile indicated suppression of folate one‑carbon metabolism (FOCM) in brain tissue, as found at embryonic stages, and reduced abundance of FOCM metabolites including betaine and choline. Neonatal administration of vector achieved reinstatement of GLDC mRNA and protein expression in GLDC-deficient mice. Treated GLDC-deficient mice showed significant lowering of plasma glycine, confirming functionality of vector expressed protein. AAV9-GLDC treatment also led to lowering of brain tissue glycine, and normalisation of the folate profile indicating restoration of glycine-derived one‑carbon supply. These findings support the hypothesis that AAV-mediated gene therapy may offer potential in treatment of NKH.
摘要：
非酮症性高血糖症（NKH）是一种罕见的先天性代谢错误，由甘氨酸裂解系统（GCS）的功能受损引起，其特征是甘氨酸在体液和组织中的积累。NKH是常染色体隐性疾病,并且大多数受影响的个体在GLDC(甘氨酸脱羧酶)中携带突变。目前对NKH的治疗效果有限，无法治愈。作为已知遗传原因的单基因条件，NKH可能适合基因治疗。设计基于AAV9的表达载体以靶向GCS活性的位点。使用普遍存在的启动子驱动GFP报告基因的表达，在对新生小鼠进行静脉内和/或脑室内给药后，证实了肝脏和大脑的转导。使用相同的衣壳和启动子与转基因表达小鼠或人类GLDC，然后在提供NKH模型的GLDC缺陷小鼠中测试载体。如先前观察到的,缺乏GLDC的小鼠表现出升高的血浆甘氨酸浓度和甘氨酸在肝脏和脑组织中的积累。此外，叶酸谱表明抑制脑组织中的叶酸一碳代谢（FOCM），在胚胎阶段发现的，和减少FOCM代谢物的丰度，包括甜菜碱和胆碱。新生儿施用载体实现了GLDC缺陷小鼠中GLDCmRNA和蛋白质表达的恢复。经治疗的GLDC缺陷小鼠显示血浆甘氨酸显着降低，证实载体表达的蛋白质的功能性。AAV9-GLDC治疗还导致脑组织甘氨酸的降低，叶酸谱的正常化表明甘氨酸衍生的一碳供应的恢复。这些发现支持AAV介导的基因治疗可能在NKH的治疗中提供潜力的假设。
