Inherited Disorders of Copper Transport

1.门克斯病小鼠模型的ATP7A基因治疗门克斯病是一种致命的婴儿铜代谢神经退行性疾病，由P型ATPase ATP7A突变引起。目前可用的治疗方法对大多数受影响的人无效，死亡率很高。斑驳斑纹(mo-br)小鼠概括了Menkes的表型，包括由于小鼠同源基因ATP7A的突变而将铜异常转运到大脑，并在14天前死亡。我们记录了C57BL/6背景的mo-br小鼠不能通过外周给铜拯救，并使用这个模型来评估脑导向治疗。新生mo-br小鼠接受侧脑室注射携带缩小尺寸的人ATP7A(RsATP7A)互补DNA(CDNA)的5型腺相关病毒(AAV5)或氯化铜，或两者兼而有之。AAV5-rsATP7A可选择性转导脉络丛上皮细胞，AAV5-rsATP7A+铜联合治疗组小鼠存活至断奶(21天)者占86%，中位生存期增至43天，超过100天者占37%，至研究终点(300天)者占22%。这种协同治疗效果与脑铜水平的增加、铜依赖的酶-多巴胺-β-羟基酶的活性增强以及大脑病理的纠正有关。这些发现提供了第一个确凿的证据，表明基因疗法可能在治疗门克斯病方面具有临床实用价值。关于具有神经元和神经胶质细胞转导能力的AAV血清型(AAV9，AAVRH10)的进一步临床前概念验证调查正在进行中。 2.与铜代谢紊乱有关的新分子缺陷。在与其他人的合作中，我们描述了来自五个家庭的患有未知铜代谢障碍的患者。我们发现SLC33A1编码高度保守的乙酰辅酶A转运蛋白(AT-1)的缺陷，该转运蛋白是多种神经节苷脂和糖蛋白乙酰化所必需的。这些突变被发现导致AT-1表达减少或缺失，以及该蛋白在细胞内的异常定位。我们发现，在HepG2细胞中，AT-1基因敲除导致铜蓝蛋白分泌减少。这一发现揭示了AT-1在许多蛋白质的适当翻译后修饰中发挥的关键作用，如果没有这些修饰，正常的大脑发育就会中断。我们还参与了Mednik综合征的基础科学和临床描述，该综合征是由适配器蛋白1亚单位突变引起的，该亚单位影响ATP7A和ATP7B的细胞内运输。

1. ATP7A gene therapy in murine models of Menkes disease. Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment is ineffective in a majority of affected individuals and mortality is high. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice; 86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-beta-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. These findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease. Further preclinical proof-of-concept investigations involving AAV serotypes with the capacity for neuronal and glial cell transduction (AAV9, AAVrh10) are under investigation. 2. Novel molecular defects associated with disordered copper metabolism. In collaboration with others, we characterized patients from five families with an unknown disorder of copper metabolism. We documented defects in in SLC33A1 that encodes a highly conserved acetylCoA transporter (AT-1), required for acetylation of multiple gangliosides and glycoproteins. The mutations were found to cause reduced or absent AT-1 expression and abnormal intracellular localization of the protein. We showed that AT-1 knockdown in HepG2 cells led to reduced ceruloplasmin secretion. The finding revealed an essential role for AT-1 in proper post-translational modification of numerous proteins, without which normal brain development is interrupted. We have also participated in the basic science and clinical delineation of MEDNIK syndrome, caused by mutations in an adaptor protein 1 subunit that affects intracellular trafficking of ATP7A and ATP7B.