Disorders of Copper Transport

1.门克斯病小鼠模型中的ATP 7A基因治疗。Menkes病是一种致命的婴儿铜代谢神经退行性疾病，由P型ATP酶（ATP 7A）突变引起。目前可用的治疗对大多数受影响的人无效，死亡率很高。斑纹小鼠（mo-br）重现了Menkes表型，包括由于小鼠同源物Atp 7a突变导致的铜向大脑的异常转运，并在14日龄时死亡。我们记录了C57 BL/6背景下的mo-br小鼠不能通过外周铜给药来挽救，并使用该模型来评估脑定向治疗。新生mo-br小鼠接受侧脑室注射腺相关病毒血清型5（AAV 5），携带大小减小的人ATP 7A（rsATP 7A）互补DNA（cDNA），氯化铜，或两者兼而有之。AAV 5-rsATP 7A显示脉络丛上皮的选择性转导，并且AAV 5-rsATP 7A加铜组合治疗拯救了mo-br小鼠; 86%存活至断奶（21天），中位存活增加至43天，37%存活超过100天，并且22%存活至研究终点（300天）。这种协同治疗效应与脑铜水平增加、多巴胺-β-羟化酶（一种铜依赖性酶）活性增强和脑病理学纠正相关。这些发现提供了第一个明确的证据，基因治疗可能在治疗门克斯病的临床效用。涉及在全身施用后具有穿过血脑屏障的能力的AAV血清型（AAV 9，AAVrh 10）的进一步临床前概念验证研究正在研究中。 2.与铜代谢紊乱相关的新型分子缺陷。在与其他人的合作，我们的特点是从五个家庭与一个未知的铜代谢紊乱的患者。我们记录了SLC 33 A1中的缺陷，该SLC 33 A1编码高度保守的乙酰辅酶A转运蛋白（AT-1），该转运蛋白是多种神经节苷脂和糖蛋白乙酰化所必需的。发现突变导致AT-1表达减少或缺失以及蛋白质的异常细胞内定位。我们发现，AT-1敲低HepG 2细胞导致血浆铜蓝蛋白分泌减少。这一发现揭示了AT-1在许多蛋白质的适当翻译后修饰中的重要作用，没有这些蛋白质，正常的大脑发育就会中断。

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 to cross the blood-brain barrier after systemic administration (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.