Inherited 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X-连锁隐性致死性婴儿神经退行性疾病，由铜转运蛋白基因ATP 7A突变引起。未经治疗的患者通常在三岁之前死亡。在治疗方面，Cyprium Therapeutics，Inc.正在开发组氨酸铜（每日皮下注射），以获得FDA新药批准（NDA）。(New约克，纽约）基于该科在过去25年中进行的先前I/II期和当前III期临床试验的结果数据。目前还没有针对这种罕见孤儿病的获批治疗方法，我们已经证明，与未接受治疗相比，早期组氨酸铜治疗显著降低了受影响患者的三岁以下死亡率，即使是在患有严重功能丧失的ATP 7A突变的受试者中。 为了开发针对这种疾病的更完整的治疗方法，我们还在门克斯病的斑点（mo-br）小鼠模型中开发病毒基因治疗方法，以提供尺寸减小、密码子优化版本的ATP 7A的工作拷贝，其可以被AAV骨架容纳并表达高水平的功能性铜转运蛋白。我们先前表明，AAV血清型5（AAV 5）与氯化铜共同施用到脑脊液（CSF）中可以拯救突变的mo-br小鼠。在最近的一项研究中，我们在剂量范围CSF导向的范例中测试了更有效的AAV血清型（AAV 9，AAVrh 10），并转换为 在小鼠中皮下施用临床级组氨酸铜治疗。我们比较了与皮下Cu组合的三种不同的AAV 9和rhlO剂量，并且发现中等（5.0e9 vg）和高（1.6e10 vg）剂量的AAV 9-rsATP 7A与最高的存活率相关。CSF导向的AAV 9加皮下Cu使躯体生长和神经行为结果正常化。脑区的电子显微照片和H&E染色反映了组合处理的动物中神经病理学的显著改善。与未治疗的突变小鼠相比，这种协同治疗效应显著改善了脑铜代谢的生物标志物，并与病毒基因组拷贝数相关。X射线荧光显微镜检查结果与脉络丛介导的铜向大脑的递送一致。与我们先前使用AAV 5的研究相比，我们的研究结果为患有Menkes病的人类受试者的CSF导向的AAV 9病毒基因治疗提供了支持。 2.与铜代谢紊乱相关的新型分子缺陷。在与其他人的合作中，我们已经描述了Huppke-Brendel综合征患者的特征，这是一种新型的铜代谢紊乱。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，在HepG 2细胞中AT-1敲低导致血浆铜蓝蛋白分泌减少，并且（最近）在HEK 293 T细胞中响应于铜的ATP 7A运输受损。我们的研究结果揭示了AT-1在铜ATP酶的适当翻译后修饰中的重要作用。 3.我们还在研究MEDNIK综合征的基础科学和临床描述，MEDNIK综合征是由衔接蛋白1亚基（sigma 1A）突变引起的，该突变影响ATP 7 B的细胞内运输，并在较小程度上影响ATP 7A。与细胞生物学和神经生物学分支的成员合作，我们已经表征了三种sigma亚型（A，B，C）的功能冗余，这些发现对患有这种罕见而有趣的疾病的受试者具有翻译意义。

1. ATP7A gene therapy in murine models of Menkes disease. Menkes disease is a X-linked recessive lethal infantile neurodegenerative disorder caused by mutations in a copper transporter gene, ATP7A. Untreated patients typically die by three years of age. In terms of treatment, Copper Histidinate (delivered by daily subcutaneous injection) is being developed for FDA new drug approval (NDA) by Cyprium Therapeutics, Inc. (New York, NY) based on outcome data from the Section's previous Phase I/II and current Phase III clinical trials conducted over the past 25 years. There are currently no approved treatments for this rare orphan disease, and we have shown that early Copper Histidinate treatment significantly reduces under-three mortality in affected patients compared to no treatment, even in subjects with severe loss-of-function ATP7A mutations. To develop an even more complete treatment for this illness, we are also developing a viral gene therapy approach in the mottled-brindled (mo-br) mouse model of Menkes disease, to provide working copies of a reduced size, codon-optimized version of ATP7A, which can be accommodated by the AAV backbone and expresses high levels of a functional copper transporter. We previously showed that AAV serotype 5 (AAV5) co-administered with copper chloride into the cerebrospinal fluid (CSF) could rescue mutant mo-br mice. In a recent study, we tested more potent AAV serotypes (AAV9, AAVrh10) in a dose-ranging CSF-directed paradigm and switched to subcutaneous administration of clinical grade Copper Histidinate treatment in the mice. We compared three different AAV9 and rh10 doses in combination with subcutaneous Cu, and found that intermediate (5.0e9 vg) and high (1.6e10 vg) doses of AAV9-rsATP7A were associated with highest rates of survival. CSF-directed AAV9 plus subcutaneous Cu normalized somatic growth and neurobehavioral outcomes. Electron micrographs and H&E stain of brain regions reflected significant improvements in neuropathology in the combination-treated animals. This synergistic treatment effect markedly improved biomarkers of brain copper metabolism in comparison to untreated mutant mice, and correlated with viral genome copy number. X-ray fluorescence microscopy findings were consistent with choroid plexus-mediated copper delivery to the brain. Compared to our previous study with AAV5, our findings provide support for CSF-directed AAV9 viral gene therapy in human subjects with Menkes disease. 2. Novel molecular defects associated with disordered copper metabolism. In collaboration with others, we have characterized patients with Huppke-Brendel syndrome, a novel disorder of copper metabolism. We documented defects in in SLC33A1 that encodes a highly conserved acetyl CoA 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, and (more recently) to impaired ATP7A trafficking in response to copper in HEK293T cells. Our findings reveal an essential role for AT-1 in the proper post-translational modification of copper ATPases. 3. We are also at work on the basic science and clinical delineation of MEDNIK syndrome, which is caused by mutations in an adaptor protein 1 subunit (sigma 1A) that affects intracellular trafficking of ATP7B and to a lesser extent, ATP7A. In collaboration with members of the Cell Biology and Neurobiology Branch, we have characterized a functional redundancy of three sigma isoforms (A, B, C), findings that have translational implications for subjects with this rare and interesting condition.