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由铜转运蛋白基因ATP7A突变引起的X连锁隐性致死性婴儿神经退行性疾病。未经治疗的患者通常会在三岁前死亡。在治疗方面，塞浦路斯治疗公司(纽约)正在根据该部门过去25年来进行的前一期I/II期和目前的III期临床试验的结果数据，为FDA新药批准(NDA)开发Histidinate铜(每日皮下注射)。目前还没有被批准的治疗这种罕见的孤儿疾病的方法，我们已经证明，与没有治疗的患者相比，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，即使在ATP7A基因突变严重丧失功能的受试者中也是如此。 为了开发一种更全面的治疗这种疾病的方法，我们还在Menkes病的斑驳斑纹(mo-br)小鼠模型中开发一种病毒基因治疗方法，以提供尺寸缩小、密码子优化的ATP7A的工作副本，该ATP7A可被AAV骨架容纳并表达高水平的功能性铜转运蛋白。我们先前的研究表明，AAV5血清(AAV5)与氯化铜联合脑脊液(CSF)可以挽救突变的mo-br小鼠。在最近的一项研究中，我们在剂量范围内的脑脊液导向的范例中测试了更有效的AAV血清型(AAV9，AAVRH10)，并改用 临床级组氨酸铜对小鼠的皮下注射。我们比较了三种不同剂量的AAV9和RH10联合皮下注射铜，发现中剂量(5.0e9 Vg)和高剂量(1.6e10 Vg)的AAV9-rsATP7A与最高的存活率相关。脑脊液导向的AAV9加皮下铜可使躯体生长和神经行为结果正常化。脑区的电子显微镜和H&E染色反映了联合治疗动物神经病理的显著改善。与未经处理的突变小鼠相比，这种协同治疗效果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进入脑内的结果一致。与我们之前关于AAV5的研究相比，我们的发现为脑脊液导向的AAV9病毒基因治疗提供了支持。 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Huppke-Brendel综合征患者的特征，这是一种新的铜代谢障碍。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，并且(最近)导致HEK293T细胞对铜的反应中ATP7A转运受损。我们的发现揭示了AT-1在铜ATPase适当的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，该综合征是由适配器蛋白1亚单位(Sigma 1A)突变引起的，该突变影响ATP7B和ATP7A的细胞内转运，程度较小。在与细胞生物学和神经生物学分会成员的合作下，我们表征了三种sigma亚型(A、B、C)的功能冗余，这一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.