Disorders of Copper Transport

Menkes病是一种铜转运的x连锁隐性疾病，由编码一种进化上保守的铜转运atp酶的基因缺陷引起。在哺乳动物中，该基因产物作为细胞内泵，将铜输送到反式高尔基空间，以整合到需要铜的酶中，并介导铜从细胞中逸出。这种疾病在婴儿期表现为发育迟缓、发育不良、神经退行性变和过早死亡（通常在3岁前）。我们在这种疾病上的工作包括开发快速可靠的神经化学和分子技术，用于早期诊断，这些努力与对患病婴儿进行早期铜组氨酸治疗的临床试验相吻合。我们使用细胞生物学、分子和生化方法来描述入组患者的特征，并与神经发育结果相关联。患者成纤维细胞的共聚焦成像用于评估突变Menkes基因产物的数量和定位。在许多Menkes疾病患者中，血脑屏障是一个具有挑战性的治疗障碍，我们假设了少数患者（约1 / 5）对早期铜组氨酸成功反应（正常神经发育结果）的治疗反应的分子基础。这些患者的基因突变至少能使一些残留的铜转运到发育中的大脑。因此，我们正在开发替代治疗方法，包括绕过血脑屏障的基因治疗。

Menkes disease is an X-linked recessive disorder of copper transport caused by defects in a gene that encodes an evolutionarily conserved copper-transporting ATPase. In mammals, this gene product functions as an intracellular pump to transport copper into trans-Golgi spaces for incorporation into copper-requiring enzymes, and also mediates copper exodus from cells. The disorder presents in infancy with delayed development, failure to thrive, neurodegeneration, and premature death (typically by 3 years of age). Our work on this disorder includes development of rapid and reliable neurochemical and molecular techniques for very early diagnosis, efforts which dovetail with a clinical trial of very early copper histidine treatment for affected infants. We use cell biological, molecular, and biochemical approaches to characterize enrolled patients and to correlate with neurodevelopmental outcomes. Confocal imaging of patient fibroblasts is used to assess quantity and localization of mutant Menkes gene products. The blood-brain barrier poses a challenging treatment obstacle in many Menkes disease patients, and we hypothesized a molecular basis for treatment responsivity in the minority of patients (about 1 in 5) who respond successfully (normal neurodevelopmental outcomes) to early copper histidine. These patients have mutations that enable at least some residual copper transport to the developing brain. Consequently, we are developing alternative therapeutic approaches, including gene therapy, that bypass the blood-brain barrier. As above, although mean survival is significantly enhanced, only a fraction of patients with Menkes disease (~30%) show good or excellent neurological outcomes when treated with copper injections beginning very early in life. The main reason for the disparate outcomes appears to be the amount of residual Atp7a function in these individuals. Those with less Atp7a function are unlikely to respond optimally to copper injection treatment. Therefore, new therapeutic strategies need to be developed for the large percentage of patients who have little or no residual Atp7a function and currently have no ideal treatment options. Toward this goal, the Unit devised an animal study proposal, recently approved, to assess the efficacy of adeno-associated virus (AAV) gene therapy in mouse models of Menkes disease. Gene therapy offers an alternative method for treating genetic disorders, such as Menkes disease, that result from the loss of a protein function. This method provides a way to restore the lost protein function in an affected individual and has been successfully employed to treat a number of genetic disorders in animal models of disease and in at least one human disease. Several mouse models of Menkes disease exist. Like the human patients, the severity of the disease varies substantially from model to model, and only some models respond to injections of copper. Thus, when used in combination, these models provide an effective tool to evaluate the effects of disease severity on the effectiveness of novel treatments. The goal of our study is to evaluate the use of recombinant adeno-associated virus serotype 5 (rAAV5) as a gene therapy vector in two mouse models of Menkes disease, one responsive to early copper therapy and the other which does not respond to early copper therapy. Efficacy will be evaluated by examining life span and several biochemical parameters that are abnormal in Menkes disease mice and Menkes disease patients. If successful, these experiments will lay the groundwork for more effective therapies for a higher percentage of human patients with Menkes disease.