Disorders of Copper Transport

门克斯病是一种由编码进化保守的铜转运ATPase基因缺陷引起的X连锁隐性铜转运障碍。在哺乳动物中，该基因产物起到细胞内泵的作用，将铜输送到跨高尔基体间隙，并结合到需要铜的酶中，也介导铜从细胞中外流。这种疾病出现在婴儿期，表现为发育迟缓、无法茁壮成长、神经退化和过早死亡(通常在3岁之前)。我们在这种疾病方面的工作包括开发快速可靠的神经化学和分子技术进行非常早期的诊断，这些努力与对受影响婴儿进行非常早期的组氨酸铜治疗的临床试验相吻合。我们使用细胞生物学、分子和生化方法来确定入选患者的特征，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的Menkes基因产物的数量和定位。血脑屏障在许多Menkes病患者中构成了一个具有挑战性的治疗障碍，我们假设了少数对早期组氨酸铜有成功反应(正常神经发育结果)的患者(约1/5)的治疗反应性存在分子基础。这些患者的突变至少使一些残留的铜能够运输到发育中的大脑。因此，我们正在开发包括基因治疗在内的替代治疗方法，绕过血脑屏障。 如上所述，尽管平均存活率显著提高，但只有一小部分门克斯病患者(约30%)在生命早期开始注射铜时表现出良好或优秀的神经预后。结果不同的主要原因似乎是这些个体的ATP7A功能残留量。那些ATP7A功能较差的人不太可能对铜注射治疗做出最佳反应。因此，需要为大部分患者开发新的治疗策略，这些患者几乎没有或没有残留的ATP7A功能，目前没有理想的治疗选择。为了实现这一目标，该单位设计了一项动物研究提案，最近获得批准，以评估腺相关病毒(AAV)基因治疗在Menkes病小鼠模型中的疗效。 基因疗法为治疗遗传性疾病提供了另一种方法，如门克斯病，这种疾病是由蛋白质功能丧失引起的。这种方法提供了一种在受影响的个体中恢复丢失的蛋白质功能的方法，并已成功地用于治疗动物疾病模型中的一些遗传疾病和至少一种人类疾病。存在几种门克斯病的小鼠模型。与人类患者一样，不同型号的疾病严重程度有很大差异，只有部分型号对注射铜有反应。因此，当结合使用时，这些模型提供了一种有效的工具来评估疾病严重程度对新疗法有效性的影响。本研究的目的是评价重组腺相关病毒血清型5(RAAV5)作为基因治疗载体在两种Menkes病小鼠模型中的应用，其中一种对早期铜治疗有效，另一种对早期铜治疗无效。疗效将通过检测寿命和在孟克斯病小鼠和门克斯病患者中异常的几个生化参数来评估。如果成功，这些实验将为更高比例的门克斯病患者提供更有效的治疗奠定基础。

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.