Inherited Disorders of Copper Transport

1. ATP7A基因治疗门克斯病小鼠模型。门克斯病是一种由铜转运体基因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3岁前死亡

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 3 years of age. Currently, the only available treatment is daily subcutaneous copper histidine injections, which however, is not effective in 75% of affected individuals, due to severe loss-of-function mutations. To develop a more complete treatment for this illness, we previously developed a viral gene therapy approach in the mottled-brindled mouse model of Menkes, to provide working copies of a reduced size version of ATP7A (rsATP7A), which can be accommodated by the AAV backbone. We showed that AAV serotype 5 (AAV5) co-administered with copper chloride into the cerebrospinal fluid (CSF) could rescue mutant mice. In the current 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. We compared three different AAV9 and rh10 doses in combination with subcutaneous Cu, and found that intermediate (5.0x109 vg) and high (1.6x1010 vg) doses of AAV9-rsATP7A were associated with highest rates of survival. CSF-directed AAV9 plus subcutaneous Cu normalized somatic growth and neurobehavioral outcomes (Wire hang and rotarod). 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 further 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 impaired ATP7A trafficking in response to copper in cells. These findings revealed an essential role for AT-1 in the proper post-translational modification of numerous proteins, without which normal brain development is interrupted. 3. We have also at work on the basic science and clinical delineation of MEDNIK syndrome, caused by mutations in an adaptor protein 1 subunit that affects intracellular trafficking of ATP7B and to a lesser extent, ATP7A.