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.
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