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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 Annual Report of the Division of Intramural Research

Viral Gene Therapy for Neurometabolic Disorders

Stephen G. Kaler
  • Stephen G. Kaler, MD, Head, Section on Translational Neuroscience
  • Ling Yi, PhD, Staff Scientist
  • Eun-Young Choi, PhD, Postdoctoral Fellow
  • Marie-Reine Haddad, PhD, Postdoctoral Fellow
  • Diego Martinelli, MD, PhD, Visiting Fellow
  • Kristen Stevens, RN, CPNP, Research Nurse Practitioner

The Section of Translational Neuroscience strives to dissect and understand mechanisms of human neurometabolic disease and to use the knowledge gained to develop new treatments, including gene therapy, for difficult illnesses. Core values including integrity, humility, hard work with purpose, moving forward rapidly, concern for patients and their families, and mutual support among laboratory members guide the Section’s efforts. In addition to molecular genetics, we employ model organisms (mouse, zebrafish, yeast) and cellular, biochemical, and biophysical approaches, and we conduct clinical trials. Preclinical work in the laboratory currently focuses on viral gene therapy in mouse models of Menkes disease and lysosomal storage disease. On the basic neuroscience side, we pursue the molecular mechanisms responsible for certain forms of motor neuron degeneration.

Adeno-associated viral (AAV) gene therapy for neurometabolic diseases

Brain-directed intracerebroventricular (ICV) recombinant adeno-associated virus serotype 5 (rAAV5) gene therapy in mo-br male mice, a mouse model of Menkes disease, resulted in rescue from early lethality by efficient transduction of choroid plexus (CP) epithelia. Rescued animals manifested elevation of brain copper concentrations and improved activity of dopamine-beta-hydroxylase, a copper-dependent enzyme. CP tissues are highly vascularized neuroectoderm-derived structures that project into the ventricles of the brain. Besides creating the blood–CSF (cerebrospinal fluid) barrier, the polarized epithelia of the CP produce CSF by transporting water, ions, and proteins into the ventricles from the blood. We hypothesized that lysosomal storage diseases, a different category of neurometabolic diseases, would benefit from a CP–targeted gene therapy approach, given that recombinant rAAV transduction results in sustained episomal transgene expression and that CP epithelia have a negligible turnover rate. Furthermore, lysosomal enzyme secreted into CSF should reach the entire brain, with delivery enhanced by cross-correction.

To further refine the mo-br rescue and plan a path forward to human application, we are currently pursuing an approach that combines brain-directed ICV administration of rAAV9 or rAAVrh10, more potent AAV serotypes than rAAV5, with subcutaneous injections of copper histidinate, the compound and mode of administration we employ in our Menkes disease clinical trial. Results suggest improved survival and performance quality using rAAV9 and subcutaneous copper and thus augur well for future FDA IND (investigational new drug) approval, which will permit a first-in-human clinical trial of this approach in Menkes disease patients with complete loss-of-function mutations in the ATP7A copper transporter.

Choroid plexus–targeted gene therapy may be especially relevant to gene therapy of lysosomal storage diseases (LSDs) that impact the CNS. Intrathecal delivery (by injecting enzyme into the cerebrospinal fluid during a spinal tap) of recombinant lysosomal enzymes has been successful in ameliorating LSDs in some animal studies and in human clinical trials. However, a major drawback to this approach is the need for repeated (e.g., monthly) intrathecal injections owing to the short half-lives of recombinant enzymes. An alternative strategy is to remodel CP epithelial cells with an AAV vector containing the cDNA for the enzyme of interest. Given the extremely low turnover rate of CP epithelia, the approach could generate a permanent source of enzyme production for secretion into the CSF and penetration into cerebral and cerebellar structures. For the project supported by our 2014 NIH U01 Award, entitled “Choroid plexus-directed gene therapy for Alpha-mannosidosis," in collaboration with John Wolfe, we will use both mouse and cat models of alpha-mannosidosis to evaluate choroid plexus transduction by several rAAV vectors as well as post-treatment alpha-mannosidase concentration and distribution in brain. Studies in the mouse model (obtained by NICHD through a Material-CRADA [Cooperative Research and Development Agreement] with the University of Kiel, Germany) will require less virus and the mice will be easier to breed. The cat model (housed at the University of Pennsylvania) features a gyrencephalic brain more similar to the human brain. Thus, the study of these two models will be complementary. In a related study, we are collaborating with Patricia Dickson to compare the efficiency of CP–mediated lysosomal enzyme production with intrathecal enzyme replacement in a mouse models of mucopolysaccharidosis type 3B (Sanfilippo syndrome).

Disease mechanisms that underlie ATP7A–related distal motor neuron degeneration

The P-type ATPase ATP7A regulates cellular copper homeostasis by its activity at the trans-Golgi network (TGN) and plasma membrane (PM), with its location normally governed by intracellular copper concentration. In addition to causing Menkes disease, defects in ATP7A may lead to the disease variants occipital horn syndrome and ATP7A–related distal motor neuropathy, a newly discovered adult-onset condition for which the precise pathophysiology has been obscure. We characterized the two ATP7A motor-neuropathy mutations (T994I, P1386S) and identified molecular mechanisms for abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence (TIRF) microscopy indicated a shift in steady-state equilibrium of ATP7AT994I and ATP7AP1386S, with excess PM accumulation. Transfection of 293T cells and NSC-34 motor neurons with the mutant alleles tagged with Venus fluorescent protein also showed enhanced PM localization and delayed endocytic retrieval of the mutant alleles to the TGN.

Immunoprecipitation assays revealed an abnormal interaction between ATP7AT994I and p97/VCP (valosin-containing protein), a protein that normally associates with the endocytic trafficking proteins clathrin and early endosomal autoantigen 1 (EEA1) and which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (siRNA) knockdown of p97/VCP corrected ATP7AT994I mislocalization. VCP did not interact significantly with ATP7AP1386S, the other mutant allele associated with the motor-neuropathy phenotype. However, flow cytometry documented that non-permeabilized ATP7AP1386S fibroblasts bound to a carboxyl-terminal ATP7A antibody, a finding consistent with partially destabilized insertion of the eighth transmembrane helix and relocation of the di-leucine endocytic retrieval signal from the cytosolic to the extracellular face of the PM. The findings illuminated mechanisms underlying ATP7A–related distal motor neuropathy, established a common link between genetically distinct forms of motor neuron disease, clarified the normal process of ATP7A endocytosis, and highlighted the possible functional role of ATP7A in the peripheral nervous system.

We recently extended our studies on this topic to elucidate the specific association of adaptor protein complexes 1 and 2 (AP-1, AP-2) with normal trafficking of ATP7A. We are also investigating the MEDNIK syndrome (caused by mutations in an AP-1 subunit) with animal models (mouse, zebrafish) and clinical studies of affected patients. The role of post-translational modifications, such as acetylation and palmitoylation, are among other topics under active investigation. We hope that these studies will help resolve unanswered questions concerning the molecular mechanisms of altered copper ATPase intracellular trafficking.

Clinical protocols

  1. Principal Investigator, 90-CH-0149: Early copper histidine treatment in Menkes disease: relationship of molecular defects to neurodevelopmental outcomes
  2. Associate Investigator, 02-CH-0023: Studies of pediatric patients with metabolic or other genetic disorders
  3. Principal Investigator, 09-CH-0059: Molecular bases of response to copper treatment in Menkes disease, related phenotypes, and unexplained copper deficiency
  4. Principal Investigator, 14-CH-0106: Clinical Biomarkers in Alpha-mannosidosis
  5. Associate Investigator, Partnership for Research on Ebola Virus in Liberia PREVAIL III (15-I-N122); Monrovia, Liberia
  6. Sub-Investigator, Partnership for Research on Ebola Virus in Liberia PREVAIL I (15-I-N071); Monrovia, Liberia
  7. Associate Investigator; Phase II Study of AAV9-GAA Gene Transfer in Pompe Disease (NHLBI U01 Award, Co-PIs: B. Byrne/A. Arai)

Patents filed

  • Patent 4239-81164-01: Identification of subjects likely to benefit from copper treatment. International Filing Date: 06 October, 2008
  • Patent 62/244,594: Codon-optimized reduced-size ATP7A cDNA and uses for treatment of copper transport disorders. Filing date: 21 October, 2015

Additional Funding

  • 2015 NIH Bench-to-Bedside Award (Kaler/Petris/Feldman)
  • U01-CH-079066-01. Choroid plexus-directed gene therapy for alpha-mannosidosis
  • U01-HL121842-01A1. Phase II Study of AAV9-GAA Gene Transfer in Pompe Disease


  1. Haddad MR, Donsante A, Zerfas P, Kaler SG. Fetal mouse brain-directed AAV gene therapy results in rapid, robust, and persistent transduction of choroid plexus epithelia. Mol Ther Nucleic Acids 2013; 2:101-108.
  2. Kaler SG. Neurodevelopment and brain growth in classic Menkes disease is influenced by age and symptomatology at initiation of copper treatment. J Trace Elem Med Biol 2014; 28:427-430.
  3. Bandmann O, Weiss KH, Kaler SG. Wilson's disease and other neurological copper disorders. Lancet Neurol 2015; 14:103-113.
  4. Choi EY, Patel K, Haddad MR, Yi L, Kaler SG. Duplication of ATP7A exons 1-7 neither impairs gene expression nor causes a Menkes disease phenotype. JIMD Rep 2015; 20:57-63.
  5. Ling Y, Kaler SG. Direct interactions of adaptor protein complexes 1 and 2 with the copper transporter ATP7A mediate its anterograde and retrograde trafficking. Hum Mol Genet 2015; 24:2411-2425.


  • Eva Baker, MD, PhD, Radiology and Imaging Sciences, NIH Clinical Center, Bethesda, MD
  • Andy Bhattacharjee, PhD, Parabase Genomics, Boston, MA
  • Lauren Brinster, VMD, Division of Veterinary Resources, Office of Research Services, NIH, Bethesda, MD
  • Sara Cathey, MD, Greenwood Genetics Center, Greenwood, SC
  • Jose Centeno, PhD, Walter Reed Army Medical Center, Silver Spring, MD
  • John Chiorini, PhD, Molecular Physiology and Therapeutics Branch, NIDCR, Bethesda, MD
  • John Christodoulou, MD, University of Sydney, Sydney, Australia
  • Patricia Dickson, MD, Harbor-UCLA Medical Center, Los Angeles, California
  • David S. Goldstein, MD, PhD, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • Courtney Holmes, CMT, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • Peter Huppke, MD, Georg August Universität, Göttingen, Germany
  • Marina L. Kennerson, PhD, University of Sydney, Sydney, Australia
  • Robert Kotin, PhD, University of Massachusetts Medical Center, Worcester, MA
  • Julian Mercer, PhD, Deakin University, Melbourne, Australia
  • Avindra Nath, MD, Section of Infections of the Nervous System, NINDS, Bethesda, MD
  • Richard Parad, MD, MPH, Brigham and Women's Hospital, Harvard Medical School, Boston, MA
  • Nicholas Patronas, MD, Diagnostic Radiology Department, Clinical Center, NIH, Bethesda, MD
  • Michael Petris, PhD, University of Missouri-Columbia Columbia, MO
  • Joseph Prohaska, PhD, University of Minnesota, Duluth, MN
  • Martina Ralle, PhD, Oregon Health Sciences University, Portland, OR
  • Evan Sadler, MD, PhD, Washington University, St. Louis, MO
  • Paul Saftig, PhD, Christian-Albrechts-Universität, Kiel, Germany
  • Alan N. Schechter, MD, Molecular Medicine Branch, NIDDK, Bethesda, MD
  • Judith Starling, RPh, Pharmaceutical Development Section, Clinical Center, NIH, Bethesda, MD
  • Peter Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
  • Wen-Hann Tan, MD, Boston Children's Hospital, Boston, MA
  • John Wolfe, VMD, PhD, University of Pennsylvania, Philadelphia, PA

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