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Viral Gene Therapy for Neurometabolic Disorders

Stephen G. Kaler, MD, MPH
  • 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
  • Stanley Ewala, African Leadership Academy Summer Student

The Section of Translational Neuroscience strives to dissect and understand mechanisms of neurometabolic disease and to use the knowledge gained to develop new treatments, including gene therapy, for difficult illnesses. Patients and families affected by inborn errors of metabolism provide the impetus for scientific inquiry. In addition to molecular genetics, the laboratory employs model organisms (mouse, zebrafish, yeast) and cellular, biochemical, and biophysical approaches and conducts clinical trials. The laboratory currently focuses on (i) viral gene therapy in two mouse models of human monogenic neurometabolic disease (Menkes disease and alpha-mannosidosis); and (ii) molecular mechanisms responsible for isolated motor neuron degeneration associated with the copper transporter ATP7A.

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

Kaler lab members

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Section on Translational Neuroscience

Left to right: Diego Martinelli, MD, PhD; Reina Haddad, PhD; Stephen Kaler, MD; Eun-Young Choi, PhD; Ling Yi, PhD

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 structures that project into the ventricles of the brain. Besides creating the blood-CSF barrier, the polarized epithelia of the CP produce CSF by transporting water, ions, and proteins into the ventricles from the blood. We hypothesize that lysosomal storage diseases, a different category of neurometabolic disease, will benefit from a CP–targeted gene therapy approach, given that recombinant rAAV transduction results in sustained episomal transgene expression, that CP epithelia have a slow turnover rate, and that CSF flow extends throughout the ventricular system to the subarachnoid space, from which molecules ultimately reach the entire brain.

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 histidine, the compound and mode of administration we employ in our Menkes disease clinical trial. Preliminary results suggest improved performance on tests of motor coordination and muscle strength compared with rAAV5 and augur well for future FDA IND (investigational new drug) approval, which will permit a clinical trial of this approach in selected Menkes disease patients with profound 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. Our 2012 NIH Bench-to-Bedside Award, entitled “Choroid plexus-directed gene therapy: Toward novel clinical management of lysosomal storage disease,” supported evaluation of this hypothesis.

In collaboration with John Wolfe, we will now extend these studies through a recently awarded U01 grant in animal models of alpha-mannosidosis, a prototypical LSD. 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 binding regions between p97/VCP and ATP7AT994I. We are also investigating the trafficking of ATP7A in conjunction with bench and clinical studies of MEDNIK syndrome. Interaction with clathrin-coated vesicles and adaptor protein complexes, as well as post-translational modification by palmitoylation, are among topics under active investigation. We hope that such studies will 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

Patent filed

Patent 4239-81164-01, Identification of subjects likely to benefit from copper treatment. International Filing Date: 06 October 2008

Additional Funding

  • National Mucopolysaccharidosis Society
  • NIH Bench-to-Bedside Award
  • U01-CH-079066-01. Choroid plexus-directed gene therapy for alpha-mannosidosis (pending)


  1. Yi L, Donsante A, Kennerson ML, Mercer JFB, Garbern JY, Kaler SG. Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy. Hum Mol Genet 2012;21:1794-1807.
  2. Donsante A, Sullivan P, Goldstein DS, Brinster LR, Kaler SG. Systemic L-threo dihydroxyphenylserine corrects neurochemical abnormalities in a mouse model of Menkes disease. Ann Neurol 2013;73:259-265.
  3. 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.
  4. 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;1-4.
  5. Bandmann O, Weiss KH, Kaler SG. Wilson's disease and other copper disorders. Lancet Neurol 2015;in press.


  • Lauren Brinster, VMD, Division of Veterinary Resources, Office of Research Services, NIH, Bethesda, MD
  • Jose Centeno, PhD, Walter Reed Army Medical Center, Silver Spring, MD
  • Christopher Chang, PhD, University of California-Berkeley, Berkeley, CA
  • John Chiorini, PhD, Molecular Physiology and Therapeutics Branch, NIDCR, Bethesda, MD
  • John Christodoulou, MD, University of Sydney, Sydney, Australia
  • Laurence Colleaux, PhD, INSERM, Paris, France
  • Soma Das, PhD, University of Chicago, Chicago, IL
  • Patricia Dickson, MD, Harbor-UCLA Medical Center, Los Angeles, California
  • Tohru Fukai, MD, PhD, University of Illinois at Chicago, Chicago, IL
  • David S. Goldstein, MD, PhD, Clinical Neurosciences Program, NINDS, Bethesda, MD
  • George Grimes, RPh, Pharmaceutical Development Service, Clinical Center, NIH, 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
  • Nicholas Patronas, MD, Diagnostic Radiology Department, Clinical Center, NIH, Bethesda, MD
  • Joseph Prohaska, PhD, University of Minnesota, Duluth, MN
  • Yulia Pushkar, PhD, Purdue University, West Lafayette, IN
  • Paul Saftig, PhD, Christian-Albrechts-Universität, Kiel, Germany
  • Alan N. Schechter, MD, Molecular Medicine Branch, NIDDK, Bethesda, MD
  • Judith Starling, RPh, Pharmaceutical Development Service, Clinical Center, NIH, Bethesda, MD
  • Peter Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
  • Bryan J. Traynor, MD, PhD, Laboratory of Neurogenetics, NIA, Bethesda, MD
  • Vinzenz Unger, PhD, Northwestern University, Evanston, IL
  • John Wolfe, VMD, PhD, University of Pennsylvania, Philadelphia, PA
  • Wei Zheng, PhD, Purdue University, West Lafayette, IN


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