title: A peptide-linked recombinant glucocerebrosidase for targeted neuronal delivery: Design, production, and assessment.
abstract: Although recombinant glucocerebrosidase (GCase) is the standard therapy for the inherited lysosomal storage disease Gaucher's disease (GD), enzyme replacement is not effective when the central nervous system is affected. We created a series of recombinant genes/proteins where GCase was linked to different membrane binding peptides including the Tat peptide, the rabies glycoprotein derived peptide (RDP), the binding domain from tetanus toxin (TTC), and a tetanus like peptide (Tet1). The majority of these proteins were well-expressed in a mammalian producer cell line (HEK 293F). Purified recombinant Tat-GCase and RDP-GCase showed similar GCase protein delivery to a neuronal cell line that genetically lacks the functional enzyme, and greater delivery than control GCase, Cerezyme (Genzyme). This initial result was unexpected based on observations of superior protein delivery to neurons with RDP as a vector. A recombinant protein where a fragment of the flexible hinge region from IgA (IgAh) was introduced between RDP and GCase showed substantially enhanced GCase neuronal delivery (2.5 times over Tat-GCase), suggesting that the original construct resulted in interference with the capacity of RDP to bind neuronal membranes. Extended treatment of these knockout neuronal cells with either Tat-GCase or RDP-IgAh-GCase resulted in an >90% reduction in the lipid substrate glucosylsphingosine, approaching normal levels. Further in vivo studies of RDP-IgAh-GCase as well as Tat-GCase are warranted to assess their potential as treatments for neuronopathic forms of GD. These peptide vectors are especially attractive as they have the potential to carry a protein across the blood-brain barrier, avoiding invasive direct brain delivery.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA; Research Service, Veterans Affairs Maryland Health Care Service, Baltimore, MD, USA. Electronic address: pgramli1@jhu.edu.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Gramlich
authors.affiliation: Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Wendy
authors.initials: W
authors.lastname: Westbroek
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, MD, USA.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Feldman
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, MD, USA.
authors.forename: Ola
authors.initials: O
authors.lastname: Awad
authors.affiliation: Research Service, Veterans Affairs Maryland Health Care Service, Baltimore, MD, USA; Department of Molecular Medicine, University of Maryland School of Medicine, MD, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Mello
authors.affiliation: Research Service, Veterans Affairs Maryland Health Care Service, Baltimore, MD, USA.
authors.forename: Mary P
authors.initials: MP
authors.lastname: Remington
authors.affiliation: Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Division of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
authors.forename: Wujuan
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Ellen
authors.initials: E
authors.lastname: Sidransky
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
authors.affiliation: Research Service, Veterans Affairs Maryland Health Care Service, Baltimore, MD, USA; Department of Neurology, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Fishman
mesh_terms: D001812:Blood-Brain Barrier / Q000187:drug effects; D002478:Cells, Cultured; D015195:Drug Design; D005962:Glucosylceramidase / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D010446:Peptide Fragments / Q000378:metabolism*; D011609:Psychosine / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D011609:Psychosine; C035742:sphingosyl beta-glucoside; D005962:Glucosylceramidase
keywords: Enzyme-replacement therapy; Gaucher’s disease (GD); Glucocerebrosidase; Linker design; Rabies-derived peptide