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: Paul A Gramlich, Wendy Westbroek, Ricardo A Feldman, Ola Awad, Nicholas Mello, Mary P Remington, Ying Sun, Wujuan Zhang, Ellen Sidransky, Michael J Betenbaugh, Paul S Fishman
• Journal: Journal of biotechnology (J Biotechnol) Vol. 221 Pg. 1-12 (Mar 10 2016) ISSN: 1873-4863 [Electronic] Netherlands
• PMID: 26795355 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2016 Elsevier B.V. All rights reserved.

Chemical References

• Peptide Fragments
• Recombinant Proteins
• Psychosine
• sphingosyl beta-glucoside
• Glucosylceramidase

Topics

• Blood-Brain Barrier (drug effects)
• Cells, Cultured
• Drug Design
• Glucosylceramidase (genetics, metabolism)
• HEK293 Cells
• Humans
• Neurons (cytology, drug effects)
• Peptide Fragments (metabolism)
• Psychosine (analogs & derivatives, metabolism)
• Recombinant Proteins (genetics, metabolism, pharmacology)