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.

The Complicated Relationship between Gaucher Disease and Parkinsonism: Insights from a Rare Disease

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Targeted therapy of intracranial glioma model mice with curcumin nanoliposomes

“…Among the targeting carriers, peptide derivatives of rabies virus glycoprotein (RVG) are suggested as potential delivery ligands to transport nanocarriers for treating various brain diseases, such as Alzheimer's disease, ischemic injury, protein misfolding diseases, and so on. 10 We previously used an RVG peptide derivative, RDP, to deliver proteins and nanoparticles into the brain for disease treatment and cell imaging. 11,12 The peptide shows high BBB-penetrating capability when it is conjugated to macromolecular cargoes.…”

Current Strategies for the Delivery of Therapeutic Proteins and Enzymes to Treat Brain Disorders

“…More specifically, large screenings of different ligands (i.e., small molecules, peptides, proteins, etc.) have been analyzed for the ability to deliver enzyme cargo into various cell types (Acosta, Ayala, Dolan, & Cramer, 2015;Eiamphungporn, Yainoy, & Prachayasittikul, 2014;Gramlich et al, 2016). One such example used fluorescent-labeled cholera toxin B (CTB) to detect GM1-gangliosidosis and intercellular adhesion molecule 1 (icam1) to deliver enzymes into fibroblasts (Rappaport, Garnacho, & Muro, 2014).…”