Targeting Macromolecules to CNS and Other Hard-to-Treat Organs Using Lectin-Mediated Delivery

Abstract

The greatest challenges for therapeutic efficacy of many macromolecular drugs that act on intracellular are delivery to key organs and tissues and delivery into cells and subcellular compartments. Transport of drugs into critical cells associated with disease, including those in organs protected by restrictive biological barriers such as central nervous system (CNS), bone, and eye remains a significant hurdle to drug efficacy and impacts commercial risk and incentives for drug development for many diseases. These limitations expose a significant need for the development of novel strategies for macromolecule delivery. RTB lectin is the non-toxic carbohydrate-binding subunit B of ricin toxin with high affinity for galactose/galactosamine-containing glycolipids and glycoproteins common on human cell surfaces. RTB mediates endocytic uptake into mammalian cells by multiple routes exploiting both adsorptive-mediated and receptor-mediated mechanisms. In vivo biodistribution studies in lysosomal storage disease models provide evidence for the theory that the RTB-lectin transports corrective doses of enzymes across the blood-brain barrier to treat CNS pathologies. These results encompass significant implications for protein-based therapeutic approaches to address lysosomal and other diseases having strong CNS involvement.

Keywords: BBB; RTB; enzyme replacement therapy (ERT); lectin; lysosomal storage diseases, CNS; macromolecule transport.

Figure 1

Pathways utilized by RTB lectin across the blood–brain barrier. Blood-borne lectin:cargo fusion product reaches the cerebral endothelial membrane or blood–brain barrier (BBB) when administered intravenously. BBB is a capillary endothelial layer formed by specialized cells with highly selective expression of receptors and transporters designed to maintain the integrity of the chemical environment within the brain. These cells are glued together with tight junctions that restrict the penetration of water-soluble compounds. RTB lectin binds to any glycoprotein or glycolipids with exposed galactose residues, which are highly abundant on the apical surface of cell membranes, triggering endocytosis and transcytosis. This typically takes the form of adsorptive-mediated transcytosis (AMT). However, because RTB also binds to the glycan component of an existing surface receptor, RTB can exploit receptor-mediated endocytosis/transcytosis (RMT) mechanisms to deliver cargo across the BBB.

Figure 2

Delivery of IDUA:RTB to CNS. (A) Comparative imaging of brains following treatment with labeled product. Brains of mice harvested 24 h after intravenous administration of 2mg/kg of IRDye^® 800CW-labelled IDUA:RTB or mammalian-cell-derived IDUA (mcd-IDUA, R&D Systems) were analyzed by infrared fluorescence (800nm) imaging. A non-treated mouse was processed in parallel as control. (B–E). Immunostaining of brain using anti-RTB antibodies. Brain slices from IDUA^-/- mice treated intravenously with 2mg/kg of IDUA:RTB or PBS were stained with anti-RTB antibodies (Green) and counterstained with DAPI (Blue). Images were acquired and processed using identical settings. RTB protein was detected in hippocampus (B) and cerebellum (D) of treated animals. Sections from PBS treated animals were carried in parallel as controls (C,E).