title: Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion.
abstract: New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid β-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid β-galactosidase enzyme were produced using a plant-based bioproduction platform. β-gal:RTB and RTB:β-gal fusion products retained both lectin activity and β-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived β-galactosidase. In contrast, plant-derived β-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native β-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both β-gal:RTB and RTB:β-gal were processed to the ~64kDa "activated" β-gal form; the RTB lectin was cleaved and rapidly degraded. The activated β-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of β-gal:RTB supported significantly greater accumulation of β-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived β-gal. These data demonstrate that plant-made β-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA.
authors.forename: Jose
authors.initials: J
authors.lastname: Condori
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA.
authors.forename: Walter
authors.initials: W
authors.lastname: Acosta
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Ayala
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA.
authors.forename: Varun
authors.initials: V
authors.lastname: Katta
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Flory
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA; Arkansas Biosciences Institute & Dept. Biological Sciences, P.O Box 639, Arkansas State University-Jonesboro, State University, AR 72467, USA.
authors.forename: Reid
authors.initials: R
authors.lastname: Martin
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Radin
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA; Arkansas Biosciences Institute & Dept. Biological Sciences, P.O Box 639, Arkansas State University-Jonesboro, State University, AR 72467, USA. Electronic address: cramer@biostrategies-lc.com.
authors.forename: Carole L
authors.initials: CL
authors.lastname: Cramer
authors.affiliation: BioStrategies LC, P.O. Box 2428, State University, AR 72467, USA.
authors.forename: David N
authors.initials: DN
authors.lastname: Radin
mesh_terms: D002478:Cells, Cultured; D056947:Enzyme Replacement Therapy; D005347:Fibroblasts / Q000201:enzymology; D016537:Gangliosidosis, GM1 / Q000188:drug therapy*; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D037121:Plant Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; C061901:GLB1 protein, human; D001616:beta-Galactosidase
keywords: GM1-gangliosidosis; Lectin-mediated delivery; Lysosomal β-galactosidase; Plant-based bioproduction; RTB lectin