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-treats 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 1–4 h, both β-gal:RTB and RTB:β-gal were processed to the ~64 kDa “activated” β-gal form; the RTB lectin was cleaved and rapidly degraded. The activated β-gal was still detected at 48 h 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.

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

“…Maximal uptake capacity, calculated using Michaelis-Menten analysis (V max ), is defined as the maximal intracellular activity achieved after saturation of the system. As expected based on mechanisms of adsorptive-mediated endocytosis, the enzyme:RTB products showed much lower uptake saturability and higher uptake capacity compared to the analogous mammalian-cell-derived human enzymes that depend on the M6P receptor to mediate endocytosis (e.g 20-fold higher IDUA intracellular activity on IDUA -/fibroblast) [58][59][60]. Upon delivery of the enzyme:RTB product to lysosomes, the RTB lectin component is rapidly cleaved and degraded; the respective enzyme precursors are processed to their typical mature forms.…”

“…In vitro uptake studies using human patient fibroblasts confirmed efficient cell uptake and lysosomal delivery by a mechanism independent of MMR and M6PR. However, adding lactose to the culture media inhibited the uptake of fusion protein by 99%, confirming that the RTB's lectin activity is the main uptake driver [58][59][60]. Uptake kinetics of recombinant enzymes can be measured on cultured cells by measuring intracellular activity after incubation with increasing concentrations of protein.…”

“…These enzymes include α-L-iduronidase (IDUA:RTB) for MPS I -Hurler, N-sulfoglucosamine sulfohydrolase (SGSH:RTB) for MPS IIIA, and β-galactosidase (β-gal:RTB) for GM1 gangliosidosis. The RTB:enzyme fusions were synthesized in a plant-based bioproduction system [58][59][60], which supports N-linked glycosylation for both the RTB carrier (2 sites) and its human glycoprotein cargo. While plants synthesize both complex and mannose-terminated glycans, they do not produce M6P-glycans.…”

“…The two carbohydrate recognition domains of RTB are separated by approximately 75Å [73], making it difficult for a single antibody to occlude both domains simultaneously [55]. Rapid RTB degradation within lysosomes may also contribute to low immunogenicity [59,63,70]. Of significance for ERT strategies, the presence of ADA directed against the cargo protein did not block RTB-mediated uptake of the fusion protein (in contrast to mcd-enzyme) into patient fibroblasts or in enzyme-immunized KO mice [64].…”

Infantile gangliosidoses: Mapping a timeline of clinical changes

“…Research is underway in animal models evaluating gene therapy technologies and intravenous enzyme replacement therapies (ERT) [5,11]. Palliative care approaches for patients with gangliosidoses, meanwhile, continue to improve.…”