Citation

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 -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 -galactosidase enzyme were produced using a plant-based bioproduction platform. -gal:RTB and RTB:-gal fusion products retained both lectin activity and -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 -galactosidase. In contrast, plant-derived -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 -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 -gal:RTB and RTB:-gal were processed to the ~64kDa activated -gal form; the RTB lectin was cleaved and rapidly degraded. The activated -gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of -gal:RTB supported significantly greater accumulation of -galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived -gal. These data demonstrate that plant-made -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.