Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion
Introduction
GM1-gangliosidosis is a lysosomal storage disease (LSD) linked to mutations in the GLB1 gene that encodes lysosomal acid β-D-galactosidase (β-gal; EC 3.2.1.23) [1]. The hallmark of GM1-gangliosidosis (GM1) is the progressive buildup of glycosphingolipid GM1 ganglioside, particularly in neurons, which results in widespread neurodegeneration. Some therapeutic modalities (e.g. substrate reduction therapy, and ex vivo and in vivo gene therapies) have been implemented in the animal model [2], [3], [4], [5], [6]. Enzyme replacement therapies (ERTs) have been efficacious for many LSDs [7], [8], [9], an effective ERT has not yet been developed or tested for this disease. Barriers for development of effective ERTs for GM1 and other lysosomal diseases with strong central nervous systems (CNS) involvement remain a) the need to deliver corrective enzyme doses to the brain and b) the continued high cost of developing recombinant protein-drugs and bringing them to patients (reviewed [10], [11]). In this study, we tested a lectin-based “enzyme delivery module” providing receptor-independent routes of cell entry and used a rapid transient plant-based expression platform with the potential to address bioproduction costs.
All currently approved lysosomal disease ERTs exploit either the mannose-6-phosphate receptor (M6PR) or the high-mannose receptor (MMR) to direct endocytosis and lysosomal delivery of the corrective enzyme. While effective in treating visceral disease manifestations, these products do not effectively cross the blood–brain-barrier (BBB) to treat pathologies of the CNS and show limited correction in other so-called “hard-to-treat” tissues such as heart, lung and eye. In contrast to receptor-mediated endo/transcytosis, the plant lectin RTB initiates endocytotic uptake primarily by binding to the abundant galactose-terminated glycoproteins and glycolipids present on mammalian cell surfaces, exploiting all known endocytic mechanisms to access cells [12], [13]. This non-specific adsorptive-mediated endocytosis may provide an alternative enzyme delivery strategy with broader biodistribution and the potential to impact pathologies of these “hard-to-treat” tissues [14]. The lectin RTB (ricin toxin B-subunit) is the non-toxic carbohydrate-binding subunit of ricin that is responsible for mediating transport both into and within target cells. The uptake and trafficking of RTB, a galactose/galactosamine-specific lectin from Ricinus communis, has been extensively studied [15], [16], [17], [18]. RTB has high affinity for surface glycolipids and glycoproteins providing access to a broad array of cells and enters cells by multiple endocytotic routes (e.g., clathrin-dependent and -independent; dynamin-dependent and -independent; caveolae-dependent and -independent; macropinocytosis; MMR-mediated) [15], [16], [17], [18]. Upon endocytosis, RTB traverses preferentially to lysosomes or cycles back to the cell membrane (transcytosis pathway), with less than 5% moving “retrograde” to the endoplasmic reticulum (route for delivery of the RTA ribosome-inactivating toxic subunit) [15], [19], [20]. In vivo toxicity studies with the ricin toxin show injected or ingested ricin is quickly mobilized to both blood and lymphatic systems and accumulates in the spleen, liver, kidneys, heart, thymus, brain, and other tissues [21], [22] (reviewed in [23]). Additional studies show that the non-toxic RTB subunit (i.e., without the RTA subunit) mediates uptake into cells and tissue [24], [25], [26]. We therefore hypothesize that RTB will facilitate delivery of genetically associated human β-galactosidase to the critical sites of GM1 pathology. As a first step in testing this, we produced fusion proteins of RTB and human β-gal in plants and characterized their activities in plant tissues and in GM1 patient fibroblasts.
β-gal functions as an exoglycosidase, removing β-1,4-linked galactose residues from glycoproteins sphingolipids such as GM1 ganglioside, and keratan sulfate. Depending on the genetic mutation, β-gal deficiencies can lead to the primarily neurodegenerative GM1 gangiolosidosis or to Morquio B disease [27], [28]. β-gal can be isolated from mammalian tissues as a monomer, dimer, tetramer and as part of a multi-enzyme complex with N-acetyl-α-neuraminidase (Neu1) and the serine peptidase protective protein/cathepsin A (PPCA) [29], [30], [31]. This multi-enzyme complex is critical for enzyme stability and function within lysosomes, as defects in PPCA lead to galactosialidosis disease and deficiencies in both Neu1 and β-gal levels. β-gal is synthesized as an 85 kDa glycoprotein and is proteolytically processed within the lysosome to a 64 kDa “active” form by cleavage of 134 a.a. from the C-terminal end [30]. In PPCA deficient cells, β-gal is synthesized and traffics to lysosomes. However, it is rapidly degraded in lysosomes suggesting that PPCA plays a critical role in stabilizing β-gal in a way that allows the specific catalytic activation events but prevents further degradation [32], [33]. To date, neither plant-based production of functional human β-gal nor production of bioactive β-gal fusions with large heterologous protein domains (mammalian cell-derived HIS-tagged version are available) has been reported.
Plant-based bioproduction provides advantages in safety (no adventitious viral contamination) and cost of manufacture compared to mammalian cell-based technologies [34]. It was initially demonstrated that plants can produce fully functional human lysosomal hydrolases in the early 1990s [70]. The first commercialized plant-made pharmaceutical protein to receive FDA approval for systemic administration was the glucocerebrosidase ERT for Gaucher disease produced in carrot cells (Protalix′ Elelyso, approved in 2012). This product has been used for patient treatment since 2007 [35] with no increase in immunogenicity compared to CHO-cell derived product [36], [37], [38]. In this study, we employ plant-based biomanufacture using transient expression in Nicotiana benthamiana. Briefly, gene vectoring components are infiltrated into leaves of intact plants and recombinant proteins are recovered in 3–7 days (e.g. [39], [40], [41]). Both monoclonal antibodies (e.g. the ZMapp MAb cocktail for treatment for Ebola) and human virus-like particles (e.g. VLP for influenza A) made with the N. benthamiana system are currently in human clinical trials highlighting its potential to make and assemble complex protein products at scale and purity that meet regulatory standards [34], [40], [41], [42], [43], [44]. Thus, this production platform may facilitate more cost-effective development of complex biologics for rare diseases.
We recently reported plant-based production of RTB fusions to the human α-L-iduronidase enzyme (ERT for mucopolysacchardosis I; MPS I) [24]. The fusion product retained both RTB lectin selectivity and iduronidase enzyme activity. The RTB:iduronidase fusion was efficiently taken up into MPS I patient fibroblasts, corrected the lysosomal phenotype, and reduced the glycosaminoglycan disease substrate to “normal” levels by MMR- and M6PR-independent mechanisms [24]. In the present study, β-gal, with and without RTB, was expressed in plants and purified proteins were analyzed for activity, catalytic processing and lysosomal disease correction of GM1 fibroblasts. RTB fusion orientations were developed that supported the complex post-translational processing and multiple protein–protein interaction required for full β-gal functionality within lysosomes and effectively corrected GM1-gangliosidosis at the cell level.
Section snippets
GLB1 cloning
The native GLB1 coding sequence (GLB1, GenBank accession no. NM 000404) and tobacco-codon optimized GLB1 were synthesized (GeneArt®, Life Technologies). Synthesized genes included an adapter carrying a hexa-histidine tag (His) and a stop codon at the 3′-end. These constructs, which harbor the endogenous human signal peptide (hSP), were cloned into a pMK-RQ vector. GLB1 sequences encoding the mature β-gal were also cloned into a pBC-SK(−) vector with an in-house patatin optimized signal peptide
Production of β-gal and β-gal fusions with RTB in N. benthamiana leaves
In lysosomes, β-gal activity is based both on interactions with diverse substrates (e.g., sphingolipids, glycoproteins, keratan sulfates) as well as other proteins, including Neu1, PPCA, and the proteinase responsible for catalytic activation. Although the protein structure of β-gal has been determined [57], it is difficult to predict the structural impact of introducing a large fusion partner such as RTB. RTB itself is a 34 kDa glycoprotein with two distinct galactose/galactosamine binding
Discussion
ERTs have revolutionized patient care and greatly enhanced patient quality of life for a number of lysosomal storage diseases. However, the inability of current ERT approaches to address neurological pathologies limits application to GM1-gangliosidosis, which presents with severe CNS involvement. The plant lectin RTB directs uptake primarily by adsorptive-mediated endocytosis, providing the potential to mediate ERT delivery into tissues and cell types with fundamentally different
Author contributions
C.L.C, D.N.R., J.C. and W.A. participated in project conceptualization and research strategy development; J.C. and V.K designed and produced the gene constructs; J.A. and A.F. expressed, purified, and characterized the plant-produced proteins with assistance in biochemical characterization from J.R.; W.A. developed the Dual bioactivity assay, enzyme kinetic studies, and all human cell-based assays with assistance in cell culture and related assays from R.M.; C.L.C., W.A., J.C., and J.A. wrote
Conflict of interest
C.L.C and D.N.R. are co-founders of biotech start-up BioStrategies LC. The other authors declare no competing financial interests.
Acknowledgments
We wish to thank Drs. Alessandra d'Azzo, Erik Bonten and Ida Annunziata at St. Jude Children's Research Hospital for valuable insights, discussion and support in the development of this work. We also thank Samantha Davis for her help in plant growth and transfection. This research was supported by funding to BioStrategies LC from an NIH/NINDS Phase I SBIR grant (1R43NS084565-01) and the National Tay-Sachs and Allied Diseases Association.
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J.C., W.A., and J.A. contributed equally and should be consider co-first authors on this work.
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Present address: Hematology Department, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.