Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands.

Since 2000, transgenic silkworms have been developed to produce recombinant proteins with therapeutic potential for future clinical use, including antibody preparations. Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by mutations of lysosomal enzymes associated with excessive accumulation of natural substrates and neurovisceral symptoms. Over the past few years, enzyme replacement therapy (ERT) with human lysosomal enzymes produced by genetically engineered mammalian cell lines has been used clinically to treat several patients with an LSD involving multi-organ symptoms. ERT is based on the incorporation of recombinant glycoenzymes by their binding to glycan receptors on the surface of target cells and their subsequent delivery to lysosomes. However, ERT has several disadvantages, including difficulty mass producing human enzymes, dangers of pathogen contamination, and high costs. Recently, the current authors have succeeded in producing transgenic silkworms overexpressing human lysosomal enzymes in the silk glands and the authors have purified catalytically active enzymes from the middle silk glands. Silk gland-derived human enzymes carrying high-mannose and pauci-mannose N-glycans were endocytosed by monocytes via the mannose receptor pathway and were then delivered to lysosomes. Conjugates with cell-penetrating peptides were also taken up by cultured fibroblasts derived from patients with enzyme deficiencies to restore intracellular catalytic activity and reduce the excessive accumulation of substrates in patient fibroblasts. Transgenic silkworms overexpressing human lysosomal enzymes in the silk glands could serve as future bioresources that provide safe therapeutic enzymes for the treatment of LSDs. Combining recent developments in transglycosylation technology with microbial endoglycosidases will promote the development of therapeutic glycoproteins as bio-medicines.

Short-term, high dose enzyme replacement therapy in sialidosis mice.

Given the success of enzyme replacement therapy (ERT) in treating the systemic manifestations in a number of lysosomal storage disorders (LSDs), we evaluated the effect of ERT on the mouse model of sialidosis. This glycoproteinosis, which affects primarily the reticuloendothelial (RE) system, is caused by deficiency of lysosomal neuraminidase (NEU1) and consequent accumulation of sialylated glycoconjugates. NEU1 lacks a functional mannose-6-phosphate recognition marker and is not endocytosed by mammalian cells. However, the enzyme produced in insect cells has features that allow its effective uptake by RE cells and macrophages via the mannose receptor, and therefore represent an alternative method of therapy. In this study we tested the therapeutic efficacy of baculovirus (BV) expressed mouse neuraminidase (Neu1) in sialidosis mice. Four-week-old Neu1-/- mice were first injected intravenously with a single dose of the recombinant enzyme for assessment of the half-life of mannosylated Neu1 in vivo. Afterwards, a short-term ERT with a total of five enzyme injections over a 2-week period was performed for evaluation of phenotype correction. Neu1 infused alone or co-administered with its associated protein, protective protein/cathepsin A (PPCA) was effectively taken up by resident macrophages in many tissues. Restored Neu1 activity persisted for up to 4 days, depending on the tissue, and resulted in a significant reduction of lysosomal storage. However, beyond 2 weeks of treatment, ERT mice developed a severe immune response towards the exogenous Neu1 enzyme. These results may have important implications for ERT in sialidosis patients.

Targeting macrophages with baculovirus-produced lysosomal enzymes: implications for enzyme replacement therapy of the glycoprotein storage disorder galactosialidosis.

Lysosomal storage diseases (LSDs) are monogenic disorders of metabolism caused by deficiency of hydrolytic enzymes. In several LSDs, cells of the reticuloendothelial (RE) system are the primary targets of the disease. Exogenous administration of recombinant enzymes overproduced in mammalian cells has proved effective for treating the systemic phenotype in nonneuropathic patients with LSDs. However, for the treatment of diseases with primary involvement of the RE system, the production of the therapeutic enzyme in insect cells could be an alternative and advantageous method because glycoproteins expressed in insect cells carry carbohydrates of the pauci-mannose or core-type. These recombinant enzymes are in principle already poised to be internalized by cells that express mannose receptors, including macrophages. Here, we demonstrate that three baculovirus-expressed enzymes, protective protein/cathepsin A (PPCA), neuraminidase (Neu1), and beta-glucosidase, were readily taken up and restored lysosomal function in enzyme-deficient mouse macrophages. The capacity of recombinant PPCA and Neu1 to clear the lysosomal storage in target cells was assessed in PPCA-/- mice, a model of galactosialidosis. Intravenously injected PPCA-/- mice efficiently internalized the corrective enzymes in resident macrophages of many organs. In addition, treated mice showed overall clearance of lysosomal storage in the most affected systemic organs, kidney, liver, and spleen. Our results suggest that ERT with baculovirus-expressed enzymes might be an effective treatment for nonneuropathic patients with galactosialidosis and possibly for others with LSDs that primarily involve the RE system.