Therapeutic Enzymes Research Articles

THERAPEUTIC L-ASPARAGINASE ACTIVITY OF BACTERIA ISOLATED FROM MARINE SEDIMENTS

L-Asparaginase, a therapeutic enzyme used in lymphoblastic leukemia and lymphosarcoma chemotherapy which is derived mostly from the bacterial sources Escherichia coli and Erwinia sp. The long term administration of the drug leads to the development of resistant tumours and anaphylactic shock in certain individuals. Hence serologically different L-Asparaginase from novel microbial sources with enhanced therapeutic potential and immunological characteristics is an essential requirement. The marine bacteria having diverse range of potential enzymes might be a source for L-Asparaginase with novel properties, which are still unexplored. In this study, we have screened marine bacteria isolated from the coastal regions of Kerala which showed both intra and extra cellular L-Asparaginase activity. Bacillus sp. (Accession no KF142395) was found to have the highest extracellular enzyme activity (2.31 IU/ml) while Shewanella sp. (Accession no KF142390) showed maximum intracellular Asparaginase activity (2.16 IU/ml).The crude extracellular enzyme preparation from Bacillus sp. had cytotoxic effect on HL60 cell line with an IC50 value of 12.5µg/ml.

Albumin/asparaginase capsules prepared by ultrasound to retain ammonia.

Asparaginase reduces the levels of asparagine in blood, which is an essential amino acid for the proliferation of lymphoblastic malign cells. Asparaginase converts asparagine into aspartic acid and ammonia. The accumulation of ammonia in the bloodstream leads to hyperammonemia, described as one of the most significant side effects of asparaginase therapy. Therefore, there is a need for asparaginase formulations with the potential to reduce hyperammonemia. We incorporated 2% of therapeutic enzyme in albumin-based capsules. The presence of asparaginase in the interface of bovine serum albumin (BSA) capsules showed the ability to hydrolyze the asparagine and retain the forming ammonia at the surface of the capsules. The incorporation of Poloxamer 407 in the capsule formulation further increased the ratio aspartic acid/ammonia from 1.92 to 2.46 (and 1.10 from the free enzyme), decreasing the levels of free ammonia. This capacity to retain ammonia can be due to electrostatic interactions and retention of ammonia at the surface of the capsules. The developed BSA/asparaginase capsules did not cause significant cytotoxic effect on mouse leukemic macrophage cell line RAW 264.7. The new BSA/asparaginase capsules could potentially be used in the treatment of acute lymphoblastic leukemia preventing hyperammonemia associated with acute lymphoblastic leukemia (ALL) treatment with asparaginase.

Production and Optimization of Site-Specific monoPEGylated Uricase Conjugates Using mPEG-Maleimide Through RP–HPLC Methodology

Uricase (Uc), a therapeutic enzyme, is widely used in its PEGylated form to treat hyperuricemia and is largely manufactured by means of random/first generation PEGylation approach. Currently available randomly PEGylated uricase conjugates exhibit inadequacies like reduced uricolytic activity, risk of inducing immunogenic reactions, lack of selectivity, and molecular heterogeneity. In the present study, site-specific/second generation PEGylation strategy involving modification of specific and rare amino acids by means of terminally functionalized PEG polymers was applied. Uricase was conjugated with methoxypolyethyelenglycol-maleimide (mPEG-mal) by means of thiol PEGylation to synthesize monoPEGylated uricase conjugates. For enhancing the yield of monoPEGylated uricase conjugates, response surface methodology was employed to determine the yield of monoPEGylated conjugates using reverse phase high performance liquid chromatography. Using the optimized conditions, the developed method was validated for the production of monoPEGylated uricase conjugates which were further purified by size exclusion fast protein liquid chromatography (SE-FPLC). The molecular weights of the purified conjugates were determined by sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE). The optimum values of reaction conditions were determined as 1:12 concentration ratio of Uc to mPEG-mal, 2.76 kDa as mPEG-mal molecular weight and 3.55 mM EDTA concentration which resulted in a very high conjugate yield of 95.16 %. The conjugate synthesized using the optimized method retained a residual uricolytic activity of 84 % and a thiol group modification extent of 68.3 %. The PEGylation reaction was optimized using OVAT and statistical methods. Using the optimized conditions very high yield of conjugates were obtained and RP–HPLC method was used to quantify the PEGylated uricase.

Targeted Polymeric Nanoparticles for Brain Delivery of High Molecular Weight Molecules in Lysosomal Storage Disorders.

Lysosomal Storage Disorders (LSDs) are a group of metabolic syndromes, each one due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and overall about 75% of the patients present neurological impairment. Enzyme Replacement Therapy, although determining some systemic clinical improvements, is ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB). With the aim to deliver the therapeutic enzymes across the BBB, we here assayed biodegradable and biocompatible PLGA-nanoparticles (NPs) in two murine models for LSDs, Mucopolysaccharidosis type I and II (MPS I and MPS II). PLGA-NPs were modified with a 7-aminoacid glycopeptide (g7), yet demonstrated to be able to deliver low molecular weight (MW) molecules across the BBB in rodents. We specifically investigated, for the first time, the g7-NPs ability to transfer a model drug (FITC-albumin) with a high MW, comparable to the enzymes to be delivered for LSDs brain therapy. In vivo experiments, conducted on wild-type mice and knockout mouse models for MPS I and II, also included a whole series of control injections to obtain a broad preliminary view of the procedure efficiency. Results clearly showed efficient BBB crossing of albumin in all injected mice, underlying the ability of NPs to deliver high MW molecules to the brain. These results encourage successful experiments with enzyme-loaded g7-NPs to deliver sufficient amounts of the drug to the brain district on LSDs, where exerting a corrective effect on the pathological phenotype.

The family of venom and mammalian sPLA2s: From toxic to therapeutic enzymes

The family of venom and mammalian sPLA2s: From toxic to therapeutic enzymes

47. Gene Transfer-Mediated Diversion Towards Non-Toxic Metabolites for Therapy of Primary Hyperoxaluria Type 1

Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to mutations of the AGXT gene encoding the peroxisomal enzyme alanine: glyoxylate-aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. In PH1 patients, glyoxylate cannot be efficiently converted into glycine and is instead oxidized to oxalate resulting in systemic oxalosis with deposition of insoluble calcium oxalate in kidneys and in other tissues, leading to nephrolithiasis, nephrocalcinosis, kidney failure, and systemic tissue damage. Combined liver/kidney transplantation is the only therapeutic strategy available to prevent disease progression. We hypothesize that overexpression of specific genes encoding enzymes involved in glyoxalate metabolism will steer glyoxylate towards alternative pathways to diminish oxalate production. To test this hypothesis, we overexpressed murine glyoxylate reductase/hydroxypyruvate reductase (GRHPR), that converts glyoxylate into glycolate, by a helper-dependent adenoviral vector (HDAd-GRHPR) in livers of Agxt−/− mice. The intravenous injection of HDAd-GRHPR resulted in significant reduction of hyperoxaluria and concomitant increase of serum glycolate that was not associated with signs of toxicity. Glutamate-pyruvate transaminase (GPT) in the cytosol transaminate glyoxylate using glutamate and alanine as amino-group donors. We hypothesize that GPT overexpression will steer glyoxylate towards transamination to diminish oxalate production. The intravenous injection of a helper-dependent adenoviral vector expressing murine GPT (HDAd-GPT) in Agxt−/− mice also resulted in significant and sustained reduction of hyperoxaluria. Interestingly, co-administration of both HDAd-GRHPR and HDAd-GPT resulted in further reduction and normalization of hyperoxaluria. Glycolate is one of the substrates leading to glyoxylate production, via peroxisomal glycolate oxidase (GO). We also show that an HDAd expressing a short hairpin RNA against GO resulted in reduction of hyperoxaluria in Agxt−/− mice. In summary, the results of this study show that metabolic diversion towards non-toxic metabolites has potential for treatment of PH1 and potentially other forms of hyperoxalurias, both primary and secondary. The metabolic diversion could be also obtained by RNA-based molecules expressing GRHPR and/or GPT or inhibiting GO activity. In addition, this study shows that HDAd vectors can be used to functionally validate therapeutic enzyme targets in inherited metabolic diseases.

364. Neurological Correction of Mucopolysaccharidosis IIIB Mice by Haematopoietic Stem Cell Gene Therapy

Mucopolysaccharidosis Type IIIB (MPSIIIB) is a paediatric, autosomal recessive Lysosomal Storage Disease (LSD) caused by deficiency of α-N-acetylglucosaminidase (NAGLU), an enzyme in the heparan sulfate (HS) degradation pathway. Absence of NAGLU leads to the accumulation of partially degraded HS glycosaminoglycan in cell lysosomes, giving rise to cellular dysfunction with devastating clinical consequences. Individuals affected by this fatal disease exhibit severe central nervous system degeneration with progressive cognitive impairment and behavioural problems, alongside more attenuated somatic symptoms. There are currently no effective treatments available. Enzyme replacement therapy with recombinant NAGLU enzyme is ineffective for MPSIIIB since enzyme cannot cross the blood brain barrier (BBB) to where it is needed. Modified recombinant NAGLU enzymes that utilise the insulin growth factor II (IGFII) peptide to facilitate improved uptake across the BBB are currently in development. Haematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy that can circumvent the BBB via monocyte trafficking and engraftment in the brain, allowing delivery of enzyme by cross correction. We have developed a novel stem cell gene therapy approach to investigate the therapeutic potential of HSCGT for MPSIIIB. We designed two lentiviral vectors expressing therapeutic enzyme; the first vector expressing codon optimised NAGLU, and the second expressing a NAGLU.IGFII fusion to aid cellular uptake, both driven by the myeloid specific promoter CD11b and compared these in autologous MPSIIIB transplants against a normal WT bone marrow transplant. Here we present for the first time neurological correction of MPSIIIB mice by HSCGT. We observed correction of the MPSIIIB behavioural phenotype in treated mice to wild-type levels with normalisation of path length, average speed, frequency entering the centre and duration of speed >100mm/s in open field tests. In addition, we observed a significant correction of astrogliosis and lysosomal compartment size in the brains of CD11b. NAGLU LV treated mice with an accompanied normalisation of inflammatory cytokines TNFα, IL1B and IL1RN. Furthermore, NAGLU enzyme activity was substantially increased in the brain. Interestingly, WT transplant alone was able to mediate a partial brain correction, although levels of inflammation and lysosomal storage remain high.

Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags.

Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in “Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans” (Kang et al., 2016 [1]).

Molecular basis and current strategies of therapeutic arginine depletion for cancer.

Renewed interest in the use of therapeutic enzymes combined with an improved knowledge of cancer cell metabolism, has led to the translation of several arginine depletion strategies into early phase clinical trials. Arginine auxotrophic tumors are reliant on extracellular arginine, due to the downregulation of arginosuccinate synthetase or ornithine transcarbamylase-key enzymes for intracellular arginine recycling. Engineered arginine catabolic enzymes such as recombinant human arginase (rh-Arg1-PEG) and arginine deiminase (ADI-PEG) have demonstrated cytotoxicity against arginine auxotrophic tumors. In this review, we discuss the molecular events triggered by extracellular arginine depletion that contribute to tumor cell death.

Synthesis of NAM-thiazoline derivatives as novel O-GlcNAcase inhibitors

Human O-GlcNAcase (GH 84) and human β-N-acetyl-D-hexosaminidase (GH 20) from Homo sapiens are two therapeutic enzyme targets that share the same catalytic mechanism but play different physiological roles in vivo. Selective inhibition toward one of these enzymes is therefore of importance to regulate the corresponding bioprocess. Here ten new NAM-thiazoline derivatives were synthesized and subsequently characterized by NMR and HRMS. A preliminary bioassay showed that most of the synthesized compounds exhibited obvious selective inhibition against human O-GlcNAcase over human β-N-acetyl-D-hexosaminidase. Among the compounds tested, compound 7d (IC50 = 6.4 µM, hOGA; IC50>1 mM, hHex) and 7f (IC50 = 11.9 µM, hOGA; IC50>1 mM, hHex) proved to be a highly selective and potent inhibitor. Structure–activity relationship analysis indicated a correlation between the inhibitory activity and the size of the groups linked to the thiazoline ring.

Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non‐human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe‐affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

Nanocapsule assemblies as effective enzyme delivery systems against hyperuricemia

The uricase nanocapsule assemblies (UNAs) were developed as effective delivery systems against hyperuricemia following parenteral enzyme therapy. UNAs were characterized in terms of micromorphology, size, catalytic activity, stability, and enzymatic kinetics. The pharmacokinetics/pharmacodynamics in rats after intravenous or subcutaneous injection was investigated. Immunogenicity, hemolysis and stimulation were determined. UNA was composed of many nanocapsules, and thus had higher loading efficiencies and stabilities than a single nanocapsule. The uricase molecules entrapped inside nanocapsules were separated from the circulating bloodstream to retain catalytic activities for a longer time than free uricase. UNAs increased the bioavailability and uric acid-lowering efficacy of uricase, while the immunogenicity and hemolysis rate of uricase were decreased. The superior properties of UNAs might be ascribed to the favorable conformational changes of uricase. Nanocapsule assemblies appeared to be able to deliver uricase effectively. This study also highlighted the importance of suitable systems for therapeutic enzyme delivery.

Chitosan-modified lipid nanovesicles for efficient systemic delivery of l-asparaginase

The goal of this study was to evaluate the enhanced catalytic activity, increased stability, in vitro anti-cancer effects on H446 cells and in vivo bioavailability of novel enzyme delivery nanovesicles (l-asparaginase containing chitosan modified lipid nanovesicles, ACLNs) when administered intravenously. It was the first time for the chitosan-modified lipid nanovesicles to be fabricated to deliver l-asparaginase (ASP, a therapeutic enzyme) efficiently. It was indicated that ACLNs markedly increased the enzymatic activity, improved the temperature/acid-base/proteolytic stabilities and favorably changed the in vivo kinetic characteristics. Moreover, ACLNs exhibited higher anti-lung-cancer activity than free ASP. The possible existence status of ASP in ACLNs and the fluorescence changes of ACLNs reflecting the conformational changes after heat treatment were preliminary explored. ACLNs might be novel promising nanovesicles for effective systemic delivery of therapeutic enzyme ASP.

Effect of anti-laronidase antibodies on efficacy and safety of laronidase enzyme replacement therapy for MPS I: A comprehensive meta-analysis of pooled data from multiple studies.

Enzyme replacement therapy (ERT) with laronidase has an important role in the treatment of patients with mucopolysaccharidosis type I (MPS I). Laronidase is safe and has demonstrated effectiveness in terms of stabilizing or improving conventional clinical and laboratory markers of the disease. However, like most ERTs, laronidase produces an anti-drug IgG antibody response in more than 90% of patients during the first few months of treatment. Preclinical data from the MPS I canine model suggest that anti-drug antibodies (ADA) impair enzyme uptake in target tissues. In patients, the effects on tissue glycosaminoglycan (GAG) clearance are difficult to assess directly but data from clinical studies have suggested an association between ADA and both a reduced pharmacodynamic response and hypersensitivity reactions. This comprehensive meta-analysis of pooled data from patients in three clinical studies of laronidase (including one study with an extension) was undertaken to provide a more robust assessment of the relationship between the ADA response to laronidase, clinical and laboratory markers of MPS I, and hypersensitivity reactions. The meta-analysis demonstrated an inverse relationship between the ADA response and the percent reduction in urinary GAG (uGAG) levels. However, no relationships between the ADA response and changes in percent predicted forced vital capacity and six-minute walk test were seen. The study also re-assayed stored serum samples from the original trials with a novel method to determine the inhibitory effect of ADA. Patients with higher ADA exposure over time were found to have higher inhibition of enzyme uptake into cells. High ADA exposure can result in a commensurate level of enzyme uptake inhibition that decreases the pharmacodynamic effect of the exogenously administered therapeutic enzyme, but with no clear effect on clinical efficacy.

α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest.

Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags—2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)—were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses.

Droplet-Based Microfluidics for Measuring Enzymatic Activities: Application to L-Asparaginase used in Antileukemic Therapy

Our work aims to ameliorate catalytic properties of L-asparaginase (L-ASNase) which is a protein drug used in antileukemic therapy. Bacterial L-ASNases are FDA-approved therapeutic enzymes for use in the treatment of various blood cancers to deplete serum L-Asn. Their therapeutic efficacy is based on the fact that several hematological malignancies, in particular Acute Lymphoblastic Leukemia (ALL), depend for growth on the extracellular supply of L-Asn. To avoid various side reactions inherent to the bacterial enzymes, it would be beneficial to substitute them with human L-ASNases. In order to find variants of improved specific activities in mutated enzyme libraries, we developed a droplet-based microfluidic platform for high throughput and miniaturization of kinetic assays that can be performed not only on purified enzymes, but also in single cells. When analyzing enzyme activity in bacterial cells, critical assumptions are: (i) protein expression levels are uniform in the population, and (ii) the reaction rate is not limited by the diffusion of small-molecule substrates/products to/from the enzyme. We demonstrate here a three-step coupled-enzyme assay for measuring ASNase activity on its natural substrate Asn, using water-in-oil droplets of nL to pL reaction volumes, at throughput rates of up to 104 per sec. Catalytic rates were found to vary by a factor of less than two within a population of more than 108 bacterial cells, following the same kinetics as the purified enzyme free in solution. To increase serum half-life of the enzyme, we loaded L-ASNases onto calcium carbonate nanoparticles serving as core templates, and manufactured biocompatible microcapsules under mild conditions to preserve catalytic activity, thereby improving protein stability and preventing exposure of the enzyme to the immune system. Encapsulated and free enzymes were able to kill leukemic cells with similar efficacy.

Uricase alkaline enzymosomes with enhanced stabilities and anti-hyperuricemia effects induced by favorable microenvironmental changes

Enzyme therapy is an effective strategy to treat diseases. Three strategies were pursued to provide the favorable microenvironments for uricase (UCU) to eventually improve its features: using the right type of buffer to constitute the liquid media where catalyze reactions take place; entrapping UCU inside the selectively permeable lipid vesicle membranes; and entrapping catalase together with UCU inside the membranes. The nanosized alkaline enzymosomes containing UCU/(UCU and catalase) (ESU/ESUC) in bicine buffer had better thermal, hypothermal, acid-base and proteolytic stabilities, in vitro and in vivo kinetic characteristics, and uric acid lowering effects. The favorable microenvironments were conducive to the establishment of the enzymosomes with superior properties. It was the first time that two therapeutic enzymes were simultaneously entrapped into one enzymosome having the right type of buffer to achieve added treatment efficacy. The development of ESU/ESUC in bicine buffer provides valuable tactics in hypouricemic therapy and enzymosomal application.

Widening and Elaboration of Consecutive Research into Therapeutic Antioxidant Enzyme Derivatives.

Undiminishing actuality of enzyme modification for therapeutic purposes has been confirmed by application of modified enzymes in clinical practice and numerous research data on them. Intravenous injection of the superoxide dismutase-chondroitin sulfate-catalase (SOD-CHS-CAT) conjugate in preventive and medicative regimes in rats with endotoxin shock induced with a lipopolysaccharide bolus has demonstrated that antioxidant agents not only effectively prevent damage caused by oxidative stress (as believed previously) but also can be used for antioxidative stress therapy. The results obtained emphasize the importance of investigation into the pathogenesis of vascular damage and the role of oxidative stress in it. The effects of intravenous medicative injection of SOD-CHS-CAT in a rat model of endotoxin shock have demonstrated a variety in the activity of this conjugate in addition to prevention of NO conversion in peroxynitrite upon interaction with O2 ∙− superoxide radical. Together with the literature data, these findings offer a prospect for the study of NO-independent therapeutic effects of SOD-CHS-CAT, implying the importance of a better insight into the mechanisms of the conjugate activity in modeled cardiovascular damage involving vasoactive agents other than NO.

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.