Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

Identification of Orthosteric and Allosteric Pharmacological Chaperones for Mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.

Human recombinant lysosomal ß-Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay-Sachs fibroblasts.

GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the ß-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αß or ßß subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidoses, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant ß-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were taken-up via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant ß-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.

A perspective on research, diagnosis, and management of lysosomal storage disorders in Colombia.

Lysosomal storage diseases (LSDs) are a group of about 50 inborn errors of metabolism characterized by the lysosomal accumulation of partially or non-degraded molecules due to mutations in proteins involved in the degradation of macromolecules, transport, lysosomal biogenesis or modulators of lysosomal environment. Significant advances have been achieved in the diagnosis, management, and treatment of LSDs patients. In terms of approved therapies, these include enzyme replacement therapy (ERT), substrate reduction therapy, hematopoietic stem cell transplantation, and pharmacological chaperone therapy. In this review, we summarize the Colombian experience in LSDs thorough the evidence published. We identified 113 articles published between 1995 and 2019 that included Colombian researchers or physicians, and which were mainly focused in Mucopolysaccharidoses, Pompe disease, Gaucher disease, Fabry disease, and Tay-Sachs and Sandhoff diseases. Most of these articles focused on basic research, clinical cases, and mutation reports. Noteworthy, implementation of the enzyme assay in dried blood samples, led to a 5-fold increase in the identification of LSD patients, suggesting that these disorders still remain undiagnosed in the country. We consider that the information presented in this review will contribute to the knowledge of a broad spectrum of LSDs in Colombia and will also contribute to the development of public policies and the identification of research opportunities.

Therapeutic Options for Mucopolysaccharidoses: Current and Emerging Treatments.

Mucopolysaccharidoses (MPS) are inborn errors of metabolism produced by a deficiency of one of the enzymes involved in the degradation of glycosaminoglycans (GAGs). Although taken separately, each type is rare. As a group, MPS are relatively frequent, with an overall estimated incidence of around 1 in 20,000-25,000 births. Development of therapeutic options for MPS, including hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT), has modified the natural history of many MPS types. In spite of the improvement in some tissues and organs, significant challenges remain unsolved, including blood-brain barrier (BBB) penetration and treatment of lesions in avascular cartilage, heart valves, and corneas. Newer approaches, such as intrathecal ERT, ERT with fusion proteins to cross the BBB, gene therapy, substrate reduction therapy (SRT), chaperone therapy, and some combination of these strategies may provide better outcomes for MPS patients in the near future. As early diagnosis and early treatment are imperative to improve therapeutic efficacy, the inclusion of MPS in newborn screening programs should enhance the potential impact of treatment in reducing the morbidity associated with MPS diseases. In this review, we evaluate available treatments, including ERT and HSCT, and future treatments, such as gene therapy, SRT, and chaperone therapy, and describe the advantages and disadvantages. We also assess the current clinical endpoints and biomarkers used in clinical trials.

Characterization of Human Recombinant N-Acetylgalactosamine-6-Sulfate Sulfatase Produced in Pichia pastoris as Potential Enzyme for Mucopolysaccharidosis IVA Treatment.

Mucopolysaccharidosis IVA (MPS IVA or Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal storage of keratan sulfate and chondroitin-6-sulfate. Currently, enzyme replacement therapy using an enzyme produced in CHO cells represents the main treatment option for MPS IVA patients. As an alternative, we reported the production of an active GALNS enzyme produced in the yeast Pichia pastoris (prGALNS), which showed internalization by cultured cells through a potential receptor-mediated process and similar post-translational processing as human enzyme. In this study, we further studied the therapeutic potential of prGALNS through the characterization of the N-glycosylation structure, in vitro cell uptake and keratan sulfate reduction, and in vivo biodistribution and generation of anti-prGALNS antibodies. Taken together, these results represent an important step in the development of a P. pastoris-based platform for production of a therapeutic GALNS for MPS IVA enzyme replacement therapy.

Neural stem cells for disease modeling and evaluation of therapeutics for Tay-Sachs disease.

BACKGROUND: Tay-Sachs disease (TSD) is a rare neurodegenerative disorder caused by autosomal recessive mutations in the HEXA gene on chromosome 15 that encodes ß-hexosaminidase. Deficiency in HEXA results in accumulation of GM2 ganglioside, a glycosphingolipid, in lysosomes. Currently, there is no effective treatment for TSD. RESULTS: We generated induced pluripotent stem cells (iPSCs) from two TSD patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs). The TSD neural stem cells exhibited a disease phenotype of lysosomal lipid accumulation. The Tay-Sachs disease NSCs were then used to evaluate the therapeutic effects of enzyme replacement therapy (ERT) with recombinant human Hex A protein and two small molecular compounds: hydroxypropyl-ß-cyclodextrin (HPßCD) and δ-tocopherol. Using this disease model, we observed reduction of lipid accumulation by employing enzyme replacement therapy as well as by the use of HPßCD and δ-tocopherol. CONCLUSION: Our results demonstrate that the Tay-Sachs disease NSCs possess the characteristic phenotype to serve as a cell-based disease model for study of the disease pathogenesis and evaluation of drug efficacy. The enzyme replacement therapy with recombinant Hex A protein and two small molecules (cyclodextrin and tocopherol) significantly ameliorated lipid accumulation in the Tay-Sachs disease cell model.

Research, diagnosis and education in inborn errors of metabolism in Colombia: 20 years' experience from a reference center.

The use of specialized centers has been the main alternative for an appropriate diagnosis, management and follow up of patients affected by inborn errors of metabolism (IEM). These centers facilitate the training of different professionals, as well as the research at basic, translational and clinical levels. Nevertheless, few reports have described the experience of these centers and their local and/or global impact in the study of IEM. In this paper, we describe the experience of a Colombian reference center for the research, diagnosis, training and education on IEM. During the last 20 years, important advances have been achieved in the clinical knowledge of these disorders, as well as in the local availability of several diagnosis tests. Organic acidurias have been the most frequently detected diseases, followed by aminoacidopathies and peroxisomal disorders. Research efforts have been focused in the production of recombinant proteins in microorganisms towards the development of new enzyme replacement therapies, the design of gene therapy vectors and the use of bioinformatics tools for the understanding of IEM. In addition, this center has participated in the education and training of a large number professionals at different levels, which has contributed to increase the knowledge and divulgation of these disorders along the country. Noteworthy, in close collaboration with patient advocacy groups, we have participated in the discussion and construction of initiatives for the inclusion of diagnosis tests and treatments in the health system.

Production and characterization of a human lysosomal recombinant iduronate-2-sulfatase produced in Pichia pastoris.

Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg-1 H-1 and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.

Anaerobic sulfatase maturase AslB from Escherichia coli activates human recombinant iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS).

Maturation of type I sulfatases requires the conversion of the cysteine (Cys) or serine (Ser) present in the active site to formylglycine (FGly). This activation represents a limiting step during the production of recombinant sulfatases in bacteria and eukaryotic hosts. AslB, YdeM and YidF have been proposed to participate in the activation of sulfatases in Escherichia coli. In this study, we combined in-silico and experimental approaches to study the interaction between Escherichia coli BL21(DE3) AslB and human sulfatases, more specifically iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In-silico results show that AslB has a higher affinity for the residual motif of GALNS (-9.4kcalmol-1), Cys- and Ser-type, than for the one of IDS (-8.0kcalmol-1). However, the distance between the AslB active residue and the target motif favors the interaction with IDS (4.4Å) more than with GALNS (5.5Å). Experimental observations supported in-silico results where the co-expression of AslB with GALNS Cys- and Ser-type presented an activity increment of 2.0- and 1.5-fold compared to the control cultures, lacking overexpressed AslB. Similarly, IDS activity was increased in 4.6-fold when co-expressed with AslB. The higher sulfatase activity of AslB-IDS suggests that the distance between the AslB active residue and the motif target is a key parameter for the in-silico search of potential sulfatase activators. In conclusion, our results suggest that AslB is involve in the maturation of heterologous human sulfatases in E. coli BL21(DE3), and that it can have important implications in the production of recombinant sulfatases for therapeutic purposes and research.

Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris / Caracterización de beta-hexosaminidasas lisosomales humanas recombinantes producidas en la levadura metilotrófica Pichia pastoris / Caracterização de beta-hexosaminidases lisossomais humanas recombinantes produzidas na levedura metilotrófica Pichia pastoris

Abstract β-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded by HEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant α-hexosaminidases. In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S) were produced in the methylotrophic yeast Pichia pastoris GS115. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14,606 U .mg-1 for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at 4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and rhHex-B showed high stability. This study demonstrates P. pastoris GS115 potential for polymeric lysosomal enzyme production, and describes the characterization of recombinant β-hexosaminidases produced within the same host.

Resumen Las β-hexosaminidasas (Hex) son enzimas diméricas involucradas en la degradación lisosomal de glicolípidos y glicanos. Estas enzimas están formadas por las subunidades α- y/o β-codificadas por los genes HEXA and HEXB respectivamente. Las mutaciones de estos genes conducen a las enfermedades de Tay Sachs o Sandhoff, que son desórdenes neurodegenerativos causados por la acumulación de glicolípidos no degradados. Aunque las Hex derivadas de tejido han sido ampliamente caracterizadas, la información disponible sobre las p-hexosaminidasas recombinantes es limitada. En este estudio se produjeron Hex recombinantes lisosomales (rhHex-A, rhHex-B y rhHex-S) en la levadura metilotrófica Pichia pastoris GS115. Las actividades específicas más altas de las enzimas fueron 13.124, 12.779, 14.606 U .mg-1 para rhHex-A, rhHex-B y rhHex-S, respectivamente. Estos resultados fueron 25 a 50 veces más altos que los obtenidos de leucocitos humanos normales. Las proteínas se purificaron y se caracterizaron a diferentes condiciones de pH y temperatura. Todas las proteínas fueron estables a pH ácido y a 4°C y 37°C. A 45°C la rhHex-S se inactivó completamente, mientras que rhHex-A y rhHex-B mostraron alta estabilidad. Este estudio demuestra el potencial de P. pastoris GS115 para la producción de enzimas lisosomales poliméricas y presenta la caracterización de distintas β-hexosaminidasas recombinantes producidas en un único hospedero.

Resumen As β-hexosaminidases (Hex) são enzimas diméricas envolvidas na degradação lisossomal de glicolipídeos e glicanos. Essas enzimas são formadas por subunidades a- e/ou p-codificadas pelos genes HEXA e HEXB, respectivamente. As mutações nesses genes causam a doença de Sandhoff ou Tay Sachs, que são desordens neurodegenerativas causadas pela acumulação de glicolipídeos não degradados. Embora Hex derivadas de tecido hajam sido caracterizadas extensivamente, as informações disponíveis sobre as p-hexosaminidases recombinantes são limitadas. Esse estudo produziu Hex recombinantes lisossomais (rhHex-A, rhHex-B e rhHex-S) na levedura metilotrófica Pichia pastoris GS115. As atividades específicas mais altas das enzimas foram 13.124, 12.779, 14.606 U .mg-1 para rhHex-A, rhHex-B y rhHex-S, respectivamente. Esses resultados foram 25 a 50 vezes mais altos do que os obtidos a partir de leucócitos humanos normais. As proteínas foram purificadas e caracterizadas em diferentes condições de pH e temperatura. Todas as proteínas foram estáveis a pH ácido e a 4°C e 37°C. A 45°C a rhHex-S foi completamente inativada, enquanto rhHex rhHex-A e B se mostraram altamente estáveis. Esse estudo demonstra o potencial de P. pastoris GS115 para a produção de enzimas lisossomais poliméricas e apresenta a caracterização de diferentes p-hexosaminidases recombinantes produzidas em único hospedeiro.

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris.

Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A.

Human recombinant lysosomal enzymes produced in microorganisms.

Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD.

Impact of enzyme replacement therapy and hematopoietic stem cell transplantation in patients with Morquio A syndrome.

Patients with mucopolysaccharidosis IVA (MPS IVA) can present with systemic skeletal dysplasia, leading to a need for multiple orthopedic surgical procedures, and often become wheelchair bound in their teenage years. Studies on patients with MPS IVA treated by enzyme replacement therapy (ERT) showed a sharp reduction on urinary keratan sulfate, but only modest improvement based on a 6-minute walk test and no significant improvement on a 3-minute climb-up test and lung function test compared with the placebo group, at least in the short-term. Surgical remnants from ERT-treated patients did not show reduction of storage materials in chondrocytes. The impact of ERT on bone lesions in patients with MPS IVA remains limited. ERT seems to be enhanced in a mouse model of MPS IVA by a novel form of the enzyme tagged with a bone-targeting moiety. The tagged enzyme remained in the circulation much longer than untagged native enzyme and was delivered to and retained in bone. Three-month-old MPS IVA mice treated with 23 weekly infusions of tagged enzyme showed marked clearance of the storage materials in bone, bone marrow, and heart valves. When treatment was initiated at birth, reduction of storage materials in tissues was even greater. These findings indicate that specific targeting of the enzyme to bone at an early stage may improve efficacy of ERT for MPS IVA. Recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21 (DE3) (erGALNS) and in the methylotrophic yeast Pichia pastoris (prGALNS) has been produced as an alternative to the conventional production in Chinese hamster ovary cells. Recombinant GALNS produced in microorganisms may help to reduce the high cost of ERT and the introduction of modifications to enhance targeting. Although only a limited number of patients with MPS IVA have been treated with hematopoietic stem cell transplantation (HSCT), beneficial effects have been reported. A wheelchair-bound patient with a severe form of MPS IVA was treated with HSCT at 15 years of age and followed up for 10 years. Radiographs showed that the figures of major and minor trochanter appeared. Loud snoring and apnea disappeared. In all, 1 year after bone marrow transplantation, bone mineral density at L2-L4 was increased from 0.372 g/cm(2) to 0.548 g/cm(2) and was maintained at a level of 0.48±0.054 for the following 9 years. Pulmonary vital capacity increased approximately 20% from a baseline of 1.08 L to around 1.31 L over the first 2 years and was maintained thereafter. Activity of daily living was improved similar to the normal control group. After bilateral osteotomies, a patient can walk over 400 m using hip-knee-ankle-foot orthoses. This long-term observation of a patient shows that this treatment can produce clinical improvements although bone deformity remained unchanged. In conclusion, ERT is a therapeutic option for MPS IVA patients, and there are some indications that HSCT may be an alternative to treat this disease. However, as neither seems to be a curative therapy, at least for the skeletal dysplasia in MPS IVA patients, new approaches are investigated to enhance efficacy and reduce costs to benefit MPS IVA patients.