Recombinant Human Α-galactosidase Research Articles

Oxidative stress and its role in Fabry disease.

Fabry disease is a rare X-linked disease characterized by deficient expression and activity of alpha-galactosidase A with consequent lysosomal accumulation of glycosphingolipids, particularly globotriaosylceramide in various organs. Currently, enzyme replacement therapy with recombinant human α-galactosidase is the cornerstone of the treatment of Fabry patients, although in the long term enzyme replacement therapy fails to halt disease progression, in particular in case of late diagnosis. This suggests that the adverse outcomes cannot be justified by the lysosomal accumulation of glycosphingolipids alone, and that additional therapies targeted at further pathophysiologic mechanisms might contribute to halting the progression of cardiac, cerebrovascular and kidney disease in Fabry patients. Recent evidence points toward the involvement of oxidative stress, oxidative stress signaling and inflammation in the pathophysiology of cardio cerebrovascular and kidney damage in Fabry patients. This review reports the current knowledge of the involvement of oxidative stress in Fabry disease, which clearly points toward the involvement of oxidative stress in the pathophysiology of the medium to long-term cardio-cerebrovascular-kidney damage of Fabry patients and summarizes the antioxidant therapeutic approaches currently available in the literature. This important role played by oxidative stress suggests potential novel additional therapeutic interventions by either pharmacologic or nutritional measures, on top of enzyme replacement therapy, aimed at improving/halting the progression of cardio-cerebrovascular disease and nephropathy that occur in Fabry patients.

Does administration of hydroxychloroquine/amiodarone affect the efficacy of enzyme replacement therapy for Fabry mice?

As a standard therapy for Fabry disease, enzyme replacement therapy (ERT) with recombinant human α-galactosidase A (α-Gal) has been successfully used, and the instructions for this drug state that “it should not be co-administrated with cationic amphiphilic drugs such as hydroxychloroquine (HCQ) and amiodarone (AMI), since these drugs have the potential to inhibit intracellular α-Gal activity”. However, there would be cases in which HCQ or AMI is required for patients with Fabry disease, considering their medical efficacy and application.Thus, we examined the impact of HCQ/AMI on recombinant human α-Gal by in vitro, cellular, and animal experiments. The results revealed that HCQ/AMI affected the enzyme activity of α-Gal incorporated into cultured fibroblasts from a Fabry mouse when the cells were cultured in medium containing these drugs and the enzyme, although their direct inhibitory effect on the enzyme is not strong. These lysosomotropic drugs may be trapped and concentrated in lysosomes, followed by inhibition of α-Gal.On the other hand, no reduction of α-Gal activity incorporated into the organs and tissues, or acceleration of glycoshingolipid accumulation was observed in Fabry mice co-administered with HCQ/AMI and the enzyme, compared with in the case of usual ERT. As HCQ/AMI administered are catabolized in the liver, these drugs possibly do not affect ERT for Fabry mice, different from in the case of cultured cells in an environment isolated from the surroundings.

A Case Report of Kidney After Heart Transplant in Patient With Fabry Disease

Fabry disease is an X-linked inherited lysosomal storage disorder caused by a mutation in the gene encoding the enzyme α-galactosidase A. It is characterized by the accumulation of globotriaosylceramide in different tissues, resulting in a wide range of clinical presentations. Fabry cardiomyopathy and Fabry nephropathy are the disease's 2 most important life-threatening manifestations and can contribute to higher morbidity and mortality. Heart and kidney transplants can play a major role in patients with Fabry disease who develop end organ damage. We report a case of a successful heart transplant in a male patient with Fabry disease at the age of 62, followed by a kidney transplant later at the age of 69. He has had an uneventful post-transplant course and has been tolerating maintenance immunosuppression and enzyme replacement therapy with recombinant human α-galactosidase A.

The Relative Abundances of Human Leukocyte Antigen-E, α-Galactosidase A and α-Gal Antigenic Determinants Are Biased by Trichostatin A-Dependent Epigenetic Transformation of Triple-Transgenic Pig-Derived Dermal Fibroblast Cells.

The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from hFUT2×hGLA×HLA-E triple-transgenic pigs followed by trichostatin A (TSA)-assisted epigenetically modulating the reprogrammability of the transgenes permanently incorporated into the host genome and subsequent comprehensive analysis of molecular signatures related to proteomically profiling the generated ACFC lines. The results of Western blot and immunofluorescence analyses have proved that the profiles of relative abundance (RA) noticed for both recombinant human α-galactosidase A (rhα-Gal A) and human leukocyte antigen-E (HLA-E) underwent significant upregulations in tri-transgenic (3×TG) ACFCs subjected to TSA-mediated epigenetic transformation as compared to not only their TSA-unexposed counterparts but also TSA-treated and untreated non-transgenic (nTG) cells. The RT-qPCR-based analysis of porcine tri-genetically engineered ACFCs revealed stable expression of mRNA fractions transcribed from hFUT2, hGLA and HLA-E transgenes as compared to a lack of such transcriptional activities in non-transgenic ACFC variants. Furthermore, although TSA-based epigenomic modulation has given rise to a remarkable increase in the expression levels of Galα1→3Gal (α-Gal) epitopes that have been determined by lectin blotting analysis, their semi-quantitative profiles have dwindled profoundly in both TSA-exposed and unexposed 3×TG ACFCs as compared to their nTG counterparts. In conclusion, thoroughly exploring proteomic signatures in such epigenetically modulated ex vivo models devised on hFUT2×hGLA×HLA-E triple-transgenic ACFCs that display augmented reprogrammability of translational activities of two mRNA transcripts coding for rhα-Gal A and HLA-E proteins might provide a completely novel and powerful research tool for the panel of further studies. The objective of these future studies should be to multiply the tri-transgenic pigs with the aid of somatic cell nuclear transfer (SCNT)-based cloning for the purposes of both xenografting the porcine cutaneous bioprostheses and dermoplasty-mediated surgical treatments in human patients.

Prediction of improved therapeutics for fabry disease patients generated by mutagenesis of the α-galactosidase A active site, dimer interface, and glycosylation region

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A that induces the accumulation of the substrate globotriaosylceramide. Currently approved enzyme replacement therapy using recombinant human α-galactosidase A improves patient symptoms but a majority of patients experience adverse events due to the multiple infusions required for full therapeutic efficacy. Our approach is to use medicinal chemistry and phylogenic comparisons to introduce mutations into the human enzyme to increase catalytic activity and/or stability to generate an improved therapeutic enzyme that may require fewer infusions. We designed mutations at three regions of the human α-galactosidase A: the active site, the dimer interface, and a site for glycosylation. The M208E mutation, adjacent to the Y207 active site residue, increased enzyme activity 3.01-fold. This mutation introduced a charged Glu residue that is adjacent to the Y207 active site residue and close to a site of N-glycosylation. The W277C mutation, designed to promote dimer stability, introduced a strong thiol-aromatic interaction (Cys-Phe) at the dimer interface and increased activity 2.31-fold. The W277C and M208E mutations modify the structure of the enzyme into forms with enhanced thermal stability 3.7- and 3.9-fold, respectively and positive cooperativity resulting in increased Hill coefficient from 1.0 to 4.60 and 3.47, respectively. Enhanced thermal stability and positive cooperativity predict improved in vivo activity and superior therapeutic properties. Our results demonstrate the value of in vitro mutagenesis for α-galactosidase A and support future perspectives to validate these results in Fabry disease patients.

Could nutritional therapy take us further in our approaches to Fabry disease?

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficiency of enzyme α-galactosidase A activity. Clinical manifestation varies from mild to severe depending on the phenotype. The main clinical manifestations are cutaneous (angiokeratomas), neurologic (acroparesthesias), gastrointestinal (nausea, diarrhea, and abdominal pain), renal (proteinuria and kidney failure), cardiovascular (cardiomyopathy and arrhythmias), and cerebrovascular (stroke). Enzyme replacement therapy with recombinant human α-galactosidase is currently the therapeutic option for FD. Although enzyme replacement therapy has changed the natural history of disease, many clinical aspects of FD require an additional specific treatment. Nutritional approach is mostly indicated in case of nephropathy and gastrointestinal symptoms. Specific dietary interventions can modulate some pathogenetic mechanisms of the disease, such as the inflammation, oxidative stress, and autophagic disorders. However, to our knowledge, limited attention has been given to the nutritional aspects of FD. The aim of this review is to examine nutritional strategies that might interfere with several pathophysiologic aspects of FD, including inflammation and oxidative stress. A dietary approach should be part of the basic treatment in renal manifestations of FD. Dietary measures recommended for irritable bowel syndrome could be recommended for gastrointestinal symptoms. Dietary factors can modulate the inflammation, oxidative stress, and autophagy involved in FD. Polyphenols, ω-3 fatty acids, microbiota, and specific dietary patterns can interfere with inflammation/oxidative stress and autophagy mechanisms and could also contribute to the slowing of FD progression.

Oxidative Stress and Cardiovascular-Renal Damage in Fabry Disease: Is There Room for a Pathophysiological Involvement?

Fabry disease is an X-linked lysosomal storage disease caused by mutations in the GLA gene that lead to a reduction or an absence of the enzyme α-galactosidase A, resulting in the progressive and multisystemic accumulation of globotriaosylceramide. Clinical manifestation varies from mild to severe, depending on the phenotype. The main clinical manifestations are cutaneous (angiokeratomas), neurological (acroparesthesias), gastrointestinal (nausea, diarrhea abdominal pain), renal (proteinuria and kidney failure), cardiovascular (cardiomyopathy and arrhythmias), and cerebrovascular (stroke). A diagnosis of Fabry disease can be made with an enzymatic assay showing absent or reduced α-galactosidase A in male patients, while in heterozygous female patients, molecular genetic testing is needed. Enzyme replacement therapy (ERT) with recombinant human α-galactosidase is nowadays the most-used disease-specific therapeutic option. Despite ERT, cardiocerebrovascular-renal irreversible organ injury occurs, therefore additional knowledge and a deeper understanding of further pathophysiological mechanisms leading to end organ damage in Fabry disease are needed. Recent data point toward oxidative stress, oxidative stress signaling, and inflammation as some such mechanisms. In this short review, the current knowledge on the involvement of oxidative stress in cardiovascular-renal remodeling is summarized and related to the most recent evidence of oxidative stress activation in Fabry disease, and clearly points toward the involvement of oxidative stress in the pathophysiology of the medium- to long-term cardiovascular-renal damage of Fabry disease.

Development and Analytical Characterization of Pegunigalsidase Alfa, a Chemically Cross-Linked Plant Recombinant Human α-Galactosidase-A for Treatment of Fabry Disease.

The current treatment of Fabry disease by enzyme replacement therapy with commercially available recombinant human α-Galactosidase A shows a continuous deterioration of the disease patients. Human recombinant α-Galactosidase A is a homodimer with noncovalently bound subunits and is expressed in the ProCellEx plant cell-based protein expression platform to produce pegunigalsidase alfa. The effect of covalent bonding between two α-Galactosidase A subunits by PEG-based cross-linkers of various lengths was evaluated in this study. The results show that cross-linking by a bifunctional PEG polymer of 2000 Da produces a more stable protein with improved pharmacokinetic and biodistribution properties. The chemical modification did not influence the tertiary protein structure but led to an increased thermal stability and showed partial masking of immune epitopes. The developed pegunigalsidase alfa is currently tested in phase III clinical trials and has a potential to show superior efficacy versus the currently used enzyme replacement therapies in the treatment of Fabry disease patients.

Mutagenesis of the Human α‐Galactosidase A Active Site, Dimer Interface, and Glycosylation Region

Fabry disease is an X‐linked lysosomal storage disorder caused by the deficiency of the enzyme, α‐galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40–60 years of age. Currently approved enzyme replacement therapy using recombinant human α‐galactosidase A improves patient symptoms but storage of the toxic enzyme substrate remains in the brain and other affected organs. The structure of α‐galactosidase A has been previously determined to be a homodimer with six N‐linked glycosylation sites, and the catalytic mechanism determined to be a ping pong bi bi with the second substrate being water. The purpose of this research is to generate a more efficient enzyme replacement therapy alternative. A more efficient therapy was investigated utilizing Pichia pastoris, as well as site directed mutagenesis of the human enzyme. Three locations on the structure of α‐galactosidase A protein were targeted for mutation: the active site; the dimer interface; and the hydrophobic loop between the active site and the third glycosylation site. Viable candidates for further study into a therapeutic were determined based on catalytic efficiency (kcat/Km), thermostability, and possible glycosylation independence. Three mutations were identified, M208E (hydrophobic loop), W277C (dimer interface), and Y207W (active site) to be of potential therapeutic significance.Support or Funding InformationNIH SBIR Grants and Royalty Income From PatentsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis of (3S,4S,5S)-trihydroxylpiperidine derivatives as enzyme stabilizers to improve therapeutic enzyme activity in Fabry patient cell lines

A series of 3S,4S,5S-trihydroxylated piperidines bearing structural diversity at C-2 or C-6 positions has been synthesized and tested to determine their ability to stabilize the activity of recombinant human α-Galactosidase A (rh-α-Gal A). Hit molecules were identified by rapid inhibitory activity screening, and then further investigated for their ability to protect this enzyme from thermo-induced denaturation and enhance its activity in Fabry patient cell lines. Our study resulted in the identification of a new class of small molecules as enzyme stabilizers for the potential treatment of Fabry disease. Of these, stabilizer 21 was the most effective, showing a 12-fold increase in rh-α-Gal A activity in Fabry disease cell lines.

High yield process for the production of active human α-galactosidase a in CHO-K1 cells through lentivirus transgenesis.

Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg cell-1 d-1 ). After two purification steps, the active enzyme was recovered (2.4 × 106 U mg-1 ) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334-1345, 2017.

Fabry Disease: Pathogenesis, Clinical Symptoms, and Treatment with Enzyme Replacement Therapy

Fabry disease is an X-linked lysosomal disorder caused by a deficiency in lysosomal glycohydrolase α-galactosidase A. This defect results in the accumulation of glycosphingolipids in various organ systems. Lipid deposits occur preferentially in vascular endothelial and smooth muscle cells, leading to vascular dysfunction, which results in tissue ischemia and vessel occlusion. Clinical symptoms are divided into two categories: early symptoms and late complications. Early symptoms include acroparesthesia, bouts of pain in the hands or feet, hypohidrosis, angiokeratoma, and gastrointestinal complications, which begin in the early childhood of patients with Fabry disease. Late complications include renal and cardiac dysfunction, and cerebral infarction, which determine morbidity and mortality after age 30 years. Enzyme replacement therapy (ERT) using recombinant human α-galactosidase A has recently developed, and has been shown to improve prognosis of patients with Fabry disease. Recent reports investigating long-term outcomes with ERT have shown that early initiation before the development of irreversible organ failure is particularly effective. Diagnosis at the early stage of the disorder and the early initiation of ERT alleviate symptoms and prevent late complications. The disorder should be widely recognized to improve patients' outcome.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α‐galactosidaseA

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.

Coformulation of a Novel Human α-Galactosidase A With the Pharmacological Chaperone AT1001 Leads to Improved Substrate Reduction in Fabry Mice

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene that encodes α-galactosidase A and is characterized by pathological accumulation of globotriaosylceramide and globotriaosylsphingosine. Earlier, the authors demonstrated that oral coadministration of the pharmacological chaperone AT1001 (migalastat HCl; 1-deoxygalactonojirimycin HCl) prior to intravenous administration of enzyme replacement therapy improved the pharmacological properties of the enzyme. In this study, the authors investigated the effects of coformulating AT1001 with a proprietary recombinant human α-galactosidase A (ATB100) into a single intravenous formulation. AT1001 increased the physical stability and reduced aggregation of ATB100 at neutral pH in vitro, and increased the potency for ATB100-mediated globotriaosylceramide reduction in cultured Fabry fibroblasts. In Fabry mice, AT1001 coformulation increased the total exposure of active enzyme, and increased ATB100 levels in cardiomyocytes, cardiac vascular endothelial cells, renal distal tubular epithelial cells, and glomerular cells, cell types that do not show substantial uptake with enzyme replacement therapy alone. Notably, AT1001 coformulation also leads to greater tissue globotriaosylceramide reduction when compared with ATB100 alone, which was positively correlated with reductions in plasma globotriaosylsphingosine. Collectively, these data indicate that intravenous administration of ATB100 coformulated with AT1001 may provide an improved therapy for Fabry disease and thus warrants further investigation.

Analysis of left ventricular mass in untreated men and in men treated with agalsidase-β: data from the Fabry Registry

The aim of this study was to evaluate the progression of left ventricular hypertrophy in untreated men with Fabry disease and to assess the effects of agalsidase-β (recombinant human α-galactosidase A) on left ventricular hypertrophy. Longitudinal Fabry Registry data were analyzed from 115 men treated with agalsidase-β (1 mg/kg/2 weeks) and 48 untreated men. Measurements included baseline left-ventricular mass and at least one additional left-ventricular mass assessment over ≥ 2 years. Patients were grouped into quartiles, based on left-ventricular mass slopes. Multivariate logistic regression analyses identified factors associated with left ventricular hypertrophy progression. For men in whom treatment was initiated at the age of 18 to <30 years, mean left ventricular mass slope was -3.6 g/year (n = 31) compared with +9.5 g/year in untreated men of that age (n = 15) (P < 0.0001). Untreated men had a 3.4-fold higher risk of having faster increases in left-ventricular mass compared with treated men (odds ratio: 3.43; 95% confidence interval: 1.05-11.22; P = 0.0415). A baseline age of ≥ 40 years was also associated with left--ventricular hypertrophy progression (odds ratio: 5.03; 95% confidence interval: 1.03-24.49; P = 0.0457) compared with men younger than 30 years. Agalsidase-β treatment for ≥2 years may improve or stabilize left-ventricular mass in men with Fabry disease. Further investigations may determine whether early intervention and stabilization of LVM are correlated with clinical outcomes.

Enhanced sialylation and in vivo efficacy of recombinant human α-galactosidase through in vitro glycosylation

Human α-galactosidase A (GLA) has been used in enzyme replacement therapy for patients with Fabry disease. We expressed recombinant GLA from Chinese hamster ovary cells with very high productivity. When compared to an approved GLA (agalsidase beta), its size and charge were found to be smaller and more neutral. These differences resulted from the lack of terminal sialic acids playing essential roles in the serum half-life and proper tissue targeting. Because a simple sialylation reaction was not enough to increase the sialic acid content, a combined reaction using galactosyltransferase, sialyltransferase, and their sugar substrates at the same time was developed and optimized to reduce the incubation time. The product generated by this reaction had nearly the same size, isoelectric points, and sialic acid content as agalsidase beta. Furthermore, it had better in vivo efficacy to degrade the accumulated globotriaosylceramide in target organs of Fabry mice compared to an unmodified version. [BMB Reports 2013; 46(3): 157-162]

Co-administration With the Pharmacological Chaperone AT1001 Increases Recombinant Human α-Galactosidase A Tissue Uptake and Improves Substrate Reduction in Fabry Mice

Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by mutations in the gene (GLA) that encodes the lysosomal hydrolase α-galactosidase A (α-Gal A), and is characterized by pathological accumulation of the substrate, globotriaosylceramide (GL-3). Regular infusion of recombinant human α-Gal A (rhα-Gal A), termed enzyme replacement therapy (ERT), is the primary treatment for Fabry disease. However, rhα-Gal A has low physical stability, a short circulating half-life, and variable uptake into different disease-relevant tissues. We hypothesized that coadministration of the orally available, small molecule pharmacological chaperone AT1001 (GR181413A, 1-deoxygalactonojirimycin, migalastat hydrochloride) may improve the pharmacological properties of rhα-Gal A via binding and stabilization. AT1001 prevented rhα-Gal A denaturation and activity loss in vitro at neutral pH and 37 °C. Coincubation of Fabry fibroblasts with rhα-Gal A and AT1001 resulted in up to fourfold higher cellular α-Gal A and ~30% greater GL-3 reduction compared to rhα-Gal A alone. Furthermore, coadministration of AT1001 to rats increased the circulating half-life of rhα-Gal A by >2.5-fold, and in GLA knockout mice resulted in up to fivefold higher α-Gal A levels and fourfold greater GL-3 reduction than rhα-Gal A alone. Collectively, these data highlight the potentially beneficial effects of AT1001 on rhα-Gal A, thus warranting clinical investigation.

Fabry Disease: from Diagnosis to Therapy

Fabry disease (FD) is an accelerating, X-linked hereditary disorder of glycosphingolipid metabolism which occurs due to improper lysosomal α-galactosidase A activity. Its pan-ethnic and the annually reported occurrence may underestimate the true frequency of the disease. Classically affected hemizygous males, with no residual α-galactosidase A activity may display all the characteristics like cardiovascular (cardiomyopathy), cutaneous (angiokeratoma), renal (kidney failure), neurological (pain), cerebrovascular and cochleo-vestibular signs of the disease while heterozygous females have similar symptoms ranging from very mild to severe. Incomplete action of lysosomal α-galactosidase A results in gradual increase of globotriaosylceramide within lysosomes, supposed to set off a flow of cellular events. Enzyme analysis may occasionally help to detect heterozygote but in females results are often questionable because of random X-chromosomal inactivation that obligates the molecular testing (genotyping). Due to ethical reasons determination of enzyme activity through prenatal diagnosis is carried out only in male's fetus. The subsistence of atypical variants and the unavailability of a specific therapy complicate the genetic counseling. Enzyme replacement therapy (ERT) is the only specific therapeutic option that introduced recombinant human α-galactosidase A. End stage renal disease and critical cardiovascular or cerebrovascular complications limit the life-expectancy of untreated males and females. Oral therapy drives the research forward into active site specific chaperones. Adjunctive therapy of ERT with

Anti-α-galactosidase A antibody response to agalsidase beta treatment: Data from the Fabry Registry

Agalsidase beta, a form of recombinant human α-galactosidase A (αGAL), is approved for use as enzyme replacement therapy (ERT) for Fabry disease. An immunogenic response against a therapeutic protein could potentially impact its efficacy or safety. The development of anti-αGAL IgG antibodies was evaluated in 571 men and 251 women from the Fabry Registry who were treated with agalsidase beta. Most men developed antibodies (416 of 571, 73%), whereas most women did not (31 of 251, 12%). Women were also significantly more likely to tolerize than men; whereas 18 of 31 women tolerized (58%, 95%CI: 52%–64%), only 47 of 416 men tolerized during the observation period (11%, 95% CI: 8%–15%). Patients who eventually tolerized had lower median peak anti-αGAL IgG antibody titers than patients who remained seropositive at their most recent assessment (400 versus 3200 in men, 200 versus 400 in women, respectively). Patients with nonsense mutations in the GLA gene were more likely to develop anti-αGAL IgG antibodies than patients with missense mutations. Approximately 26% of men (151 of 571) reported infusion-associated reactions (IARs), compared to 11% of women (27 of 251). Men who developed anti-αGAL IgG antibodies were more likely to experience IARs compared to those who remained seronegative. Nine percent of seronegative men and women (34 of 375) reported IARs. The majority of IARs occurred during the first 6 to 12months of agalsidase beta treatment and decreased over time, in both seroconverted and seronegative patients.

Efficient Uptake of Recombinant α-Galactosidase A Produced with a Gene-Manipulated Yeast by Fabry Mice Kidneys

To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.