Screening of Fabry disease in patients with chronic kidney disease in Japan.

BACKGROUND: Fabry disease (FD), an X-linked lysosomal storage disorder caused by a deficiency in alfa-galactosidase A (α-Gal A) activity due to mutations in the GLA gene, has a prevalence of 0-1.69% in patients undergoing haemodialysis; however, its prevalence in patients with chronic kidney disease (CKD) Stages 1-5 is unknown. METHODS: Serum α-Gal A activity analysis and direct sequencing of GLA were used to screen for FD in 2122 male patients with CKD, including 1703 patients with CKD Stage 5D and 419 with CKD Stages 1-5. The correlation between serum α-Gal A activity and confounding factors in patients with CKD Stages 1-5 was evaluated. RESULTS: FD prevalence rates in patients with CKD Stage 5D and CKD Stages 1-5 were 0.06% (1/1703) and 0.48% (2/419), respectively. A patient with CKD Stage 5D exhibited a novel GLA mutation, p.Met208Arg, whereas two patients with CKD Stages 1-5 had c.370delG and p.Met296Ile. p. Met208Arg caused moderate structural changes in the molecular surface region near the substituted amino acid residue but did not affect the catalytic residues Asp170 and Asp231 in α-Gal A. Serum α-Gal A activity in patients with CKD Stages 1-5 was inversely correlated with age (P < 0.0001) but directly correlated with estimated glomerular filtration rate (P < 0.0001). CONCLUSIONS: FD prevalence was much higher in male patients with CKD Stages 1-5 than in those with CKD Stage 5D. FD screening in patients with CKD Stages 1-5 may improve patient survival, decreasing the number of patients with CKD Stage 5D.

Effectiveness of immunosuppressive therapy for nephrotic syndrome in a patient with late-onset Fabry disease: a case report and literature review.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations of the GLA gene, followed by deficiency in α-galactosidase A (α-gal) activity. Nephrotic syndrome, as the renal phenotype of FD, is unusual. Here, we report the rare case of a patient with FD with nephrotic syndrome whose proteinuria disappeared by immunotherapy. CASE PRESENTATION: A 67-year-old Japanese man was admitted to our hospital because of emesis, abdominal pain, and facial edema due to nephrotic syndrome. The patient was diagnosed with focal segmental glomerulosclerosis (FSGS) by renal biopsy before being diagnosed with FD, and immunotherapy was initiated. After treatment, the kidney biopsy results showed typical glycosphingolipid accumulation in the podocytes of this patient. The white blood cell α-gal activity was very low, and genetic analysis revealed a GLA gene variant (M296I), which is known as a late-onset genetic mutation of FD. Immunotherapy (steroids and cyclosporine A) dramatically improved the massive proteinuria. Currently, he has been undergoing enzyme replacement therapy, and his proteinuria has further decreased. There is the possibility that other nephrotic syndromes, such as minimal change nephrotic syndrome or FSGS, may co-exist in this patient. CONCLUSIONS: We experienced the rare case of a FD patient whose nephrotic syndrome disappeared by immunotherapy. These findings suggest that immunosuppressive treatment may be considered if nephrotic syndrome develops, even in patients with FD.

Mucopolysaccharidosis type VI (MPS VI) and molecular analysis: Review and classification of published variants in the ARSB gene.

Maroteaux-Lamy syndrome (MPS VI) is an autosomal recessive lysosomal storage disorder caused by pathogenic ARSB gene variants, commonly diagnosed through clinical findings and deficiency of the arylsulfatase B (ASB) enzyme. Detection of ARSB pathogenic variants can independently confirm diagnosis and render genetic counseling possible. In this review, we collect and summarize 908 alleles (201 distinct variants, including 3 polymorphisms previously considered as disease-causing variants) from 478 individuals diagnosed with MPS VI, identified from literature and public databases. Each variant is further analyzed for clinical classification according to American College of Medical Genetics and Genomics (ACMG) guidelines. Results highlight the heterogeneity of ARSB alleles, with most unique variants (59.5%) identified as missense and 31.7% of unique alleles appearing once. Only 18% of distinct variants were previously recorded in public databases with supporting evidence and clinical significance. ACMG recommends publishing clinical and biochemical data that accurately characterize pathogenicity of new variants in association with reporting specific alleles. Variants analyzed were sent to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and MPS VI locus-specific database (http://mps6-database.org) where they will be available. High clinical suspicion coupled with diagnostic testing for deficient ASB activity and timely submission and classification of ARSB variants with biochemical and clinical data in public databases is essential for timely diagnosis of MPS VI.

Inhibition of astrocytic adenosine receptor A2A attenuates microglial activation in a mouse model of Sandhoff disease.

Astrocyte-microglia communication influences the onset and progression of central nervous system (CNS) disorders. In this study, we determined how chronic inflammation by activated astrocytes affected and regulated CNS functions in Sandhoff disease (SD), a CNS lysosomal storage disorder. SD triggers intense CNS inflammation such as microglial activation and astrogliosis. It is caused by mutation of the HEXB gene, which reduces ß-hexosaminidase (Hex) enzymatic activity in lysosomes, leading to accumulation of the substrate GM2 ganglioside in neuronal cells. Hexb-/- mice display a phenotype similar to human patients that suffer from chronic inflammation characterized by activation of astrocytes and microglia. In Hexb-/- mice, tremors and loss of muscle coordination begins at ~12 weeks. Interestingly, we found that reactive astrocytes expressed adenosine A2A receptor in the cerebral cortices of Hexb-/- mice at the later inflammatory phase. In cultured astrocytes, expression of A2A receptor could be induced by astrocyte defined medium, and then the activation of the A2A receptor induced ccl2 expression. In Hexb-/- mice, inhibition of the A2A receptor antagonized by istradefylline decreased the number of activated microglial cells and inflammatory cytokines/chemokines at 13 weeks. Thus, the astrocytic A2A receptor is an important sensor that regulates microglial activation in the late phase of inflammation.

Plasma lyso-Gb3: a biomarker for monitoring fabry patients during enzyme replacement therapy.

BACKGROUND: Recently, globotriaosylsphingosine (lyso-Gb3) has attracted interest as a biomarker of Fabry disease. However, little is known regarding its utility for the evaluation of the therapeutic efficacy. METHOD: We measured plasma lyso-Gb3 concentration in Japanese healthy subjects and Fabry patients by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). We determined the reference interval in Japanese (UMIN000016854), and examined the effect of enzyme replacement therapy (ERT) with recombinant α-galactosidase A (GLA) and the influence of antibodies against the enzyme on the plasma lyso-Gb3 level in Fabry patients (UMIN000017152). RESULTS: The reference interval was determined to be 0.35-0.71 nmol/L, this being almost the same as the normal range in a non-Japanese population previously reported. The analysis revealed that the plasma lyso-Gb3 level was strikingly increased in classic Fabry males, and to a lesser extent in later-onset Fabry males and Fabry females. The elevation of the plasma lyso-Gb3 level was related to renal involvement in the Fabry females. ERT gave a rapid reduction in the elevated plasma lyso-Gb3 level in the classic Fabry males, and a gradual one or stabilization in most of the later-onset Fabry males and Fabry females. However, formation of antibodies against the recombinant GLA had a negative effect on the reduction of plasma lyso-Gb3. CONCLUSIONS: Regular observation of plasma lyso-Gb3 and antibodies is useful for monitoring of Fabry patients during ERT.

Differences in cleavage of globotriaosylceramide and its derivatives accumulated in organs of young Fabry mice following enzyme replacement therapy.

In Fabry disease, large amounts of globotriaosylceramide (Gb3) and related glycosphingolipids accumulate in organs due to a deficiency of α-galactosidase A (GLA) activity. Enzyme replacement therapy (ERT) with recombinant GLA is now available, and it has been reported that ERT is beneficial for patients with Fabry disease, especially those who start treatment at an early stage of the disease. However, it seems that the efficacy of ERT differs with each organ, and Gb3 accumulated in the kidneys shows resistance to ERT when it is started at a late stage. In this study, we examined the differences in cleavage of Gb3 isoforms, and lyso-Gb3 and its analogues in the kidneys, liver, and heart in young Fabry mice subjected to ERT. The results revealed that recurrent administration of recombinant GLA had prominent effects in terms of degradation of Gb3 and its derivatives accumulated in the organs. However, particular Gb3 isoforms, i.e., Gb3 (C20:0) and Gb3 (C24OH), accumulated in the kidneys largely escaped from degradation. Such Gb3 isoforms may gradually accumulate in the kidneys from a young age, which results in a reduction in the efficacy of ERT for Fabry disease.

Molecular diagnosis of 65 families with mucopolysaccharidosis type II (Hunter syndrome) characterized by 16 novel mutations in the IDS gene: Genetic, pathological, and structural studies on iduronate-2-sulfatase.

Mucopolysaccharidosis type II (MPS II: also called as Hunter syndrome) is an X-linked recessive lysosomal storage disorder characterized by the accumulation of extracellular glycosaminoglycans due to the deficiency of the enzyme iduronate-2-sulfatase (IDS). Previous observations suggested that MPS II can be classified into two distinct disease subtypes: (1) severe type of MPS II involves a decline in the cognitive ability of a patient and (2) attenuated type of MPS II exhibits no such intellectual phenotype. To determine whether such disease subtypes of MPS II could be explained by genetic diagnosis, we analyzed mutations in the IDS gene of 65 patients suffering from MPS II among the Japanese population who were diagnosed with both the accumulation of urinary glycosaminoglycans and a decrease in their IDS enzyme activity between 2004 and 2014. Among the specimens examined, we identified the following mutations: 33 missense, 8 nonsense, 7 frameshift, 4 intronic changes affecting splicing, 8 recombinations involving IDS-IDS2, and 7 other mutations including 4 large deletions. Consistent with the previous data, the results of our study showed that most of the attenuated phenotype was derived from the missense mutations of the IDS gene, whereas mutations associated with a large structural alteration including recombination, splicing, frameshift, and nonsense mutations were linked to the severe phenotype of MPS II. Furthermore, we conducted a homology modeling study of IDS P120R and N534I mutant as representatives of the causative mutation of the severe and attenuated type of MPS II, respectively. We found that the substitution of P120R of the IDS enzyme was predicted to deform the α-helix generated by I119-F123, leading to the major structural alteration of the wild-type IDS enzyme. In sharp contrast, the effect of the structural alteration of N534I was marginal; thus, this mutation was pathogenically predicted to be associated with the attenuated type of MPS II. These results suggest that a combination of the genomic diagnosis of the IDS gene and the structural prediction of the IDS enzyme could enable the prediction of a phenotype more effectively.

Human α-L-iduronidase uses its own N-glycan as a substrate-binding and catalytic module.

N-glycosylation is a major posttranslational modification that endows proteins with various functions. It is established that N-glycans are essential for the correct folding and stability of some enzymes; however, the actual effects of N-glycans on their activities are poorly understood. Here, we show that human α-l-iduronidase (hIDUA), of which a dysfunction causes accumulation of dermatan/heparan sulfate leading to mucopolysaccharidosis type I, uses its own N-glycan as a substrate binding and catalytic module. Structural analysis revealed that the mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. A deglycosylation study showed that enzyme activity was highly correlated with the N-glycan attached to N372. The kinetics of native and deglycosylated hIDUA suggested that the N-glycan is also involved in catalytic processes. Our study demonstrates a previously unrecognized function of N-glycans.

Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant GM1 ß-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations.

A heterozygous female with Fabry disease due to a novel α-galactosidase A mutation exhibits a unique synaptopodin distribution in vacuolated podocytes.

We report the case of a 42-yearold woman diagnosed with heterozygous Fabry disease (FD) due to a novel α-galactosidase A Pro210Ser mutation and exhibiting a unique distribution of synaptopodin within podocytes. The patient was referred to our hospital with moderate proteinuria, and a renal biopsy was performed. Light microscopic examination of the specimen revealed diffuse global enlargement of podocytes, which also showed foamy changes. Electron microscopy revealed abundant myeloid bodies in podocytes and focal mitochondrial abnormalities within the tubules. The patient exhibited none of the characteristic symptoms of FD except hypohidrosis and had no obvious family history. Genetic analysis revealed a novel missense mutation (Pro210Ser) in the α-galactosidase A gene. She was ultimately diagnosed with FD based on immunohistochemical staining indicating large amounts of accumulated globotriaosylceramide in her podocytes, detection of urinary globotriaosylceramide secretion using high-performance thin-layer chromatography/ immunostaining, and structural modeling of the mutated α-galactosidase A (Pro210Ser). Immunostaining of the swollen and foamy podocytes using podocyte-associated antibodies (against podocalyxin, Wilms tumor-1, vimentin, and synaptopodin) revealed a unique distribution of synaptopodin surrounding globotriaosylceramide. To our knowledge, this is the first report of immunohistologically detected synaptopodin upregulation in foamy podocytes in a patient with FD.

Structural and clinical implications of amino acid substitutions in α-L-iduronidase: insight into the basis of mucopolysaccharidosis type I.

Allelic mutations, predominantly missense ones, of the α-l-iduronidase (IDUA) gene cause mucopolysaccharidosis type I (MPS I), which exhibits heterogeneous phenotypes. These phenotypes are basically classified into severe, intermediate, and attenuated types. We previously examined the structural changes in IDUA due to MPS I by homology modeling, but the reliability was limited because of the low sequence identity. In this study, we built new structural models of mutant IDUAs due to 57 amino acid substitutions that had been identified in 27 severe, 1 severe-intermediate, 13 intermediate, 1 attenuated-intermediate and 15 attenuated type MPS I patients based on the crystal structure of human IDUA, which was recently determined by us. The structural changes were examined by calculating the root-mean-square distances (RMSD) and the number of atoms influenced by the amino acid replacements. The results revealed that the structural changes of the enzyme protein tended to be correlated with the severity of the disease. Then we focused on the structural changes resulting from amino acid replacements in the immunoglobulin-like domain and adjacent region, of which the structure had been missing in the IDUA model previously built. Coloring of atoms influenced by an amino acid substitution was performed in each case and the results revealed that the structural changes occurred in a region far from the active site of IDUA, suggesting that they affected protein folding. Structural analysis is thus useful for elucidation of the basis of MPS I.

Comparative study on mannose 6-phosphate residue contents of recombinant lysosomal enzymes.

As most recombinant lysosomal enzymes are incorporated into cells via mannose 6-phosphate (M6P) receptors, the M6P content is important for effective enzyme replacement therapy (ERT) for lysosomal diseases. However, there have been no comprehensive reports of the M6P contents of lysosomal enzymes. We developed an M6P assay method comprising three steps, i.e., acid hydrolysis of glycoproteins, derivatization of M6P, and high-performance liquid chromatography, and determined the M6P contents of six recombinant lysosomal enzymes now available for ERT and one in the process of development. The assay is easy, specific, and reproducible. The results of the comparative study revealed that the M6P contents of agalsidase alfa, agalsidase beta, modified α-N-acetylgalactosaminidase, alglucosidase alfa, laronidase, idursulfase, and imiglucerase are 2.1, 2.9, 5.9, 0.7, 2.5, 3.2, and <0.3 mol/mol enzyme, respectively. The results were correlated with those of the biochemical analyses previously performed and that of the binding assay of exposed M6P of the enzymes with the domain 9 of the cation-independent M6P receptor. This assay method is useful for comparison of the M6P contents of recombinant lysosomal enzymes for ERT.

A case of Fabry nephropathy with histological features of oligonephropathy.

UNLABELLED: Newborn screening studies indicate the expected high incidence of later-onset Fabry disease with silent Fabry nephropathy while, with recent improved clinical care of premature infants, children with congenital oligonephropathy caused by premature embryonal development of the kidney are thought to be increasing. However, the coexistence of Fabry nephropathy and oligonephropathy has not been reported previously. We present the case of a 13-year-old boy who was diagnosed with Fabry nephropathy accompanied with histological features of oligonephropathy. He was born as a preterm baby, and a renal biopsy was performed because of mild renal dysfunction and mild proteinuria. He had neither characteristic early symptoms nor a family history of Fabry disease. Histologic findings demonstrated diffuse global enlargement and foamy change of podocytes with markedly decreased number and enlargement of the glomeruli. Both his plasma and leukocyte α-galactosidase A (GLA) activities were markedly decreased, and the plasma globotriaosylsphingosine and urine globotriaosylceramide levels were increased. Gene analysis revealed a missense mutation, R112H, in the GLA gene, which had been reported in the later-onset phenotype of Fabry patients. He is now under treatment with enzyme replacement therapy and an angiotensin-converting enzyme inhibitor. CONCLUSION: This case indicated the possible co-occurrence of Fabry nephropathy and oligonephropathy. For early diagnosis and timely management, careful examinations for proteinuria and renal function, in addition to establishing an effective screening system for Fabry disease, will be necessary.

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.

Potential Usefulness of Lifetime Globotriaosylsphingosine Exposure at Diagnosis and Baseline Modified Disease Severity Score in Early-Diagnosed Patients With Fabry Disease.

BACKGROUND: Fabry disease (FD) is a lysosomal storage disease caused by a deficit of α-galactosidase A (GAL). Recently, plasma globotriaosylsphingosine (lyso-Gb3), a pathogenic analog of a substrate of GAL, has been suggested as a potential biomarker for FD, and disease severity scores, such as the Mainz Severity Score Index (MSSI), the Disease Severity Scoring System (DS3), and FASTEX (FAbry STabilization indEX), are useful tools for evaluating the severity of signs and symptoms in symptomatic FD patients. However, a more useful method of evaluating disease severity in early-diagnosed FD patients such as children, adult females, and asymptomatic patients is needed. Here, we proposed modified MSSI and DS3 scores to which we added phenotype, urinary mulberry bodies, and history of past pain attacks and examined the clinical usefulness of lyso-Gb3 and modified scores for early-diagnosed FD patients. RESULT: In 13 early-diagnosed FD patients, we developed modified MSSI and DS3 scores and examined the correlation of lifetime lyso-Gb3 exposure at diagnosis with the conventional or modified scores. Lifetime lyso-Gb3 exposure was positively correlated only with the modified DS3 score. Additionally, we examined the long-term changes in plasma lyso-Gb3 concentration and in conventional MSSI, DS3, and FASTEX. In males, plasma lyso-Gb3 concentration decreased more rapidly than in females. In all patients, the severity scores were mild and remained nearly stable throughout the follow-up period. CONCLUSION: Our data suggest that lifetime lyso-Gb3 exposure and the modified DS3 score are useful in early-diagnosed patients.

Monitoring of anti-drug antibodies and disease-specific biomarkers in three patients from a Japanese Fabry family treated with enzyme replacement therapy.

We monitored anti-drug antibodies and disease-specific biomarkers in three patients with a nonsense mutation from a Japanese Fabry family treated with enzyme replacement therapy (ERT). In two male patients from the family, neutralizing anti-drug antibodies were induced at an early stage of ERT, the antibody titer peak being found at an earlier stage of ERT in the patient treated with 1.0 mg/kg agalsidase beta than in that treated with 0.2 mg/kg agalsidase alfa. Then, the antibody titers decreased with continuation of ERT. The formation of neutralizing anti-drug antibodies adversely affected the plasma globotriaosylsphingosine (Lyso-Gb3) level and urinary globotriaosylceramide (Gb3) excretion in both patients, the impact being greater in the patient treated with 0.2 mg/kg agalsidase alfa than in that treated with 1.0 mg/kg agalsidase beta. The difference might be explained by the different doses of the infused enzymes based on supersaturation of the antibodies. In a heterozygous Fabry female from the family, no sign of antibody formation was found, and both the plasma Lyso-Gb3 level and urinary Gb3 excretion, which were moderately increased at the baseline, decreased gradually. No deterioration of the manifestations or laboratory findings was observed during ERT in either of the patients. Thus, monitoring of anti-drug antibodies and biomarkers in these Fabry patients provided us with important information on their pathological condition during ERT.

Profiles of Globotriaosylsphingosine Analogs and Globotriaosylceramide Isoforms Accumulated in Body Fluids from Various Phenotypic Fabry Patients.

Objectives Fabry disease is characterized by the systemic accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), which are widely used as biomarkers of the disease. However, few reports have described the relationship of Lyso-Gb3 analogs and Gb3 isoforms with the disease. The present study determined the profiles of Lyso-Gb3 analogs and Gb3 isoforms accumulated in body fluids from various phenotypic Fabry patients to elucidate the basis of the disease. Methods Plasma Lyso-Gb3 and related analogs were measured in 15 classic Fabry men, 6 later-onset Fabry men, 11 Fabry women, and 36 controls, while urinary Gb3 isoforms were measured in 5 classic Fabry men, 5 later-onset Fabry men, 17 Fabry women, and 11 controls, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, these values were monitored for a classic Fabry man, in whom neutralizing anti-drug antibodies had developed following enzyme replacement therapy (ERT). Results The levels of plasma Lyso-Gb3 analogs/urinary Gb3 isoforms were higher in Fabry patients than in controls, especially in classic Fabry men. However, minor differences in the ratio of each Lyso-Gb3 analog and Gb3 isoform with respect to the total Lyso-Gb3 analogs and Gb3 isoforms, respectively, were observed among individual classic Fabry men. Their time courses were well associated with the development and attenuation of anti-drug antibodies in a patient with classic Fabry disease during ERT. Conclusion Quantification of Lyso-Gb3 analogs and Gb3 isoforms provides us with more detailed information about the substrates that accumulated in the body fluids of Fabry patients than does quantification of Lyso-Gb3 and Gb3 alone, so this approach may be useful for elucidating the basis of Fabry disease.

In Vivo Delivery of Therapeutic Molecules by Transplantation of Genome-Edited Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) have already been used in transplantation therapies. Currently, cells from healthy people are transplanted into patients with diseases. With the rapid evolution of genome editing technology, genetic modification could be applied to enhance the therapeutic effects of iPSCs, such as the introduction of secreted molecules to make the cells a drug delivery system. Here, we addressed this possibility by utilizing a Fabry disease mouse model, as a proof of concept. Fabry disease is caused by the lack of α-galactosidase A (GLA). We previously developed an immunotolerant therapeutic molecule, modified α-N-acetylgalactosaminidase (mNAGA). We confirmed that secreted mNAGA from genome-edited iPSCs compensated for the GLA activity in GLA-deficient cells using an in vitro co-culture system. Moreover, iPSCs transplanted into Fabry model mice secreted mNAGA and supplied GLA activity to the liver. This study demonstrates the great potential of genome-edited iPSCs secreting therapeutic molecules.