Yohei Sato

Disease modeling and lentiviral gene transfer in patient-specific induced pluripotent stem cells from late-onset Pompe disease patient

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid α-glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart, and liver. Hypertrophic cardiomyopathy is frequently seen in the infantile onset Pompe disease. On the other hand, cardiovascular complication of the late-onset Pompe disease is considered as less frequent and severe than that of infantile onset. There are few investigations which show cardiovascular complication of late onset Pompe disease due to the shortage of appropriate disease model. We have generated late-onset Pompe disease-specific induced pluripotent stem cell (iPSC) and differentiated them into cardiomyocytes. Differentiated cardiomyocyte shows glycogen accumulation and lysosomal enlargement. Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC. The efficacy of gene therapy is maintained following the cardiomyocyte differentiation. Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote. It is suggested that Pompe disease iPSC-derived cardiomyocyte is replicating disease-specific changes in the context of disease modeling, drug screening, and cell therapy.

The production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants.

Summary For the production of therapeutic proteins in plants, the presence of β1,2‐xylose and core α1,3‐fucose on plants’ N‐glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down‐regulate the endogenous N‐acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco‐engineered line (Nb GNTI‐RNAi) showed a strong reduction of plant‐specific N‐glycans, with the result that as much as 90.9% of the total N‐glycans were of high‐mannose type. Therefore, this Nb GNTI‐RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The Nb GNTI‐RNAi plant was cross‐pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gauchers disease, requires terminal mannose for its therapeutic efficacy. The N‐glycan structures that were presented on all of the four occupied N‐glycosylation sites of recombinant GC in Nb GNTI‐RNAi plants (GC gnt1) showed that the majority (ranging from 73.3% up to 85.5%) of the N‐glycans had mannose‐type structures lacking potential immunogenic β1,2‐xylose and α1,3‐fucose epitopes. Moreover, GC gnt1 could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gauchers disease. Notably, the Nb GNTI‐RNAi line, producing GC, was stable and the Nb GNTI‐RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N‐glycan structures.

Metabolomic Profiling of Pompe Disease-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals That Oxidative Stress Is Associated With Cardiac and Skeletal Muscle Pathology

Pompe disease (PD) is a lysosomal storage disease that is caused by a deficiency of the acid α‐glucosidase, which results in glycogen accumulation in the lysosome. The major clinical symptoms of PD include skeletal muscle weakness, respiratory failure, and cardiac hypertrophy. Based on its severity and symptom onset, PD is classified into infantile and late‐onset forms. Lysosomal accumulation of glycogen can promote many types of cellular dysfunction, such as autophagic dysfunction, endoplasmic reticulum stress, and abnormal calcium signaling within skeletal muscle. However, the disease mechanism underlying PD cardiomyopathy is not fully understood. Several researchers have shown that PD induced pluripotent stem cell (iPSC)‐derived cardiomyocytes successfully replicate the disease phenotype and are useful disease models. We have analyzed the metabolomic profile of late‐onset PD iPSC‐derived cardiomyocytes and found that oxidative stress and mitochondrial dysfunction are likely associated with cardiac complications. Furthermore, we have validated that these disease‐specific changes were also observed in the cardiomyocytes and skeletal muscle of a genetically engineered murine PD model. Oxidative stress may contribute to skeletal muscle and cardiomyocyte dysfunction in PD mice; however, NF‐E2‐related factor 2 was downregulated in cardiomyocytes and skeletal muscle, despite evidence of oxidative stress. We hypothesized that oxidative stress and an impaired antioxidative stress response mechanism may underlie the molecular pathology of late‐onset PD. Stem Cells Translational Medicine 2017;6:31–39

TFEB overexpression promotes glycogen clearance of Pompe disease iPSC-derived skeletal muscle

Pompe disease (PD) is a lysosomal disorder caused by acid α-glucosidase (GAA) deficiency. Progressive muscular weakness is the major symptom of PD, and enzyme replacement therapy can improve the clinical outcome. However, to achieve a better clinical outcome, alternative therapeutic strategies are being investigated, including gene therapy and pharmacological chaperones. We previously used lentiviral vector-mediated GAA gene transfer in PD patient-specific induced pluripotent stem cells. Some therapeutic efficacy was observed, although glycogen accumulation was not normalized. Transcription factor EB is a master regulator of lysosomal biogenesis and autophagy that has recently been associated with muscular pathology, and is now a potential therapeutic target in PD model mice. Here, we differentiated skeletal muscle from PD patient-specific induced pluripotent stem cells by forced MyoD expression. Lentiviral vector-mediated GAA and transcription factor EB gene transfer independently improved GAA enzyme activity and reduced glycogen content in skeletal muscle derived from PD-induced pluripotent stem cells. Interestingly, GAA and transcription factor EB cooperatively improved skeletal muscle pathology, both biochemically and morphologically. Thus, our findings show that abnormal lysosomal biogenesis is associated with the muscular pathology of PD, and transcription factor EB gene transfer is effective as an add-on strategy to GAA gene transfer.

Anti-BlyS antibody reduces the immune reaction against enzyme and enhances the efficacy of enzyme replacement therapy in Fabry disease model mice

Formation of antibodies against a therapeutic enzyme is an important complication during enzyme replacement therapy (ERT) for lysosomal storage diseases. Fabry disease (FD) is caused by a deficiency of alpha-galactosidase (GLA), which results in the accumulation of globotriaosylceramide (GL-3). We have shown immune tolerance induction (ITI) during ERT in FD model mice by using an anti-B lymphocyte stimulator (anti-BlyS) antibody (belimumab). A single dose of the anti-BlyS antibody temporarily lowered the percentage of B cells and IgG antibody titer against recombinant human GLA. Administration of a low maintenance dose of the anti-BlyS antibody suppressed the B cell population and immunotolerance was induced in 20% of mice, but antibody formation could not be prevented. We then increased the maintenance dose of the anti-BlyS antibody and immunotolerance was induced in 50% of mice. Therapeutic enzyme distribution and clearance of GL-3 were also enhanced by a high maintenance dose of the anti-BlyS antibody.

Residual glycosaminoglycan accumulation in mitral and aortic valves of a patient with attenuated MPS I (Scheie syndrome) after 6 years of enzyme replacement therapy: Implications for early diagnosis and therapy

Mucopolysaccharidosis (MPS) is an inherited metabolic disease caused by deficiency of the enzymes needed for glycosaminoglycan (GAG) degradation. MPS type I is caused by the deficiency of the lysosomal enzyme alpha-l-iduronidase and is classified into Hurler syndrome, Scheie syndrome, and Hurler–Scheie syndrome based on disease severity and onset. Cardiac complications such as left ventricular hypertrophy, cardiac valve disease, and coronary artery disease are often observed in MPS type I. Enzyme replacement therapy (ERT) has been available for MPS type I, but the efficacy of this treatment for cardiac valve disease is unknown. We report on a 56-year-old female patient with attenuated MPS I (Scheie syndrome) who developed aortic and mitral stenosis and coronary artery narrowing. The cardiac valve disease progressed despite ERT and she finally underwent double valve replacement and coronary artery bypass grafting. The pathology of the cardiac valves revealed GAG accumulation and lysosomal enlargement in both the mitral and aortic valves. Zebra body formation was also confirmed using electron microscopy. Our results suggest that ERT had limited efficacy in previously established cardiac valve disease. Early diagnosis and initiation of ERT is crucial to avoid further cardiac complications in MPS type I.

Systemic accumulation of undigested lysosomal metabolites in an autopsy case of mucolipidosis type II; autophagic dysfunction in cardiomyocyte

Mucolipidosis type II is an autosomal recessive lysosomal storage disease caused by N-acetylglucosamine-1-phosphotransferese deficiency. We report here pathological findings of an autopsy case of mucolipidosis type II. The patient was an 8-year-old boy with mucolipidosis type II and was complicated with hypertrophic cardiomyopathy. He suddenly developed progressive respiratory failure and finally died. At autopsy, systemic accumulation of undigested lysosomal metabolites was prominent, particularly in the heart, lungs, and dorsal root ganglion. In cardiomyocyte, LC3, an autophagy marker, was positive in the cytoplasm. Ubiquitin, p62, K48 polyubiquitin, and K63 polyubiquitin were also positive in the cytoplasm. Our findings suggest that autophagic dysfunction might be associated with the cardiomyopahty of mucolipidosis type II.

Lentiviral Gene Transfer to iPS Cells: Toward the Cardiomyocyte Differentiation of Pompe Disease-Specific iPS Cells

Pompe disease is an inherited neuromuscular disorder caused by a genetic deficiency of acid-glucosidase-alpha (GAA). The clinical symptoms of Pompe disease include progressive weakness, respiratory failure, and ventricular hypertrophy. Enzyme replacement therapy has been shown to ameliorate these symptoms. Cardiomyocytes derived from patient/disease-specific iPS cells (iPS-CMs) have been used for pathophysiological analyses, drug screening, and cell therapy. Our research goal was to generate cardiomyocytes that can be differentiated from gene-corrected Pompe disease-specific iPS cells.

375. Direct Reprogramming of Fibroblast Allows Live-Cell Imaging of Autophagic Buildup in Pompe Disease Skeletal Myoblast

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid alpha glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart and liver. Autophagic buildup is associated to skeletal muscle pathology and known to inhibit the efficacy of enzyme replacement therapy. Myogenic conversion by MyoD forced expression is one of the feasible approaches towards disease modeling of neuromascular disorders. We have generated skeletal myocyte from Pompe disease fibroblast by direct reprogramming by lentiviral MyoD transfer.We have cloned human MYOD1 by RT-PCR from normal control fibroblast (AG08498). EcoR1 site was added and then cloned into 3rd generation lentiviral vector (CSII-EF1α-MCS). High titer lentiviral vector was produced by ultracentrifuge. Next we have infected CS-EF1α-MYOD1 at MOI50 to healthy control fibroblast. 48 hours after transfection, we have confirmed MYOD1 expression in cytoplasm and nucleus of control fibroblast by immunofluorescence. Other than MyoD, other myogenic factors are expressed and confirmed by immunofluorescence (MyoD, MyoG, Myf5, Pax7 and MHC). We have also infected CS-EF1α-MYOD1 to patient derived fibroblast (GM20124) and confirmed the expressions of myogenic factors, MyoD, MyoG, Myf5, Pax7 and MHC by immunofluorescence. Moreover, we have checked gene expression by RT-PCR and confirmed myogenic markers (Pax7, Pax3, Myf5, MyoD, AchR and MHC) are positive both in healthy control and patient derived fibroblasts.To visualize autophagic accumulation, we have infected RFP-GFP-LC3B vector (Promo™ Autophagy Sensor Kit) to induced myoblasts. It allows us to detect LC-3 accumulation which is suggesting autophagic buildup in pompe disease patient compared to healthy control. We have investigated autophagic buildup seen in skeletal myoblasts of Pompe disease at the live-cell imaging. Moreover, we have cloned lentiviral vector which expresses TFEB, a master gene for lysosomal biogenesis, to treat autophagic buildup and infected to MyoD induced myoblast. Massive accumulation of LC-3 is decreased after CSII-EF1α-TFEB transfection.High titer lentiviral MyoD transfer is useful in terms of direct reprogramming of fibroblast into skeletal myoblast. In addition, disease modeling of Pompe disease allows live-cell imaging of autophagic buildup of skeletal myocyote.