Ex Vivo Gene Therapy Treats Bone Complications of Mucopolysaccharidosis Type II Mouse Models through Bone Remodeling Reactivation

Mucopolysaccharidosis type II is a disease caused by organ accumulation of glycosaminoglycans due to iduronate 2-sulfatase deficiency. This study investigated the pathophysiology of the bone complications associated with mucopolysaccharidosis II and the effect of lentivirus-mediated gene therapy of hematopoietic stem cells on bone lesions of mucopolysaccharidosis type II mouse models in comparison with enzyme replacement therapy. Bone volume, density, strength, and trabecular number were significantly higher in the untreated mucopolysaccharidosis type II mice than in wild-type mice. Accumulation of glycosaminoglycans caused reduced bone metabolism. Specifically, persistent high serum iduronate 2-sulfatase levels and release of glycosaminoglycans from osteoblasts and osteoclasts in mucopolysaccharidosis type II mice that had undergone gene therapy reactivated bone lineage remodeling, subsequently reducing bone mineral density, strength, and trabecular number to a similar degree as that observed in wild-type mice. Bone formation, resorption parameters, and mineral density in the diaphysis edge did not appear to have been affected by the irradiation administered as a pre-treatment for gene therapy. Hence, the therapeutic effect of gene therapy on the bone complications of mucopolysaccharidosis type II mice possibly outweighed that of enzyme replacement therapy in many aspects.Wada M., Shimada Y., Iizuka S., et al. Ex Vivo Gene Therapy Treats Bone Complications of Mucopolysaccharidosis Type II Mouse Models through Bone Remodeling Reactivation. Molecular Therapy - Methods and Clinical Development, 19, 261. https://doi.org/10.1016/j.omtm.2020.09.012

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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

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

Nafamostat Mesilate Enhances the Radiosensitivity and Reduces the Radiation-Induced Invasive Ability of Colorectal Cancer Cells

Neoadjuvant chemoradiotherapy followed by radical surgery is the standard treatment for patients with locally advanced low rectal cancer. However, several studies have reported that ionizing radiation (IR) activates nuclear factor kappa B (NF-κB) that causes radioresistance and induces matrix metalloproteinase (MMP)-2/-9, which promote tumor migration and invasion. Nafamostat mesilate (FUT175), a synthetic serine protease inhibitor, enhances the chemosensitivity to cytotoxic agents in digestive system cancer cells by inhibiting NF-κB activation. Therefore, we evaluated the combined effect of IR and FUT175 on cell proliferation, migration and invasion of colorectal cancer (CRC) cells. IR-induced upregulation of intranuclear NF-κB, FUT175 counteracted this effect. Moreover, the combination treatment suppressed cell viability and induced apoptosis. Similar effects were also observed in xenograft tumors. In addition, FUT175 prevented the migration and invasion of cancer cells caused by IR by downregulating the enzymatic activity of MMP-2/-9. In conclusion, FUT175 enhances the anti-tumor effect of radiotherapy through downregulation of NF-κB and reduces IR-induced tumor invasiveness by directly inhibiting MMP-2/-9 in CRC cells. Therefore, the use of FUT175 during radiotherapy might improve the efficacy of radiotherapy in patients with CRC

Correction: Hiroshi Sugano et al. Nafamostat Mesilate Enhances the Radiosensitivity and Reduces the Radiation-Induced Invasive Ability of Colorectal Cancer Cells. Cancers 2018, 10, 386

The authors would like to make a correction to their published paper [...

Correction: Adipose Tissue-Derived Mesenchymal Stem Cells in Long-Term Dialysis Patients Display Downregulation of PCAF Expression and Poor Angiogenesis Activation.

[This corrects the article DOI: 10.1371/journal.pone.0102311.]

Adipose tissue-derived mesenchymal stem cells in long-term dialysis patients display downregulation of PCAF expression and poor angiogenesis activation.

We previously demonstrated that mesenchymal stem cells (MSCs) differentiate into functional kidney cells capable of urine and erythropoietin production, indicating that they may be used for kidney regeneration. However, the viability of MSCs from dialysis patients may be affected under uremic conditions. In this study, we isolated MSCs from the adipose tissues of end-stage kidney disease (ESKD) patients undergoing long-term dialysis (KD-MSCs; mean: 72.3 months) and from healthy controls (HC-MSCs) to compare their viability. KD-MSCs and HC-MSCs were assessed for their proliferation potential, senescence, and differentiation capacities into adipocytes, osteoblasts, and chondrocytes. Gene expression of stem cell-specific transcription factors was analyzed by PCR array and confirmed by western blot analysis at the protein level. No significant differences of proliferation potential, senescence, or differentiation capacity were observed between KD-MSCs and HC-MSCs. However, gene and protein expression of p300/CBP-associated factor (PCAF) was significantly suppressed in KD-MSCs. Because PCAF is a histone acetyltransferase that mediates regulation of hypoxia-inducible factor-1α (HIF-1α), we examined the hypoxic response in MSCs. HC-MSCs but not KD-MSCs showed upregulation of PCAF protein expression under hypoxia. Similarly, HIF-1α and vascular endothelial growth factor (VEGF) expression did not increase under hypoxia in KD-MSCs but did so in HC-MSCs. Additionally, a directed in vivo angiogenesis assay revealed a decrease in angiogenesis activation of KD-MSCs. In conclusion, long-term uremia leads to persistent and systematic downregulation of PCAF gene and protein expression and poor angiogenesis activation of MSCs from patients with ESKD. Furthermore, PCAF, HIF-1α, and VEGF expression were not upregulated by hypoxic stimulation of KD-MSCs. These results suggest that the hypoxic response may be blunted in MSCs from ESKD patients