Sex Difference Leads to Differential Gene Expression Patterns and Therapeutic Efficacy in Mucopolysaccharidosis IVA Murine Model Receiving AAV8 Gene Therapy.

Adeno-associated virus (AAV) vector-based therapies can effectively correct some disease pathology in murine models with mucopolysaccharidoses. However, immunogenicity can limit therapeutic effect as immune responses target capsid proteins, transduced cells, and gene therapy products, ultimately resulting in loss of enzyme activity. Inherent differences in male versus female immune response can significantly impact AAV gene transfer. We aim to investigate sex differences in the immune response to AAV gene therapies in mice with mucopolysaccharidosis IVA (MPS IVA). MPS IVA mice, treated with different AAV vectors expressing human N-acetylgalactosamine 6-sulfate sulfatase (GALNS), demonstrated a more robust antibody response in female mice resulting in subsequent decreased GALNS enzyme activity and less therapeutic efficacy in tissue pathology relative to male mice. Under thyroxine-binding globulin promoter, neutralizing antibody titers in female mice were approximately 4.6-fold higher than in male mice, with GALNS enzyme activity levels approximately 6.8-fold lower. Overall, male mice treated with AAV-based gene therapy showed pathological improvement in the femur and tibial growth plates, ligaments, and articular cartilage as determined by contrasting differences in pathology scores compared to females. Cardiac histology revealed a failure to normalize vacuolation in females, in contrast, to complete correction in male mice. These findings promote the need for further determination of sex-based differences in response to AAV-mediated gene therapy related to developing treatments for MPS IVA.

Neonatal combination therapy improves some of the clinical manifestations in the Mucopolysaccharidosis type I murine model.

Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase enzyme, results in the progressive accumulation of glycosaminoglycans and consequent multiorgan dysfunction. Despite the effectiveness of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) in correcting clinical manifestations related to visceral organs, complete improvement of musculoskeletal and neurocognitive defects remains an unmet challenge and provides an impact on patients' quality of life. We tested the therapeutic efficacy of combining HSCT and ERT in the neonatal period. Using a mouse model of MPS-I, we demonstrated that the combination therapy improved clinical manifestations in organs usually refractory to current treatment. Moreover, combination with HSCT prevented the production of anti-IDUA antibodies that negatively impact ERT efficacy. The added benefits of combining both treatments also resulted in a reduction of skeletal anomalies and a trend towards decreased neuroinflammation and metabolic abnormalities. As currently there are limited therapeutic options for MPS-I patients, our findings suggest that the combination of HSCT and ERT during the neonatal period may provide a further step forward in the treatment of this rare disease.

Bone-Specific Drug Delivery for Osteoporosis and Rare Skeletal Disorders.

PURPOSE OF REVIEW: The skeletal system provides an important role to support body structure and protect organs. The complexity of its architecture and components makes it challenging to deliver the right amount of the drug into bone regions, particularly avascular cartilage lesions. In this review, we describe the recent advance of bone-targeting methods using bisphosphonates, polymeric oligopeptides, and nanoparticles on osteoporosis and rare skeletal diseases. RECENT FINDINGS: Hydroxyapatite (HA), a calcium phosphate with the formula Ca10(PO4)6(OH)2, is a primary matrix of bone mineral that includes a high concentration of positively charged calcium ion and is found only in the bone. This unique feature makes HA a general targeting moiety to the entire skeletal system. We have applied bone-targeting strategy using acidic amino acid oligopeptides into lysosomal enzymes, demonstrating the effects of bone-targeting enzyme replacement therapy and gene therapy on bone and cartilage lesions in inherited skeletal disorders. Virus or no-virus gene therapy using techniques of engineered capsid or nanomedicine has been studied preclinically for skeletal diseases. Efficient drug delivery into bone lesions remains an unmet challenge in clinical practice. Bone-targeting therapies based on gene transfer can be potential as new candidates for skeletal diseases.

Evading the AAV Immune Response in Mucopolysaccharidoses.

The humoral immune response elicited by adeno-associated virus (AAV)-mediated gene therapy for the treatment of mucopolysaccharidoses (MPS) poses a significant challenge to achieving therapeutic levels of transgene expression. Antibodies targeting the AAV capsid as well as the transgene product diminish the production of glycosaminoglycan (GAG)-degrading enzymes essential for the treatment of MPS. Patients who have antibodies against AAV capsid increase in number with age, serotype, and racial background and are excluded from the clinical trials at present. In addition, patients who have undergone AAV gene therapy are often excluded from the additional AAV gene therapy with the same serotype, since their acquired immune response (antibody) against AAV will limit further efficacy of treatment. Several methods are being developed to overcome this immune response, such as novel serotype design, antibody reduction by plasmapheresis and immunosuppression, and antibody evasion using empty capsids and enveloped AAV vectors. In this review, we examine the mechanisms of the anti-AAV humoral immune response and evaluate the strengths and weaknesses of current evasion strategies in order to provide an evidence-based recommendation on evading the immune response for future AAV-mediated gene therapies for MPS.

Mucopolysaccharidosis IVA: Diagnosis, Treatment, and Management.

Mucopolysaccharidosis type IVA (MPS IVA, or Morquio syndrome type A) is an inherited metabolic lysosomal disease caused by the deficiency of the N-acetylglucosamine-6-sulfate sulfatase enzyme. The deficiency of this enzyme accumulates the specific glycosaminoglycans (GAG), keratan sulfate, and chondroitin-6-sulfate mainly in bone, cartilage, and its extracellular matrix. GAG accumulation in these lesions leads to unique skeletal dysplasia in MPS IVA patients. Clinical, radiographic, and biochemical tests are needed to complete the diagnosis of MPS IVA since some clinical characteristics in MPS IVA are overlapped with other disorders. Early and accurate diagnosis is vital to optimizing patient management, which provides a better quality of life and prolonged life-time in MPS IVA patients. Currently, enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available for patients with MPS IVA. However, ERT and HSCT do not have enough impact on bone and cartilage lesions in patients with MPS IVA. Penetrating the deficient enzyme into an avascular lesion remains an unmet challenge, and several innovative therapies are under development in a preclinical study. In this review article, we comprehensively describe the current diagnosis, treatment, and management for MPS IVA. We also illustrate developing future therapies focused on the improvement of skeletal dysplasia in MPS IVA.

Critical review of current MPS guidelines and management.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders that impair degradation of glycosaminoglycans (GAG). The specific GAGs that accumulate depend on the type of MPS, leading to unique characteristic clinical features. Development of guidelines for treatment of MPS has traditionally been multifaceted and largely based on palliative care. In the last three decades, hematopoietic stem cell transplantation and enzyme replacement therapy have been developed based on experimental and clinical studies. Guidelines have been established with the accumulation of the clinical data from natural history of the disease and therapeutic consequences, mainly sponsored by pharmaceutical companies. In recent years, committees in three countries, Australia (2015), Japan (2017), and Brazil (2018) have adopted guidelines for the treatment of MPS II, sponsored and authorized by each government. As novel treatments for MPS including substrate reduction therapy, pharmacological chaperone therapy, and gene therapy become clinically available, it is increasingly necessary to establish the optimal guideline for each type of MPS, considering multiple factors including therapeutic efficacy, adverse effects, age, disease stage, prognosis, feasibility and availability of access to treatment, and cost- performance. In this article, we discuss the historical guidelines for specific MPS types and the most recently adopted guidelines for MPS II and propose the development of future guidelines without conflict of interest and bias leading to mutual benefits to all parties including patients and families, professionals, tax payers, and governments.

Enzyme replacement therapy for mucopolysaccharidoses; past, present, and future.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders, which lack an enzyme corresponding to the specific type of MPS. Enzyme replacement therapy (ERT) has been the standard therapeutic option for some types of MPS because of the ability to start immediate treatment with feasibility and safety and to improve prognosis. There are several disadvantages for current ERT, such as limited impact to the brain and avascular cartilage, weekly or biweekly infusions lasting 4-5 h, the immune response against the infused enzyme, a short half-life, and the high cost. Clinical studies of ERT have shown limited efficacy in preventing or resolving progression in neurological, cardiovascular, and skeletal diseases. One focus is to penetrate the avascular cartilage area to at least stabilize, if not reverse, musculoskeletal diseases. Although early intervention in some types of MPS has shown improvements in the severity of skeletal dysplasia and stunted growth, this limits the desired effect of ameliorating musculoskeletal disease progression to young MPS patients. Novel ERT strategies are under development to reach the brain: (1) utilizing a fusion protein with monoclonal antibody to target a receptor on the BBB, (2) using a protein complex from plant lectin, glycan, or insulin-like growth factor 2, and (3) direct infusion across the BBB. As for MPS IVA and VI, bone-targeting ERT will be an alternative to improve therapeutic efficacy in bone and cartilage. This review summarizes the effect and limitations on current ERT for MPS and describes the new technology to overcome the obstacles of conventional ERT.

Development of Substrate Degradation Enzyme Therapy for Mucopolysaccharidosis IVA Murine Model.

Mucopolysaccharidosis IVA (MPS IVA) is caused by a deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Conventional enzyme replacement therapy (ERT) is approved for MPS IVA. However, the fact that the infused enzyme cannot penetrate avascular lesions in cartilage leads to minimal impact on the bone lesion. Moreover, short half-life, high cost, instability, and narrow optimal pH range remain unmet challenges in ERT. Thermostable keratanase, endo-ß-N-acetylglucosaminidase, has a unique character of a wide optimal pH range of pH 5.0-7.0. We hypothesized that this endoglycosidase degrades keratan sulfate (KS) polymer in circulating blood and, therefore, ameliorates the accumulation of KS in multiple tissues. We propose a novel approach, Substrate Degradation Enzyme Therapy (SDET), to treat bone lesion of MPS IVA. We assessed the effect of thermostable keratanase on blood KS level and bone pathology using Galns knock-out MPS IVA mice. After a single administration of 2 U/kg (= 0.2 mg/kg) of the enzyme at 8 weeks of age via intravenous injection, the level of serum KS was significantly decreased to normal range level, and this suppression was maintained for at least 4 weeks. We administered 2 U/kg of the enzyme to MPS IVA mice every fourth week for 12 weeks (total of 3 times) at newborns or 8 weeks of age. After a third injection, serum mono-sulfated KS levels were kept low for 4 weeks, similar to that in control mice, and at 12 weeks, bone pathology was markedly improved when SDET started at newborns, compared with untreated MPS IVA mice. Overall, thermostable keratanase reduces the level of KS in blood and provides a positive impact on cartilage lesions, demonstrating that SDET is a novel therapeutic approach to MPS IVA.

Gene therapy for Mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders (LSDs) caused by a deficiency of lysosomal enzymes, leading to a wide range of various clinical symptoms depending upon the type of MPS or its severity. Enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), substrate reduction therapy (SRT), and various surgical procedures are currently available for patients with MPS. However, there is no curative treatment for this group of disorders. Gene therapy should be a one-time permanent therapy, repairing the cause of enzyme deficiency. Preclinical studies of gene therapy for MPS have been developed over the past three decades. Currently, clinical trials of gene therapy for some types of MPS are ongoing in the United States, some European countries, and Australia. Here, in this review, we summarize the development of gene therapy for MPS in preclinical and clinical trials.

Molecular genetics and metabolism, special edition: Diagnosis, diagnosis and prognosis of Mucopolysaccharidosis IVA.

Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is an autosomal recessive disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to the accumulation of specific glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), which are mainly synthesized in the cartilage. Therefore, the substrates are stored primarily in the cartilage and its extracellular matrix (ECM), leading to a direct impact on bone development and successive systemic skeletal spondylepiphyseal dysplasia. The skeletal-related symptoms for MPS IVA include short stature with short neck and trunk, odontoid hypoplasia, spinal cord compression, tracheal obstruction, obstructive airway, pectus carinatum, restrictive lung, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. The degree of imbalance of growth in bone and other organs and tissues largely contributes to unique skeletal dysplasia and clinical severity. Diagnosis of MPS IVA needs clinical, radiographic, and laboratory testing to make a complete conclusion. To diagnose MPS IVA, total urinary GAG analysis which has been used is problematic since the values overlap with those in age-matched controls. Currently, urinary and blood KS and C6S, the enzyme activity of GALNS, and GALNS molecular analysis are used for diagnosis and prognosis of clinical phenotype in MPS IVA. MPS IVA can be diagnosed with unique characters although this disorder relates closely to other disorders in some characteristics. In this review article, we comprehensively describe clinical, radiographic, biochemical, and molecular diagnosis and clinical assessment tests for MPS IVA. We also compare MPS IVA to other closely related disorders to differentiate MPS IVA. Overall, imbalance of growth in MPS IVA patients underlies unique skeletal manifestations leading to a critical indicator for diagnosis.

Contribution of equilibrative nucleoside transporters 1 and 2 to gemcitabine uptake in pancreatic cancer cells.

Hepatic arterial infusion (HAI) chemotherapy is expected to be a more effective and safer method to treat the hepatic metastasis of pancreatic cancer than intravenous (iv) administration because of higher tumor exposure and lower systemic exposure. To clarify the uptake mechanism of nucleoside anticancer drugs, including gemcitabine (GEM), in pancreatic cancer, we investigated the uptakes of radiolabeled uridine (a general substrate of nucleoside transporters) and GEM in pancreatic cancer cell lines MIA-PaCa2 and As-PC1. Uridine uptake was inhibited by non-labeled GEM and also by S-(4-nitrobenzyl)-6-thioinosine (NBMPR; an inhibitor of equilibrative nucleoside transporters, ENTs) in a concentration-dependent manner, suggesting that ENTs contribute to uridine uptake in pancreatic cancer cells. As for GEM, saturable uptake was mediated by high- and low-affinity components with Km values of micromolar and millimolar orders, respectively. Uptake was inhibited in a concentration-dependent manner by NBMPR and was sodium ion-independent. Moreover, the concentration dependence of uptake in the presence of 0.1 µM NBMPR showed a single low-affinity site. These results indicated that the high- and low-affinity sites correspond to hENT1 and hENT2, respectively. The results indicated that at clinically relevant hepatic concentrations of GEM in GEM-HAI therapy, the metastatic tumor exposure of GEM is predominantly determined by hENT2 under unsaturated conditions, suggesting that hENT2 expression in metastatic tumor would be a candidate biomarker for indicating anticancer therapy with GEM-HAI.

Mucopolysaccharidosis IVA and glycosaminoglycans.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A: OMIM 253000) is a lysosomal storage disease with an autosomal recessive trait caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to accumulation of specific glycosaminoglycans (GAGs): chondroitin-6-sulfate (C6S) and keratan sulfate (KS). C6S and KS are mainly produced in the cartilage. Therefore, the undegraded substrates are stored primarily in cartilage and in its extracellular matrix (ECM), leading to a direct impact on cartilage and bone development, and successive systemic skeletal dysplasia. Chondrogenesis, the earliest phase of skeletal formation, is maintained by cellular interactions with the ECM, growth and differentiation factors, signaling pathways, and transcription factors in a temporal-spatial manner. In patients with MPS IVA, the cartilage is disrupted at birth as a consequence of abnormal chondrogenesis and/or endochondral ossification. The unique skeletal features are distinguished by a disproportional short stature, odontoid hypoplasia, spinal cord compression, tracheal obstruction, pectus carinatum, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. In spite of many descriptions of these unique clinical features, delay of diagnosis still happens. The pathogenesis and treatment of systemic skeletal dysplasia in MPS IVA remains an unmet challenge. In this review article, we comprehensively describe historical aspect, property of GAGs, diagnosis, screening, pathogenesis, and current and future therapies of MPS IVA.

Development of Bone Targeting Drugs.

The skeletal system, comprising bones, ligaments, cartilage and their connective tissues, is critical for the structure and support of the body. Diseases that affect the skeletal system can be difficult to treat, mainly because of the avascular cartilage region. Targeting drugs to the site of action can not only increase efficacy but also reduce toxicity. Bone-targeting drugs are designed with either of two general targeting moieties, aimed at the entire skeletal system or a specific cell type. Most bone-targeting drugs utilize an affinity to hydroxyapatite, a major component of the bone matrix that includes a high concentration of positively-charged Ca2+. The strategies for designing such targeting moieties can involve synthetic and/or biological components including negatively-charged amino acid peptides or bisphosphonates. Efficient delivery of bone-specific drugs provides significant impact in the treatment of skeletal related disorders including infectious diseases (osteoarthritis, osteomyelitis, etc.), osteoporosis, and metabolic skeletal dysplasia. Despite recent advances, however, both delivering the drug to its target without losing activity and avoiding adverse local effects remain a challenge. In this review, we investigate the current development of bone-targeting moieties, their efficacy and limitations, and discuss future directions for the development of these specific targeted treatments.

Obstructive airway in Morquio A syndrome, the past, the present and the future.

Patients with severe tracheal obstruction in Morquio A syndrome are at risk of dying of sleep apnea and related complications. Tracheal obstruction also leads to life-threatening complications during anesthesia as a result of the difficulty in managing the upper airway due to factors inherent to the Morquio A syndrome, compounded by the difficulty in intubating the trachea. A detailed description of the obstructive pathology of the trachea is not available in the literature probably due to lack of a homogenous group of Morquio A patients to study at any one particular center. We present a series of cases with significant tracheal obstruction who were unrecognized due to the difficulty in interpreting tracheal narrowing airway symptoms. Our goal is to provide the guidelines in the management of these patients that allow earlier recognition and intervention of tracheal obstruction. Sagittal MRI images of the cervical spine of 28 Morquio A patients (12±8.14years) showed that19/28 (67.9%) patients had at least 25% tracheal narrowing and that narrowing worsened with age (all 8 patients over 15years had greater than 50% narrowing). Eight out of 28 patients were categorized as severe (>75%) tracheal narrowing when images were evaluated in neutral head and neck position. Of the 19 patients with tracheal narrowing, compression by the tortuous brachiocephalic artery was the most common cause (n=15). Evidence of such tracheal narrowing was evident as early as at 2years of age. The etiology of tracheal impingement by the brachiocephalic artery in Morquio A appears to be due to a combination of the narrow thoracic inlet crowding structures and the disproportionate growth of trachea and brachiocephalic artery in relationship to the chest cavity leading to tracheal tortuosity. In conclusion, tracheal narrowing, often due to impression from the crossing tortuous brachiocephalic artery, increases with age in Morquio A patients. Greater attention to the trachea is needed when evaluating cervical spine MRIs as well as other imaging and clinical investigations, with the goal of establishing a timely treatment protocol to reduce the mortality rate in this patient population.

Hematopoietic stem cell transplantation for Morquio A syndrome.

Morquio A syndrome features systemic skeletal dysplasia. To date, there has been no curative therapy for this skeletal dysplasia. No systemic report on a long-term effect of hematopoietic stem cell transplantation (HSCT) for Morquio A has been described. We conducted HSCT for 4 cases with Morquio A (age at HSCT: 4-15years, mean 10.5years) and followed them at least 10years (range 11-28years; mean 19years). Current age ranged between 25 and 36years of age (mean 29.5years). All cases had a successful full engraftment of allogeneic bone marrow transplantation without serious GVHD. Transplanted bone marrow derived from HLA-identical siblings (three cases) or HLA-identical unrelated donor. The levels of the enzyme activity in the recipient's lymphocytes reached the levels of donors' enzyme activities within two years after HSCT. For the successive over 10years post-BMT, GALNS activity in lymphocytes was maintained at the same level as the donors. Except one case who had osteotomy in both legs one year later post BMT, other three cases had no orthopedic surgical intervention. All cases remained ambulatory, and three of them could walk over 400m. Activity of daily living (ADL) in patients with HSCT was better than untreated patients. The patient who underwent HSCT at four years of age showed the best ADL score. In conclusion, the long-term study of HSCT has demonstrated therapeutic effect in amelioration of progression of the disease in respiratory function, ADL, and biochemical findings, suggesting that HSCT is a therapeutic option for patients with Morquio A.

Activity of daily living for Morquio A syndrome.

The aim of this study was to evaluate the activity of daily living (ADL) and surgical interventions in patients with mucopolysaccharidosis IVA (MPS IVA). The factor(s) that affect ADL are age, clinical phenotypes, surgical interventions, therapeutic effect, and body mass index. The ADL questionnaire comprises three domains: "Movement," "Movement with cognition," and "Cognition." Each domain has four subcategories rated on a 5-point scale based on the level of assistance. The questionnaire was collected from 145 healthy controls and 82 patients with MPS IVA. The patient cohort consisted of 63 severe and 17 attenuated phenotypes (2 were undefined); 4 patients treated with hematopoietic stem cell transplantation (HSCT), 33 patients treated with enzyme replacement therapy (ERT) for more than a year, and 45 untreated patients. MPS IVA patients show a decline in ADL scores after 10years of age. Patients with a severe phenotype have a lower ADL score than healthy control subjects, and lower scores than patients with an attenuated phenotype in domains of "Movement" and "Movement with cognition." Patients, who underwent HSCT and were followed up for over 10years, had higher ADL scores and fewer surgical interventions than untreated patients. ADL scores for ERT patients (2.5years follow-up on average) were similar with the-age-matched controls below 10years of age, but declined in older patients. Surgical frequency was higher for severe phenotypic patients than attenuated ones. Surgical frequency for patients treated with ERT was not decreased compared to untreated patients. In conclusion, we have shown the utility of the proposed ADL questionnaire and frequency of surgical interventions in patients with MPS IVA to evaluate the clinical severity and therapeutic efficacy compared with age-matched controls.

Neonatal cellular and gene therapies for mucopolysaccharidoses: the earlier the better?

Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs). The increasing interest in newborn screening procedures for LSDs underlines the need for alternative cellular and gene therapy approaches to be developed during the perinatal period, supporting the treatment of MPS patients before the onset of clinical signs and symptoms. The rationale for considering these early therapies results from the clinical experience in the treatment of MPSs and other genetic disorders. The normal or gene-corrected hematopoiesis transplanted in patients can produce the missing protein at levels sufficient to improve and/or halt the disease-related abnormalities. However, these current therapies are only partially successful, probably due to the limited efficacy of the protein provided through the hematopoiesis. An alternative explanation is that the time at which the cellular or gene therapy procedures are performed could be too late to prevent pre-existing or progressive organ damage. Considering these aspects, in the last several years, novel cellular and gene therapy approaches have been tested in different animal models at birth, a highly early stage, showing that precocious treatment is critical to prevent long-term pathological consequences. This review provides insights into the state-of-art accomplishments made with neonatal cellular and gene-based therapies and the major barriers that need to be overcome before they can be implemented in the medical community.

Therapies for the bone in mucopolysaccharidoses.

Patients with mucopolysaccharidoses (MPS) have accumulation of glycosaminoglycans in multiple tissues which may cause coarse facial features, mental retardation, recurrent ear and nose infections, inguinal and umbilical hernias, hepatosplenomegaly, and skeletal deformities. Clinical features related to bone lesions may include marked short stature, cervical stenosis, pectus carinatum, small lungs, joint rigidity (but laxity for MPS IV), kyphoscoliosis, lumbar gibbus, and genu valgum. Patients with MPS are often wheelchair-bound and physical handicaps increase with age as a result of progressive skeletal dysplasia, abnormal joint mobility, and osteoarthritis, leading to 1) stenosis of the upper cervical region, 2) restrictive small lung, 3) hip dysplasia, 4) restriction of joint movement, and 5) surgical complications. Patients often need multiple orthopedic procedures including cervical decompression and fusion, carpal tunnel release, hip reconstruction and replacement, and femoral or tibial osteotomy through their lifetime. Current measures to intervene in bone disease progression are not perfect and palliative, and improved therapies are urgently required. Enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), and gene therapy are available or in development for some types of MPS. Delivery of sufficient enzyme to bone, especially avascular cartilage, to prevent or ameliorate the devastating skeletal dysplasias remains an unmet challenge. The use of an anti-inflammatory drug is also under clinical study. Therapies should start at a very early stage prior to irreversible bone lesion, and damage since the severity of skeletal dysplasia is associated with level of activity during daily life. This review illustrates a current overview of therapies and their impact for bone lesions in MPS including ERT, HSCT, gene therapy, and anti-inflammatory drugs.

A natural Nrf2 activator glucoraphanin improves hepatic steatosis in high-fat diet-induced obese male mice associated with AMPK activation.

Genetic and pharmacological activation of the transcription factor nuclear factor, erythroid derived 2, like 2 (Nrf2) alleviates high-fat diet (HFD)-induced obesity in mice; however, synthetic Nrf2 activators are not clinically available due to safety concerns. Dietary glucoraphanin (GR), a naturally occurring compound found in cruciferous vegetables that activates Nrf2 and induces its target antioxidant genes. We previously demonstrated that GR increased thermogenesis and mitigated HFD-induced obesity in lean healthy mice. In this study, we investigated the therapeutic effects of GR on pre-existing obesity and associated metabolic disorders, such as hepatic steatosis, with or without low-fat dietary intervention. Eight-week-old male C57BL/6J mice were fed an HFD for 9 weeks to induce obesity. Subsequently, these obese mice were fed either the HFD or a normal chow diet, supplemented with or without GR, for an additional 11 weeks. GR supplementation did not decrease the body weight of HFD-fed mice; however, it significantly reduced plasma alanine aminotransferase and aspartate aminotransferase levels and hepatic triglyceride accumulation. These improvements in liver damage by GR were associated with decreased expression levels of fatty acid synthesis genes and proinflammatory chemokine genes, suppressed c-Jun N-terminal kinase activation, and reduced proinflammatory phenotypes of macrophages in the liver. Moreover, metabolome analysis identified increased hepatic levels of adenosine 5'-monophosphate (AMP) in HFD-GR mice compared with those in HFD mice, which agreed with increased phosphorylation levels of AMP-activated protein kinase. Our results show that GR may have a therapeutic potential for treating obesity-associated hepatic steatosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s13340-023-00658-6.

Liquid Biopsy for Pancreatic Cancer by Serum Extracellular Vesicle-Encapsulated Long Noncoding RNA HEVEPA.

OBJECTIVES: The role of long noncoding RNAs (lncRNAs) in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Extracellular vesicle (EV)-encapsulated RNAs could be effective targets for liquid biopsy. We aimed to identify previously unknown EV-encapsulated lncRNAs in PDAC and establish highly accurate methods for isolating EVs. MATERIALS AND METHODS: Extracellular vesicles were isolated using existing and newly developed methods, namely, PEViA-UC and PEViA-IP, from serum samples of 20 patients with PDAC, 22 patients with intraductal papillary mucinous neoplasms, and 21 healthy individuals. Extracellular vesicle lncRNA expression was analyzed using digital PCR. RESULTS: Gene expression analysis using cDNA microarray revealed a highly expressed lncRNA, HEVEPA , in serum EVs from patients with PDAC. We established PEViA-UC and PEViA-IP using PEViA reagent, ultracentrifugation, and immunoprecipitation. Although detection of EV-encapsulated HEVEPA using existing methods is challenging, PEViA-UC and PEViA-IP detected EV HEVEPA , which was highly expressed in patients with PDAC compared with non-PDAC patients. The detection sensitivity for discriminating PDAC from non-PDAC using the combination of HEVEPA and HULC , which are highly expressed lncRNAs in PDAC, and carbohydrate antigen 19-9 (CA19-9), was higher than that of HEVEPA , HULC , or CA19-9 alone. CONCLUSIONS: Extracellular vesicle lncRNAs isolated using PEViA-IP and CA19-9 together could be effective targets in liquid biopsy for PDAC diagnosis.