Disease progression in Sanfilippo type B: Case series of Brazilian patients.

Mucopolysaccharidosis type IIIB (MPS IIIB) is caused by deficiency of alpha-N-acetylglucosaminidase, leading to storage of heparan sulphate. The disease is characterized by intellectual disability and hyperactivity, among other neurological and somatic features. Here we studied retrospective data from a total of 19 MPS IIIB patients from Brazil, aiming to evaluate disease progression. Mean age at diagnosis was 7.2 years. Speech delay was one of the first symptoms to be identified, around 2-3 years of age. Behavioral alterations include hyperactivity and aggressiveness, starting around age four. By the end of the first decade, patients lost acquired abilities such as speech and ability to walk. Furthermore, as disease progresses, respiratory, cardiovascular and joint abnormalities were found in more than 50% of the patients, along with organomegaly. Most common cause of death was respiratory problems. The disease progression was characterized in multiple systems, and hopefully these data will help the design of appropriate clinical trials and clinical management guidelines.

Disturbances in mitochondrial bioenergetics and control quality and unbalanced redox homeostasis in the liver of a mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a lysosomal storage disease caused by mutations in the gene encoding the enzyme iduronate 2-sulfatase (IDS) and biochemically characterized by the accumulation of glycosaminoglycans (GAGs) in different tissues. It is a multisystemic disorder that presents liver abnormalities, the pathophysiology of which is not yet established. In the present study, we evaluated bioenergetics, redox homeostasis, and mitochondrial dynamics in the liver of 6-month-old MPS II mice (IDS-). Our findings show a decrease in the activity of α-ketoglutarate dehydrogenase and an increase in the activities of succinate dehydrogenase and malate dehydrogenase. The activity of mitochondrial complex I was also increased whereas the other complex activities were not affected. In contrast, mitochondrial respiration, membrane potential, ATP production, and calcium retention capacity were not altered. Furthermore, malondialdehyde levels and 2',7'-dichlorofluorescein oxidation were increased in the liver of MPS II mice, indicating lipid peroxidation and increased ROS levels, respectively. Sulfhydryl and reduced glutathione levels, as well as glutathione S-transferase, glutathione peroxidase (GPx), superoxide dismutase, and catalase activities were also increased. Finally, the levels of proteins involved in mitochondrial mass and dynamics were decreased in knockout mice liver. Taken together, these data suggest that alterations in energy metabolism, redox homeostasis, and mitochondrial dynamics can be involved in the pathophysiology of liver abnormalities observed in MPS II.

Effect of genistein and coenzyme Q10 in oxidative damage and mitochondrial membrane potential in an attenuated type II mucopolysaccharidosis cellular model.

Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared with WT vehicle group) and genistein at concentrations of 25 and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.

Regulatory Framework, Challenges, and Initial Strategic Planning for Advanced Therapy Products (PTAs) Development in Brazil.

Advanced Therapies are a class of innovative complex biological products used for therapeutic purposes, encompassing cell therapy, tissue engineering, and gene therapy products. These are promising therapeutic strategies for several complex diseases with low or non-existent therapeutic alternatives. The proper transposition of basic research in this area into medicinal products must comply with regulatory requirements. Here we review the main regulatory recommendations, emphasizing on the Brazilian regulation. The critical points are the manufacturing process, challenges in characterizing the product, development of non-clinical trials, lack of adequate animal models representative of the clinical situation, and absence of valid and measurable therapeutic endpoints. Based on that, we propose a framework for strategic planning of pre-clinical studies in this field. The detailed example involves producing a nonviral vector-based gene editing product, but the regulations and methods may be extrapolated for developing different types of advanced therapies.

Characterization of heart disease in mucopolysaccharidosis type II mice.

Mucopolysaccharidosis type II (MPSII) is a progressive lysosomal storage disease caused by mutations in the IDS gene, that leads to iduronate 2-sulfatase (IDS) enzyme deficiency. The enzyme catalyzes the first step of degradation of two glycosaminoglycans (GAGs), heparan sulfate (HS) and dermatan sulfate (DS). The consequences of MPSII are progressively harmful and can lead to death by cardiac failure. The aim of this study was to characterize the cardiovascular disease in MPSII mice. Thus, we evaluated the cardiovascular function of MPSII male mice at 6, 8, and 10 months of age, through functional, histological, and biochemical analyzes. Echocardiographic analyses showed a progressive loss in cardiac function, observed through parameters such as reduction in ejection fraction (46% in control versus 28% in MPS II at 10 months, P < .01) and fractional area change (31% versus 23%, P < .05). Similar results were found in parameters of vascular competence, obtained by echo Doppler. Both aortic dilatation and an increase in pulmonary resistance were observed at all time points in MPSII mice. The histological analyses showed an increase in the thickness of the heart valves (2-fold thicker than control values at 10 months). Biochemical analyzes confirmed GAG storage in these tissues, with a massive elevation of DS in the myocardium. Furthermore, an important increase in the activity of proteases such as cathepsin S and B (up to 5-fold control values) was found and could be related to the progressive loss of cardiac function observed in MPSII mice. In this work, we demonstrated that loss of cardiac function in MPSII mice started at 6 months of age, although its global cardiac capacity was still preserved at this time. Disease progressed at later time points leading to heart failure. The MPSII mice at later times reproduce many of the cardiovascular events found in patients with Hunter's disease.

Genome Editing Tools for Lysosomal Storage Disorders.

Genome editing has multiple applications in the biomedical field. They can be used to modify genomes at specific locations, being able to either delete, reduce, or even enhance gene transcription and protein expression. Here, we summarize applications of genome editing used in the field of lysosomal disorders. We focus on the development of cell lines for study of disease pathogenesis, drug discovery, and pathogenicity of specific variants. Furthermore, we highlight the main studies that use gene editing as a gene therapy platform for these disorders, both in preclinical and clinical studies. We conclude that gene editing has been able to change quickly the scenario of these disorders, allowing the development of new therapies and improving the knowledge on disease pathogenesis. Should they confirm their hype, the first gene editing-based products for lysosomal disorders could be available in the next years.

Ex vivo gene therapy for lysosomal storage disorders: future perspectives.

INTRODUCTION: Lysosomal storage disorders (LSD) are a group of monogenic rare diseases caused by pathogenic variants in genes that encode proteins related to lysosomal function. These disorders are good candidates for gene therapy for different reasons: they are monogenic, most of lysosomal proteins are enzymes that can be secreted and cross-correct neighboring cells, and small quantities of these proteins are able to produce clinical benefits in many cases. Ex vivo gene therapy allows for autologous transplant of modified cells from different sources, including stem cells and hematopoietic precursors. AREAS COVERED: Here, we summarize the main gene therapy and genome editing strategies that are currently being used as ex vivo gene therapy approaches for lysosomal disorders, highlighting important characteristics, such as vectors used, strategies, types of cells that are modified and main results in different disorders. EXPERT OPINION: Clinical trials are already ongoing, and soon approved therapies for LSD based on ex vivo gene therapy approaches should reach the market.

Inflammatory process and oxidative/nitrative stress: in vivo study in mucopolysaccharidosis type IV A patients under long-term enzyme replacement therapy.

Mucopolysaccharidosis type IV A (MPS IVA) is an inborn error of the metabolism (IEM) caused by a deficiency of the enzyme N-acetylgalactosamine 6-sulfate sulfatase (GALNS). Since 2014, enzyme replacement therapy (ERT) is the recommended treatment for these patients. It is known that the inflammatory response is closely related to antioxidant defenses and oxidative stress, and literature shows involvement of oxidative stress in the pathogenesis of IEM. The aim of this study is to investigate the mechanisms of oxidative/nitrative stress and inflammation in patients with MPS IVA under long-term ERT. In the present work we investigate parameters of oxidative/nitrative stress in plasma and urine of MPS IVA patients under long-term ERT and controls, such as plasmatic nitrate/nitrite levels using the LDH Method, urinary di-tyrosine levels by fluorometric method, plasmatic content of sulfhydryl groups, urinary oxidized guanine species by ELISA kit and the plasmatic total antioxidant status. We next evaluated the plasmatic pro and anti-inflammatory cytokines concentration (IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α) and the expression of factors and enzymes Nrf-2, NF-κß and HO-1, main mediators between inflammation and oxidative stress. In concern to the oxidative/nitrative stress parameters, there was no significant difference between the groups MPS IVA patients under long-term ERT and controls, showing that there is no overproducing of RNS, no protein damage, no DNA/RNA oxidative damage and no modification in the non-enzymatic antioxidant capacity of a tissue to prevent the damage associated to free radical processes in these patients. It was also verified no significant difference between the MPS IVA patients under long-term ERT and controls groups regarding the production of proinflammatory cytokines. About anti-inflammatory cytokines, IL 10 was shown to be elevated in MPS IVA patients under long-term ERT in comparison to the control group. We next evaluated the genic expression of Nrf-2, NF-κß and HO-1and there was no significant difference between the MPS IVA patients under long-term ERT and control groups. In conclusion, MPS IVA patients under long term ERT are not in an inflammatory state and there is no alteration in genic expression in the genes analyzed which are involved in oxidative stress and inflammatory pathways. It is,however, important to consider that absence of imbalance of antioxidant defenses in MPS IVA patients under long term ERT is so far preliminary it is supported by methodologies that are not highly sensitive nor very accurate. Further experiments in future using state-of-the-art methodologies will corroborate these findings. Nevertheless, our results demonstrated the protective effect of the treatment in relation to the parameters studied and the importance of starting treatment in the early stages of the disease.

Copy number variations in SPAST and ATL1 are rare among Brazilians.

Copy number variations (CNV) may represent a significant proportion of SPG4 and SPG3A diagnosis, the most frequent autosomal dominant subtypes of hereditary spastic paraplegias (HSP). We aimed to assess the frequency of CNVs in SPAST and ATL1 and to update the molecular epidemiology of HSP families in southern Brazil. A cohort study that included 95 Brazilian index cases with clinical suspicion of HSP was conducted between April 2011 and September 2022. Multiplex Ligation Dependent Probe Amplification (MLPA) was performed in 41 cases without defined diagnosis by different massive parallel sequencing techniques (MPS). Diagnosis was obtained in 57/95 (60%) index cases, 15/57 (26.3%) being SPG4. Most frequent autosomal recessive HSP subtypes were SPG7 followed by SPG11, SPG76 and cerebrotendinous xanthomatosis. No CNVs in SPAST and ATL1 were found. Copy number variations are rare among SPG4 and SPG3A families in Brazil. Considering the possibility of CNVs detection by specific algorithms with MPS data, we consider that this is likely the most cost-effective approach to investigate CNVs in these genes in low-risk populations, with MLPA being reserved as an orthogonal confirmatory test.

Evaluation of oxidative stress and mitochondrial function in a type II mucopolysaccharidosis cellular model: in vitro effects of genistein and coenzyme Q10.

Mucopolysaccharidosis type II (MPS II or Hunter Syndrome) is a lysosomal disease caused by deficient degradation of glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate due to the deficiency of the enzyme iduronate-2-sulfatase. The main treatment for MPS II is the administration of the recombinant form of the enzyme, in a process known as enzyme replacement therapy (ERT). Oxidative damage can contribute to the pathophysiology of MPS II and treatment with ERT can reduce the effects of oxidative stress. For a better understanding of pathophysiology of MPS II, we evaluated biomarkers of mitochondrial dysfunction, DNA (Deoxyribonucleic acid) damage, antioxidant defenses, reactive species production and lysosomal size in IDS-deficient HEK 293 cells and investigate the in vitro effect of genistein and coenzyme Q10 (CoQ) on these biomarkers. An increase in the production of reactive species was demonstrated, as well as an increase in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Also, an increase in lysosomal volume and oxidative damage to DNA were verified. There was no evidence of a change in mitochondrial function in this cell model. In the HEK 293 (human embryonic kidney 293) knockout (KO) HP10 cell model we found that genistein at concentrations of 25 and 50 µm decreased in vitro the production of reactive species and the activity of the SOD enzyme, showing an antioxidant protective effect. Still, in these cells we verified that the coenzyme Q10 in the concentrations of 5 and 10 µm decreased in vitro the activity of the SOD enzyme and in the concentration of 10 µm decreased in vitro the DNA damage, also demonstrating antioxidant protection. In conclusion, MPS II knockout cells demonstrated oxidative stress and DNA damage and genistein, as well as coenzyme Q10, have been shown to have an important protective effect in vitro against these oxidative damages.

Gene editing strategies to treat lysosomal disorders: The example of mucopolysaccharidoses.

Lysosomal storage disorders are a group of progressive multisystemic hereditary diseases with a combined incidence of 1:4,800. Here we review the clinical and molecular characteristics of these diseases, with a special focus on Mucopolysaccharidoses, caused primarily by the lysosomal storage of glycosaminoglycans. Different gene editing techniques can be used to ameliorate their symptoms, using both viral and nonviral delivery methods. Whereas these are still being tested in animal models, early results of phase I/II clinical trials of gene therapy show how this technology may impact the future treatment of these diseases. Hurdles related to specific hard-to-reach organs, such as the central nervous system, heart, joints, and the eye must be tackled. Finally, the regulatory framework necessary to advance into clinical practice is also discussed.

Improved engraftment and therapeutic efficacy by human genome-edited hematopoietic stem cells with Busulfan-based myeloablation.

Autologous hematopoietic stem cell transplantation using genome-edited cells can become a definitive therapy for hematological and non-hematological disorders with neurological involvement. Proof-of-concept studies using human genome-edited hematopoietic stem cells have been hindered by the low efficiency of engraftment of the edited cells in the bone marrow and their modest efficacy in the CNS. To address these challenges, we tested a myeloablative conditioning regimen based on Busulfan in an immunocompromised model of mucopolysaccharidosis type 1. Compared with sub-lethal irradiation, Busulfan conditioning enhanced the engraftment of edited CD34+ cells in the bone marrow, as well the long-term homing and survival of bone-marrow-derived cells in viscera, and in the CNS, resulting in higher transgene expression and biochemical correction in these organs. Edited cell selection using a clinically compatible marker resulted in a population with low engraftment potential. We conclude that conditioning can impact the engraftment of edited hematopoietic stem cells. Furthermore, Busulfan-conditioned recipients have a higher expression of therapeutic proteins in target organs, particularly in the CNS, constituting a better conditioning approach for non-hematological diseases with neurological involvement.

Brain and visceral gene editing of mucopolysaccharidosis I mice by nasal delivery of the CRISPR/Cas9 system.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha-l-iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice. METHODS: Liposomes were prepared by microfluidization, and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30 days in young MPS I mice, which were euthanized at 6 months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated. RESULTS: Monodisperse mucoadhesive complexes around 110 nm, which are able to efficiently permeate the PNM. In animals, the treatment led to a modest increase in IDUA activity in the lung, heart, and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues, and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage. CONCLUSION: Nonviral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly for other neurological disorders.

Sanfilippo syndrome type B: Analysis of patients diagnosed by the MPS Brazil Network.

Mucopolysaccharidosis type IIIB is a rare autosomal recessive disorder characterized by deficiency of the enzyme N-acetyl-alpha-d-glucosaminidase (NAGLU), caused by biallelic pathogenic variants in the NAGLU gene, which leads to storage of heparan sulfate and a series of clinical consequences which hallmark is neurodegeneration. In this study clinical, epidemiological, and biochemical data were obtained from MPS IIIB patients diagnosed from 2004-2019 by the MPS Brazil Network ("Rede MPS Brasil"), which was created with the goal to provide an easily accessible and comprehensive investigation of all MPS types. One hundred and ten MPS IIIB patients were diagnosed during this period. Mean age at diagnosis was 10.9 years. Patients were from all over Brazil, with a few from abroad, with a possible cluster of MPS IIIB identified in Ecuador. All patients had increased urinary levels of glycosaminoglycans and low NAGLU activity in blood. Main clinical symptoms reported at diagnosis were coarse facies and neurocognitive regression. The most common variant was p.Leu496Pro (30% of alleles). MPS IIIB seems to be relatively frequent in Brazil, but patients are diagnosed later than in other countries, and reasons for that probably include the limited awareness about the disease by health professionals and the difficulties to access diagnostic tests, factors that the MPS Brazil Network is trying to mitigate.

Thioredoxin system activation is associated with the progression of experimental pulmonary arterial hypertension.

In addition to being an antioxidant, thioredoxin (Trx) is known to stimulate signaling pathways involved in cell proliferation and to inhibit apoptosis. The aim of this study was to explore the role of Trx in some of these pathways along the progression of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Male rats were first divided into two groups: monocrotaline (MCT - 60 mg/kg i.p.) and control (received saline), that were further divided into three groups: 1, 2, and 3 weeks. Animals were submitted to echocardiographic analysis. Right and left ventricles were used for the measurement of hypertrophy, through morphometric and histological analysis. The lung was prepared for biochemical and molecular analysis. One week after MCT injection, there was an increase in thioredoxin reductase (TrxR) activity, a reduction in glutathione reductase (GR) activity, and an increase in Trx-1 and vitamin D3 up-regulated protein-1 (VDUP-1) expression. Two weeks after MCT injection, there was an increase in VDUP-1, Akt and cleaved caspase-3 activation, and a decrease in Trx-1 and Nrf2 expression. PAH-induced by MCT promoted a reduction in Nrf2 and Trx-1 expression as well as an increase in Akt and VDUP-1 expression after three weeks. The increase in pulmonary vascular resistance was accompanied by increased TrxR activity, suggesting an association between the Trx system and functional changes in the progression of PAH. It seems that Trx-1 activation was an adaptive response to MCT administration to cope with pulmonary remodeling and disease progression, suggesting a potential new target for PAH therapeutics.

Schizophreniform presentation and abrupt neurologic decline in a patient with late-onset mucopolysaccharidosis type IIIB.

Due to their low frequency and some atypical presentations, inborn errors of metabolism are frequently misdiagnosed or underdiagnosed, which hinders the correct management of these patients. To illustrate that, here we present a patient that, at early school age, had learning disabilities compared to her classmates, especially for writing. She completed basic education in a regular school and was transferred to a secondary school for students with special needs. At 18 years of age, she presented a first psychiatric abrupt outbreak: she spent a month screaming and without sleeping. Behavioral problems then became apparent, especially hyperactivity, destructive and chaotic behavior, anxiety, and auto-aggressivity and hetero-aggressivity. A diagnosis of schizophreniform disorder was established. Clinical genetic evaluation revealed coarse face, macroglossia, coarse thick hair, and mild hepatomegaly, and the hypothesis of mucopolysaccharidosis-III was raised. Laboratory tests indicated high levels of urinary glycosaminoglycans and almost undetectable NAGLU activity, confirming the diagnosis. Sequencing of the NAGLU gene revealed the c.1318G>C (p.Gly440Arg) and c.1834A>G (p.Ser612Gly) mutations.

Creating cell lines for mimicking diseases.

Cell lines can be good models for the disease they are derived from but can also be used to study general physiological and pathological processes. They can also be used to generate cell models of diseases when primary cultures are not available. Recent genome editing tools have been very promising tools toward creating cell models to mimic diseases in vitro. In this chapter, we highlight techniques used to obtain genome-edited cell lines, including cell line selection, transfection and gene editing tools available, together with methods of phenotype characterization and, lastly, a few examples of how in vitro disease models were created using CRISPR-Cas9.

Genome editing in lysosomal disorders.

Lysosomal disorders are a group of heterogenous diseases caused by mutations in genes that encode for lysosomal proteins. With exception of some cases, these disorders still lack both knowledge of disease pathogenesis and specific therapies. In this sense, genome editing arises as a technique that allows both the creation of specific cell lines, animal models and gene therapy protocols for these disorders. Here we explain the main applications of genome editing for lysosomal diseases, with examples based on the literature. The ability to rewrite the genome will be of extreme importance to study and potentially treat these rare disorders.