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BACKGROUND AND OBJECTIVE: Weight stigma has negative consequences for both physiological and psychological health. Studies on weight stigma in adolescence, particularly from general populations, are scarce in the Mediterranean area. The main aim of this study is to describe the prevalence of experienced and internalized weight stigma among a representative sample of adolescents from the Spanish city of Terrassa, and to determine its association with sociodemographic variables and weight status. METHODS: Drawing on data from the initial assessment of a longitudinally funded project on weight stigma in adolescents, a cross-sectional survey-based study was conducted using random multistage cluster sampling. Weight stigma experiences, their frequency and sources, and weight bias internalization with the Modified Weight Bias Internalization Scale (WBISM) were assessed in a sample of 1016 adolescents. Adjusted odds ratios (AOR) between sociodemographic variables, weight status and having experienced weight stigma, and having reported high scores of WBISM (WBISM ≥ 4) were estimated by multiple logistic regression models. RESULTS: The prevalence of weight-related stigma experiences was 43.2% in the sample (81.8 in adolescents with obesity) and the prevalence of high levels of weight bias internalization was 19.4% (50.7 in adolescents with obesity). Other kids and school were the most prevalent sources of weight stigma, with society and family being other significant sources of stigma reported by girls. A significantly higher risk of having experienced weight stigma was observed in girls (AOR = 2.6) and in older adolescents (AOR = 1.9). Compared to normal weight adolescents, all weight statuses showed higher risk, being 3.4 times higher in adolescents with underweight and reaching 11.4 times higher risk in those with obesity. Regarding high levels of weight bias internalization, girls had a risk 6.6 times higher than boys. Once again, a "J-shaped" pattern was observed, with a higher risk at the lowest and highest weight statuses. The risk was 6.3 times higher in adolescents with underweight, and 13.1 times higher in adolescents with obesity compared to those with normal weight. CONCLUSIONS: Considering the high prevalence of experienced and internalized weight stigma among adolescents in Spain, especially in adolescents with obesity and girls, it seems important to implement preventive strategies in different settings and address all sources of stigma.
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Estigma Social , Humanos , Adolescente , Femenino , Masculino , España/epidemiología , Estudios Transversales , Prevalencia , Peso Corporal , Obesidad/epidemiología , Obesidad/psicologíaRESUMEN
PURPOSE: Weight Bias Internalization (WBI) is pervasive and potentially damaging for health. Little is known about WBI in youth. As negative effects of WBI have been observed when controlling for BMI, measures that allow WBI to be assessed across different weight categories are needed. The Modified Weight Bias Internalization Scale (WBIS-M) is one of the most frequently used scales in this field. Our purpose was to obtain a Spanish validated version of the WBIS-M for adolescents across different weight statuses. METHODS: The data were collected from 298 secondary students (mean age 14.31; 48.32% girls; 18.8% were overweight and 6.4% had obesity). Internal structure was examined by a cross-validation analysis, using both exploratory and confirmatory factor analyses in different subsamples. RESULTS: Item 1 showed a psychometric anomalous functioning and was deleted. The one-factor structure of the 10-item version was confirmed with adequate fit ([EFA (KMO = 0.915, χ2(55) = 1075.633, p < 0.0001)]; [CFA (χ2(35) = 200.515; GFI = 0.995; PGFI = 0.992; NFI = 0.991; SRMR = 0.060)]). Internal consistency was high [Formula: see text] ω = 0.93). Significant correlations with the same set of external variables assessed in the original version (anti-fat bias, self-esteem, mood, body dissatisfaction, drive for thinness, binge eating), all of them correlates of WBI in adolescents, were found. Girls and participants with obesity obtained higher scores. CONCLUSION: The results provide support for the validity and reliability of our WBIS-M version for use with adolescents across weight categories in Spain. LEVEL OF EVIDENCE: Level III, evidence obtained from well-designed cohort studies.
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Imagen Corporal , Prejuicio de Peso , Femenino , Humanos , Adolescente , Masculino , Reproducibilidad de los Resultados , Encuestas y Cuestionarios , Obesidad , PsicometríaRESUMEN
Delivery of adeno-associated viral vectors (AAVs) to cerebrospinal fluid (CSF) has emerged as a promising approach to achieve widespread transduction of the central nervous system (CNS) and peripheral nervous system (PNS), with direct applicability to the treatment of a wide range of neurological diseases, particularly lysosomal storage diseases. Although studies in small animal models have provided proof of concept and experiments in large animals demonstrated feasibility in bigger brains, there is not much information on long-term safety or durability of the effect. Here, we report a 7-year study in healthy beagle dogs after intra-CSF delivery of a single, clinically relevant dose (2 × 1013 vg/dog) of AAV9 vectors carrying the canine sulfamidase, the enzyme deficient in mucopolysaccharidosis type IIIA. Periodic monitoring of CSF and blood, clinical and neurological evaluations, and magnetic resonance and ultrasound imaging of target organs demonstrated no toxicity related to treatment. AAV9-mediated gene transfer resulted in detection of sulfamidase activity in CSF throughout the study. Analysis at tissue level showed widespread sulfamidase expression and activity in the absence of histological findings in any region of encephalon, spinal cord, or dorsal root ganglia. Altogether, these results provide proof of durability of expression and long-term safety for intra-CSF delivery of AAV-based gene transfer vectors encoding therapeutic proteins to the CNS.
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Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.
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Condroitinsulfatasas/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Terapia Genética/métodos , Mucopolisacaridosis IV/terapia , Sistema Musculoesquelético/metabolismo , Animales , Cartílago Articular/metabolismo , Cartílago Articular/patología , Cartílago Articular/ultraestructura , Condroitinsulfatasas/deficiencia , Condroitinsulfatasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Vectores Genéticos/genética , Humanos , Masculino , Microscopía Electrónica de Transmisión , Mucopolisacaridosis IV/enzimología , Mucopolisacaridosis IV/genética , Sistema Musculoesquelético/patología , Sistema Musculoesquelético/ultraestructura , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resultado del TratamientoRESUMEN
Prevalence of type 2 diabetes (T2D) and obesity is increasing worldwide. Currently available therapies are not suited for all patients in the heterogeneous obese/T2D population, hence the need for novel treatments. Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic agent for T2D/obesity. Native FGF21 has, however, poor pharmacokinetic properties, making gene therapy an attractive strategy to achieve sustained circulating levels of this protein. Here, adeno-associated viral vectors (AAV) were used to genetically engineer liver, adipose tissue, or skeletal muscle to secrete FGF21. Treatment of animals under long-term high-fat diet feeding or of ob/ob mice resulted in marked reductions in body weight, adipose tissue hypertrophy and inflammation, hepatic steatosis, inflammation and fibrosis, and insulin resistance for > 1 year. This therapeutic effect was achieved in the absence of side effects despite continuously elevated serum FGF21. Furthermore, FGF21 overproduction in healthy animals fed a standard diet prevented the increase in weight and insulin resistance associated with aging. Our study underscores the potential of FGF21 gene therapy to treat obesity, insulin resistance, and T2D.
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Diabetes Mellitus Tipo 2/terapia , Factores de Crecimiento de Fibroblastos/genética , Terapia Genética , Resistencia a la Insulina , Obesidad/terapia , Adipocitos/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Peso Corporal , Diabetes Mellitus Tipo 2/genética , Dieta Alta en Grasa , Metabolismo Energético , Hígado Graso/terapia , Factores de Crecimiento de Fibroblastos/metabolismo , Fibrosis/terapia , Técnicas de Transferencia de Gen , Hiperplasia/terapia , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Músculo Esquelético/metabolismo , Obesidad/genética , Pancreatitis/terapiaRESUMEN
Gene therapy is a promising therapeutic alternative for Lysosomal Storage Disorders (LSD), as it is not necessary to correct the genetic defect in all cells of an organ to achieve therapeutically significant levels of enzyme in body fluids, from which non-transduced cells can uptake the protein correcting their enzymatic deficiency. Animal models are instrumental in the development of new treatments for LSD. Here we report the generation of the first mouse model of the LSD Muccopolysaccharidosis Type IIID (MPSIIID), also known as Sanfilippo syndrome type D. This autosomic recessive, heparan sulphate storage disease is caused by deficiency in N-acetylglucosamine 6-sulfatase (GNS). Mice deficient in GNS showed lysosomal storage pathology and loss of lysosomal homeostasis in the CNS and peripheral tissues, chronic widespread neuroinflammation, reduced locomotor and exploratory activity and shortened lifespan, a phenotype that closely resembled human MPSIIID. Moreover, treatment of the GNS-deficient animals with GNS-encoding adeno-associated viral (AAV) vectors of serotype 9 delivered to the cerebrospinal fluid completely corrected pathological storage, improved lysosomal functionality in the CNS and somatic tissues, resolved neuroinflammation, restored normal behaviour and extended lifespan of treated mice. Hence, this work represents the first step towards the development of a treatment for MPSIIID.
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Terapia Genética , Enfermedades por Almacenamiento Lisosomal/terapia , Mucopolisacaridosis III/terapia , Sulfatasas/genética , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Ratones , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/patología , Fenotipo , Sulfatasas/administración & dosificaciónRESUMEN
Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disease characterized by severe neurologic and somatic disease caused by deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes the glycosaminoglycans heparan and dermatan sulphate. Intravenous enzyme replacement therapy (ERT) currently constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier (BBB) limits ERT efficacy in treating neurological symptoms. Here, we report a gene therapy approach for MPSII through direct delivery of vectors to the CNS. Through a minimally invasive procedure, we administered adeno-associated virus vectors encoding IDS (AAV9-Ids) to the cerebrospinal fluid of MPSII mice with already established disease. Treated mice showed a significant increase in IDS activity throughout the encephalon, with full resolution of lysosomal storage lesions, reversal of lysosomal dysfunction, normalization of brain transcriptomic signature, and disappearance of neuroinflammation. Moreover, our vector also transduced the liver, providing a peripheral source of therapeutic protein that corrected storage pathology in visceral organs, with evidence of cross-correction of nontransduced organs by circulating enzyme. Importantly, AAV9-Ids-treated MPSII mice showed normalization of behavioral deficits and considerably prolonged survival. These results provide a strong proof of concept for the clinical translation of our approach for the treatment of Hunter syndrome patients with cognitive impairment.
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Terapia Genética , Iduronato Sulfatasa/genética , Mucopolisacaridosis II/terapia , Animales , Dependovirus , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BLRESUMEN
Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe lysosomal storage disease caused by deficiency in activity of the transmembrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT) that catalyses the N-acetylation of α-glucosamine residues of heparan sulfate. Enzyme deficiency causes abnormal substrate accumulation in lysosomes, leading to progressive and severe neurodegeneration, somatic pathology and early death. There is no cure for MPSIIIC, and development of new therapies is challenging because of the unfeasibility of cross-correction. In this study, we generated a new mouse model of MPSIIIC by targeted disruption of the Hgsnat gene. Successful targeting left LacZ expression under control of the Hgsnat promoter, allowing investigation into sites of endogenous expression, which was particularly prominent in the CNS, but was also detectable in peripheral organs. Signs of CNS storage pathology, including glycosaminoglycan accumulation, lysosomal distension, lysosomal dysfunction and neuroinflammation were detected in 2-month-old animals and progressed with age. Glycosaminoglycan accumulation and ultrastructural changes were also observed in most somatic organs, but lysosomal pathology seemed most severe in liver. Furthermore, HGSNAT-deficient mice had altered locomotor and exploratory activity and shortened lifespan. Hence, this animal model recapitulates human MPSIIIC and provides a useful tool for the study of disease physiopathology and the development of new therapeutic approaches.
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Progresión de la Enfermedad , Mucopolisacaridosis III/patología , Acetiltransferasas/deficiencia , Acetiltransferasas/metabolismo , Animales , Conducta Animal , Encéfalo/enzimología , Encéfalo/patología , Modelos Animales de Enfermedad , Glicosaminoglicanos/metabolismo , Homeostasis , Humanos , Inflamación/patología , Longevidad , Lisosomas/metabolismo , Lisosomas/patología , Lisosomas/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/patología , Mucopolisacaridosis III/enzimología , Especificidad de Órganos , Análisis de SupervivenciaRESUMEN
Gene therapy is an attractive tool for the treatment of monogenic disorders, in particular for lysosomal storage diseases (LSD) caused by deficiencies in secretable lysosomal enzymes in which neither full restoration of normal enzymatic activity nor transduction of all affected cells are necessary. However, some LSD such as Mucopolysaccharidosis Type IIIB (MPSIIIB) are challenging because the disease's main target organ is the brain and enzymes do not efficiently cross the blood-brain barrier even if present at very high concentration in circulation. To overcome these limitations, we delivered AAV9 vectors encoding for α-N-acetylglucosaminidase (NAGLU) to the Cerebrospinal Fluid (CSF) of MPSIIIB mice with the disease already detectable at biochemical, histological and functional level. Restoration of enzymatic activity in Central Nervous System (CNS) resulted in normalization of glycosaminoglycan content and lysosomal physiology, resolved neuroinflammation and restored the pattern of gene expression in brain similar to that of healthy animals. Additionally, transduction of the liver due to passage of vectors to the circulation led to whole-body disease correction. Treated animals also showed reversal of behavioural deficits and extended lifespan. Importantly, when the levels of enzymatic activity were monitored in the CSF of dogs following administration of canine NAGLU-coding vectors to animals that were either naïve or had pre-existing immunity against AAV9, similar levels of activity were achieved, suggesting that CNS efficacy would not be compromised in patients seropositive for AAV9. Our studies provide a strong rationale for the clinical development of this novel therapeutic approach as the treatment for MPSIIIB.
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Acetilglucosaminidasa/genética , Terapia Genética/métodos , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/terapia , Acetilglucosaminidasa/líquido cefalorraquídeo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Dependovirus/genética , Dependovirus/metabolismo , Femenino , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mucopolisacaridosis III/líquido cefalorraquídeo , Mucopolisacaridosis III/enzimologíaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disease worldwide, and evidence suggests that it promotes insulin resistance and type 2 diabetes. Caloric restriction (CR) is the only available strategy for NAFLD treatment. The protein deacetylase Sirtuin1 (SIRT1), which is activated by CR, increases catabolic metabolism and decreases lipogenesis and inflammation, both involved in the development of NAFLD. Here we show that adeno-associated viral vectors of serotype 8 (AAV8)-mediated liver-specific Sirt1 gene transfer prevents the development of NAFLD induced by a high carbohydrate (HC) diet. Long-term hepatic SIRT1 overexpression led to upregulation of key hepatic genes involved in ß-oxidation, prevented HC diet-induced lipid accumulation and reduced liver inflammation. AAV8-Sirt1-treated mice showed improved insulin sensitivity, increased oxidative capacity in skeletal muscle and reduced white adipose tissue inflammation. Moreover, HC feeding induced leptin resistance, which was also attenuated in AAV8-Sirt1-treated mice. Therefore, AAV-mediated gene transfer to overexpress SIRT1 specifically in the liver may represent a new gene therapy strategy to counteract NAFLD and related diseases such as type 2 diabetes.
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Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglucemia/metabolismo , Mitocondrias/metabolismo , Animales , Dependovirus , Diabetes Mellitus Tipo 2/genética , Metabolismo Energético/genética , Ingeniería Genética , Hiperglucemia/genética , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Endogámicos NOD , Ratones Obesos , Mitocondrias/genéticaRESUMEN
For most lysosomal storage diseases (LSDs) affecting the CNS, there is currently no cure. The BBB, which limits the bioavailability of drugs administered systemically, and the short half-life of lysosomal enzymes, hamper the development of effective therapies. Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomic recessive LSD caused by a deficiency in sulfamidase, a sulfatase involved in the stepwise degradation of glycosaminoglycan (GAG) heparan sulfate. Here, we demonstrate that intracerebrospinal fluid (intra-CSF) administration of serotype 9 adenoassociated viral vectors (AAV9s) encoding sulfamidase corrects both CNS and somatic pathology in MPS IIIA mice. Following vector administration, enzymatic activity increased throughout the brain and in serum, leading to whole body correction of GAG accumulation and lysosomal pathology, normalization of behavioral deficits, and prolonged survival. To test this strategy in a larger animal, we treated beagle dogs using intracisternal or intracerebroventricular delivery. Administration of sulfamidase-encoding AAV9 resulted in transgenic expression throughout the CNS and liver and increased sulfamidase activity in CSF. High-titer serum antibodies against AAV9 only partially blocked CSF-mediated gene transfer to the brains of dogs. Consistently, anti-AAV antibody titers were lower in CSF than in serum collected from healthy and MPS IIIA-affected children. These results support the clinical translation of this approach for the treatment of MPS IIIA and other LSDs with CNS involvement.
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Insulin-like growth factor I (IGF-I) exerts multiple effects on different retinal cell types in both physiological and pathological conditions. Despite the growth factor's extensively described neuroprotective actions, transgenic mice with increased intraocular levels of IGF-I showed progressive impairment of electroretinographic amplitudes up to complete loss of response, with loss of photoreceptors and bipolar, ganglion, and amacrine neurons. Neurodegeneration was preceded by the overexpression of genes related to retinal stress, acute-phase response, and gliosis, suggesting that IGF-I altered normal retinal homeostasis. Indeed, gliosis and microgliosis were present from an early age in transgenic mice, before other alterations occurred, and were accompanied by signs of oxidative stress and impaired glutamate recycling. Older mice also showed overproduction of pro-inflammatory cytokines. Our results suggest that, when chronically increased, intraocular IGF-I is responsible for the induction of deleterious cellular processes that can lead to neurodegeneration, and they highlight the importance that this growth factor may have in the pathogenesis of conditions such as ischemic or diabetic retinopathy.
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Gliosis/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Enfermedades de la Retina/metabolismo , Células Amacrinas/metabolismo , Células Amacrinas/patología , Animales , Apoptosis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Ácido Glutámico/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patología , Enfermedades Neurodegenerativas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Células Fotorreceptoras de Vertebrados/patología , Retina/metabolismo , Retina/patología , Retina/fisiopatología , Transducción de Señal , Técnicas de Cultivo de Tejidos , TranscriptomaRESUMEN
In type 1 diabetes, loss of tolerance to ß-cell antigens results in T-cell-dependent autoimmune destruction of ß cells. The abrogation of autoreactive T-cell responses is a prerequisite to achieve long-lasting correction of the disease. The liver has unique immunomodulatory properties and hepatic gene transfer results in tolerance induction and suppression of autoimmune diseases, in part by regulatory T-cell (Treg) activation. Hence, the liver could be manipulated to treat or prevent diabetes onset through expression of key genes. IGF-I may be an immunomodulatory candidate because it prevents autoimmune diabetes when expressed in ß cells or subcutaneously injected. Here, we demonstrate that transient, plasmid-derived IGF-I expression in mouse liver suppressed autoimmune diabetes progression. Suppression was associated with decreased islet inflammation and ß-cell apoptosis, increased ß-cell replication, and normalized ß-cell mass. Permanent protection depended on exogenous IGF-I expression in liver nonparenchymal cells and was associated with increased percentage of intrapancreatic Tregs. Importantly, Treg depletion completely abolished IGF-I-mediated protection confirming the therapeutic potential of these cells in autoimmune diabetes. This study demonstrates that a nonviral gene therapy combining the immunological properties of the liver and IGF-I could be beneficial in the treatment of the disease.
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Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Terapia Genética , Factor I del Crecimiento Similar a la Insulina/genética , Hígado/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Apoptosis/genética , Apoptosis/inmunología , División Celular/genética , División Celular/inmunología , Humanos , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/patología , Hígado/inmunología , Ratones , Ratones Transgénicos , Pancreatitis/genética , Pancreatitis/inmunología , Plásmidos/genéticaRESUMEN
Mucopolysaccharidosis type IIIA (MPSIIIA) is a rare lysosomal storage disorder caused by mutations in the sulfamidase gene. Accumulation of glycosaminoglycan (GAG) inside the lysosomes is associated with severe neurodegeneration as well as peripheral organ pathological changes leading to death of affected individuals during adolescence. There is no cure for MPSIIIA. Due to the limitation of the blood-brain barrier, enzyme replacement therapy and gene therapy strategies attempted thus far have not achieved whole-body correction of the disease. After the systemic administration of an adeno-associated virus 9 (AAV9) vector encoding for sulfamidase under the control of a ubiquitous promoter, we were able to obtain widespread expression of the therapeutic transgene in brain and in peripheral organs, and sulfamidase activity in serum of both male and female MPSIIIA mice. This was accompanied by the normalization of GAG storage levels in most peripheral organs. In brain, decrease in GAG tissue content following AAV9 gene transfer of sulfamidase was associated with the resolution of neuroinflammation. Finally, correction of disease phenotype resulted in a remarkable prolongation of survival of both male and female AAV-treated MPSIIIA mice. This proof-of-concept study will be relevant to the future development of therapies for MPSIIIA.
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Terapia Genética/métodos , Vectores Genéticos/farmacología , Glicosaminoglicanos/metabolismo , Hidrolasas/genética , Mucopolisacaridosis III/terapia , Animales , Sistema Nervioso Central/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Vectores Genéticos/administración & dosificación , Hígado/metabolismo , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/patología , TransgenesRESUMEN
Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders.
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Envejecimiento/patología , Retinopatía Diabética/patología , Retinopatía Diabética/prevención & control , Proteínas del Ojo/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Retina/metabolismo , Neovascularización Retiniana/patología , Neovascularización Retiniana/prevención & control , Serpinas/metabolismo , Animales , Hipoxia de la Célula , Dependovirus/genética , Retinopatía Diabética/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Inyecciones Intravítreas , Ratones , Ratones Transgénicos , Retina/patología , Desprendimiento de Retina/metabolismo , Desprendimiento de Retina/patología , Neovascularización Retiniana/metabolismo , Factores de Tiempo , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mucopolysaccharidosis type IIIA (MPSIIIA) is an inherited lysosomal storage disease caused by deficiency of sulfamidase, resulting in accumulation of the glycosaminoglycan (GAG) heparan sulfate. It is characterized by severe progressive neurodegeneration, together with somatic alterations, which lead to death during adolescence. Here, we tested the ability of adeno-associated virus (AAV) vector-mediated genetic modification of either skeletal muscle or liver to revert the already established disease phenotype of 2-month-old MPSIIIA males and females. Intramuscular administration of AAV-Sulfamidase failed to achieve significant therapeutic benefit in either gender. In contrast, AAV8-mediated liver-directed gene transfer achieved high and sustained levels of circulating active sulfamidase, which reached normal levels in females and was fourfold higher in males, and completely corrected lysosomal GAG accumulation in most somatic tissues. Remarkably, a 50% reduction of GAG accumulation was achieved throughout the entire brain of males, which correlated with a partial improvement of the pathology of cerebellum and cortex. Liver-directed gene transfer expanded the lifespan of MPSIIIA males, underscoring the importance of reaching supraphysiological plasma levels of enzyme for maximal therapeutic benefit. These results show how liver-directed gene transfer can reverse somatic and ameliorate neurological pathology in MPSIIIA.
Asunto(s)
Sistema Nervioso Central/patología , Terapia Genética , Hidrolasas/genética , Hígado/metabolismo , Mucopolisacaridosis III/terapia , Animales , Cerebelo/ultraestructura , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Orden Génico , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/farmacocinética , Hidrolasas/metabolismo , Inyecciones Intramusculares , Inyecciones Intravenosas , Hígado/ultraestructura , Lisosomas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/mortalidad , Músculo Esquelético/metabolismo , Análisis de Supervivencia , Transducción Genética , Corteza Visual/patología , Corteza Visual/ultraestructuraRESUMEN
Blood-retinal barrier (BRB) breakdown is a key event in diabetic retinopathy and other ocular disorders that leads to increased retinal vascular permeability. This causes edema and tissue damage resulting in visual impairment. Insulin-like growth factor-I (IGF-I) is involved in these processes, although the relative contribution of increased systemic versus intraocular IGF-I remains controversial. Here, to elucidate the role of this factor in BRB breakdown, transgenic mice with either local or systemic elevations of IGF-I have been examined. High intraocular IGF-I, resulting from overexpression of IGF-I in the retina, increased IGF-I receptor content and signaling and led to accumulation of vascular endothelial growth factor. This was parallel to up-regulation of vascular Intercellular adhesion molecule I and retinal infiltration by bone marrow-derived microglial cells. These alterations resulted in increased vessel paracellular permeability to both low and high molecular weight compounds in IGF-I-overexpressing retinas and agreed with the loss of vascular tight junction integrity observed by electron microscopy and the altered junctional protein content. In contrast, mice with chronically elevated serum IGF-I did not show alterations in the retinal vasculature structure and permeability, indicating that circulating IGF-I cannot initiate BRB breakdown. Consistent with a key role of IGF-I signaling in retinal diseases, a strong up-regulation of the IGF-I receptor in human retinas with marked gliosis was also observed. Thus, this study demonstrates that intraocular IGF-I, but not systemic IGF-I, is sufficient to trigger processes leading to BRB breakdown and increased retinal vascular permeability. Therefore, therapeutic interventions designed to counteract local IGF-I effects may prove successful to prevent BRB disruption.