AAV-mediated BMP7 gene therapy counteracts insulin resistance and obesity.

Type 2 diabetes, insulin resistance, and obesity are strongly associated and are a major health problem worldwide. Obesity largely results from a sustained imbalance between energy intake and expenditure. Therapeutic approaches targeting metabolic rate may counteract body weight gain and insulin resistance. Bone morphogenic protein 7 (BMP7) has proven to enhance energy expenditure by inducing non-shivering thermogenesis in short-term studies in mice treated with the recombinant protein or adenoviral vectors encoding BMP7. To achieve long-term BMP7 effects, the use of adeno-associated viral (AAV) vectors would provide sustained production of the protein after a single administration. Here, we demonstrated that treatment of high-fat-diet-fed mice and ob/ob mice with liver-directed AAV-BMP7 vectors enabled a long-lasting increase in circulating levels of this factor. This rise in BMP7 concentration induced browning of white adipose tissue (WAT) and activation of brown adipose tissue, which enhanced energy expenditure, and reversed WAT hypertrophy, hepatic steatosis, and WAT and liver inflammation, ultimately resulting in normalization of body weight and insulin resistance. This study highlights the potential of AAV-BMP7-mediated gene therapy for the treatment of insulin resistance, type 2 diabetes, and obesity.

[Surveillance of rare diseases in Spain: Spanish Registry of Rare Diseases (ReeR).] / Vigilancia de las enfermedades raras en España: el Registro Estatal de Enfermedades Raras (ReeR).

Traditionally, epidemiological surveillance has focused on infectious diseases, but the concept of Public Health surveillance, introduced in Spain with the Law 33/2011, is broader and includes chronic diseases. Health strategies for these diseases need epidemiological information to improve understanding of socio-health needs and to facilitate the efficient management of resources. The European Union defines rare diseases (RD) as those that, being life-threatening or chronically debilitating, have a prevalence of less than 5 cases per 10,000 inhabitants. The RD Strategy of the National Health System, approved in 2009 and updated in 2014, recommends the development of regional registries of rare diseases (RAER), in addition to a national registry. The REpIER and Spain-RDR projects of the Institute of Health Carlos III (ISCIII) promoted the creation and regulation of 94% of the RAER. After more than 10 years of initiatives and work to improve the knowledge of RD's epidemiology in Spain, it was possible to implement the Spanish Registry of Rare Diseases (ReeR) in 2015, becoming one of the first population surveillance systems for chronic diseases of state scope. The ReeR procedures manual is the result of consensus between the RAER, the Ministry of Health, the ISCIII and the patient associations. The participatory methodology used for the implementation and launching of ReeR is considered an added value. The information system implemented will allow improving knowledge about the prevalence and distribution of RD in Spain.

Tradicionalmente la vigilancia epidemiológica se ha centrado en enfermedades transmisibles, pero el concepto de vigilancia en Salud Pública, incorporado en España con la Ley 33/2011, es más amplio e incluye las enfermedades crónicas. Las estrategias de salud para estas enfermedades necesitan disponer de información epidemiológica para mejorar el conocimiento de las necesidades sociosanitarias y facilitar la gestión eficiente de recursos. La Unión Europea define las enfermedades raras (ER) como aquellas que, con peligro de muerte o invalidez crónica, presentan una prevalencia inferior a 5 casos por cada 10.000 habitantes. La Estrategia en ER del Sistema Nacional de Salud, aprobada en 2009 y actualizada en 2014, recomienda desarrollar registros autonómicos de enfermedades raras (RAER) y uno estatal. Los proyectos REpIER y Spain-RDR del Instituto de Salud Carlos III (ISCIII) impulsaron la creación y regulación del 94% de los RAER; y tras más de 10 años de iniciativas y trabajos para mejorar el conocimiento de la epidemiología de las ER en España, se logró implementar el Registro Estatal de Enfermedades Raras (ReeR) en 2015, convirtiéndose en uno de los primeros sistemas de vigilancia poblacional de enfermedades crónicas de ámbito estatal. El manual de procedimientos del ReeR es el resultado del consenso entre los RAER, Ministerio de Sanidad, ISCIII y asociaciones de pacientes. La metodología participativa empleada para la implementación y puesta en funcionamiento del ReeR es considerada un valor añadido. El sistema de información implementado va a permitir mejorar el conocimiento sobre la prevalencia y distribución de las ER en España.

BMP7 overexpression in adipose tissue induces white adipogenesis and improves insulin sensitivity in ob/ob mice.

BACKGROUND/OBJECTIVES: During obesity, hypertrophic enlargement of white adipose tissue (WAT) promotes ectopic lipid deposition and development of insulin resistance. In contrast, WAT hyperplasia is associated with preservation of insulin sensitivity. The complex network of factors that regulates white adipogenesis is not fully understood. Bone morphogenic protein 7 (BMP7) can induce brown adipogenesis, but its role on white adipogenesis remains to be elucidated. Here, we assessed BMP7-mediated effects on white adipogenesis in ob/ob mice. METHODS: BMP7 was overexpressed in either WAT or liver of ob/ob mice using adeno-associated viral (AAV) vectors. Analysis of gene expression, histological and morphometric alterations, and metabolites and hormones concentrations were carried out. RESULTS: Overexpression of BMP7 in adipocytes of subcutaneous and visceral WAT increased fat mass, the proportion of small-size adipocytes and the expression of adipogenic and mature adipocyte genes, suggesting induction of adipogenesis irrespective of fat depot. These changes were associated with reduced hepatic steatosis and improved insulin sensitivity. In contrast, liver-specific overproduction of BMP7 did not promote WAT hyperplasia despite BMP7 circulating levels were similar to those achieved after genetic engineering of WAT. CONCLUSIONS: This study unravels a new autocrine/paracrine role of BMP7 on white adipogenesis and highlights that BMP7 may modulate WAT plasticity and increase insulin sensitivity.

Vitamin D Receptor Overexpression in ß-Cells Ameliorates Diabetes in Mice.

Vitamin D deficiency has been associated with increased incidence of diabetes, both in humans and in animal models. In addition, an association between vitamin D receptor (VDR) gene polymorphisms and diabetes has also been described. However, the involvement of VDR in the development of diabetes, specifically in pancreatic ß-cells, has not been elucidated yet. Here, we aimed to study the role of VDR in ß-cells in the pathophysiology of diabetes. Our results indicate that Vdr expression was modulated by glucose in healthy islets and decreased in islets from both type 1 diabetes and type 2 diabetes mouse models. In addition, transgenic mice overexpressing VDR in ß-cells were protected against streptozotocin-induced diabetes and presented a preserved ß-cell mass and a reduction in islet inflammation. Altogether, these results suggest that sustained VDR levels in ß-cells may preserve ß-cell mass and ß-cell function and protect against diabetes.

"Glándulas floridas profundas" en cáncer de cérvix, ¿es un precursor del adenocarcinoma mucinoso endocervical de tipo gástrico? / "Florid deep glands" in cervical cancer, a precursor of mucinous gastric-type endocervical adenocarcinoma?

OBJETIVO: se presentan dos casos clínicos de adenocarcinoma endocervical, uno del tipo adenocarcinoma mucinoso endocervical de tipo gástrico y otro adenocarcinoma endocervical in situ, con el fin de aportar la evolución de este tipo de adenocarcinomas desde lesiones premalignas. MATERIAL Y MÉTODOS: caso clínico. RESULTADOS: no procede. CONCLUSIONES: podría existir cierto vínculo entre la lesión "glándulas floridas profundas" y el adenocarcinoma mucinoso de desviación mínima o adenoma maligno; este conlleva un diagnóstico difícil debido a la que la mayoría de biopsias suelen ser poco representativas

OBJECTIVE: Two clinical cases of endocervical adenocarcinoma are presented, the first one is a mucinous gastric-type endocervical adenocarcinoma and the second one is endocervical adenocarcinoma in situ; in order to provide the evolution of this type of adenocarcinomas from premalignant lesions. MATERIAL AND METHODS: Clinical case. RESULTS: Not applicable. CONCLUSIONS: There could be some link between the lesion "florid deep glands" and the minimal deviation adenocarcinoma of mucinous type or adenoma malignum; this leads to a difficult diagnosis because most biopsies are usually unrepresentative

FGF21 gene therapy as treatment for obesity and insulin resistance.

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.

AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice.

OBJECTIVE: Type 1 diabetes is characterized by autoimmune destruction of ß-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding ß-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for ß-cells with immunomodulatory properties. METHODS: Transgenic NOD mice overexpressing IGF1 specifically in ß-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28-30 weeks. RESULTS: In transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of ß-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved ß-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice. CONCLUSIONS: Local expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.

Insulin-like Growth Factor 2 Overexpression Induces ß-Cell Dysfunction and Increases Beta-cell Susceptibility to Damage.

The human insulin-like growth factor 2 (IGF2) and insulin genes are located within the same genomic region. Although human genomic studies have demonstrated associations between diabetes and the insulin/IGF2 locus or the IGF2 mRNA-binding protein 2 (IGF2BP2), the role of IGF2 in diabetes pathogenesis is not fully understood. We previously described that transgenic mice overexpressing IGF2 specifically in ß-cells (Tg-IGF2) develop a pre-diabetic state. Here, we characterized the effects of IGF2 on ß-cell functionality. Overexpression of IGF2 led to ß-cell dedifferentiation and endoplasmic reticulum stress causing islet dysfunction in vivo. Both adenovirus-mediated overexpression of IGF2 and treatment of adult wild-type islets with recombinant IGF2 in vitro further confirmed the direct implication of IGF2 on ß-cell dysfunction. Treatment of Tg-IGF2 mice with subdiabetogenic doses of streptozotocin or crossing these mice with a transgenic model of islet lymphocytic infiltration promoted the development of overt diabetes, suggesting that IGF2 makes islets more susceptible to ß-cell damage and immune attack. These results indicate that increased local levels of IGF2 in pancreatic islets may predispose to the onset of diabetes. This study unravels an unprecedented role of IGF2 on ß-cells function.

Pancreatic transduction by helper-dependent adenoviral vectors via intraductal delivery.

Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue.

In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice.

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.

Ablation of TRIP-Br2, a regulator of fat lipolysis, thermogenesis and oxidative metabolism, prevents diet-induced obesity and insulin resistance.

Obesity develops as a result of altered energy homeostasis favoring fat storage. Here we describe a new transcription co-regulator for adiposity and energy metabolism, SERTA domain containing 2 (TRIP-Br2, also called SERTAD2). TRIP-Br2-null mice are resistant to obesity and obesity-related insulin resistance. Adipocytes of these knockout mice showed greater stimulated lipolysis secondary to enhanced expression of hormone sensitive lipase (HSL) and ß3-adrenergic (Adrb3) receptors. The knockout mice also have higher energy expenditure because of increased adipocyte thermogenesis and oxidative metabolism caused by upregulating key enzymes in their respective processes. Our data show that a cell-cycle transcriptional co-regulator, TRIP-Br2, modulates fat storage through simultaneous regulation of lipolysis, thermogenesis and oxidative metabolism. These data, together with the observation that TRIP-Br2 expression is selectively elevated in visceral fat in obese humans, suggests that this transcriptional co-regulator is a new therapeutic target for counteracting the development of obesity, insulin resistance and hyperlipidemia.

Vascular endothelial growth factor-mediated islet hypervascularization and inflammation contribute to progressive reduction of ß-cell mass.

Type 2 diabetes (T2D) results from insulin resistance and inadequate insulin secretion. Insulin resistance initially causes compensatory islet hyperplasia that progresses to islet disorganization and altered vascularization, inflammation, and, finally, decreased functional ß-cell mass and hyperglycemia. The precise mechanism(s) underlying ß-cell failure remain to be elucidated. In this study, we show that in insulin-resistant high-fat diet-fed mice, the enhanced islet vascularization and inflammation was parallel to an increased expression of vascular endothelial growth factor A (VEGF). To elucidate the role of VEGF in these processes, we have genetically engineered ß-cells to overexpress VEGF (in transgenic mice or after adeno-associated viral vector-mediated gene transfer). We found that sustained increases in ß-cell VEGF levels led to disorganized, hypervascularized, and fibrotic islets, progressive macrophage infiltration, and proinflammatory cytokine production, including tumor necrosis factor-α and interleukin-1ß. This resulted in impaired insulin secretion, decreased ß-cell mass, and hyperglycemia with age. These results indicate that sustained VEGF upregulation may participate in the initiation of a process leading to ß-cell failure and further suggest that compensatory islet hyperplasia and hypervascularization may contribute to progressive inflammation and ß-cell mass loss during T2D.