Miquel Garcia

Correction of pathological accumulation of glycosaminoglycans in central nervous system and peripheral tissues of MPSIIIA mice through systemic AAV9 gene transfer.

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.

Liver Production of Sulfamidase Reverses Peripheral and Ameliorates CNS Pathology in Mucopolysaccharidosis IIIA Mice

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.

ALOX5AP Overexpression in Adipose Tissue Leads to LXA4 Production and Protection Against Diet-Induced Obesity and Insulin Resistance.

Eicosanoids, such as leukotriene B4 (LTB4) and lipoxin A4 (LXA4), may play a key role during obesity. While LTB4 is involved in adipose tissue inflammation and insulin resistance, LXA4 may exert anti-inflammatory effects and alleviate hepatic steatosis. Both lipid mediators derive from the same pathway, in which arachidonate 5-lipoxygenase (ALOX5) and its partner, arachidonate 5-lipoxygenase–activating protein (ALOX5AP), are involved. ALOX5 and ALOX5AP expression is increased in humans and rodents with obesity and insulin resistance. We found that transgenic mice overexpressing ALOX5AP in adipose tissue had higher LXA4 rather than higher LTB4 levels, were leaner, and showed increased energy expenditure, partly due to browning of white adipose tissue (WAT). Upregulation of hepatic LXR and Cyp7a1 led to higher bile acid synthesis, which may have contributed to increased thermogenesis. In addition, transgenic mice were protected against diet-induced obesity, insulin resistance, and inflammation. Finally, treatment of C57BL/6J mice with LXA4, which showed browning of WAT, strongly suggests that LXA4 is responsible for the transgenic mice phenotype. Thus, our data support that LXA4 may hold great potential for the future development of therapeutic strategies for obesity and related diseases.

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

Background: Human and animal studies have shown associations between insulin growth factor 2 (IGF2) and diabetes. Results: Overexpression of insulin growth factor 2 in β-cells leads to β-cell dysfunction and makes islets more vulnerable to β-cell damage and immune attack. Conclusion: IGF2 may play an important role in the predisposition and development of diabetes. Significance: This study unravels an unprecedented role of IGF2 on physiology of β-cells. 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 β-cells (Tg-IGF2) develop a pre-diabetic state. Here, we characterized the effects of IGF2 on β-cell functionality. Overexpression of IGF2 led to β-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 β-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 β-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 β-cells function.

HMGA1 overexpression in adipose tissue impairs adipogenesis and prevents diet-induced obesity and insulin resistance

High-Mobility-Group-A1 (HMGA1) proteins are non-histone proteins that regulate chromatin structure and gene expression during embryogenesis, tumourigenesis and immune responses. In vitro studies suggest that HMGA1 proteins may be required to regulate adipogenesis. To examine the role of HMGA1 in vivo, we generated transgenic mice overexpressing HMGA1 in adipose tissues. HMGA1 transgenic mice showed a marked reduction in white and brown adipose tissue mass that was associated with downregulation of genes involved in adipogenesis and concomitant upregulation of preadipocyte markers. Reduced adipogenesis and decreased fat mass were not associated with altered glucose homeostasis since HMGA1 transgenic mice fed a regular-chow diet exhibited normal glucose tolerance and insulin sensitivity. However, when fed a high-fat diet, overexpression of HMGA1 resulted in decreased body-weight gain, reduced fat mass, but improved insulin sensitivity and glucose tolerance. Although HMGA1 transgenic mice exhibited impaired glucose uptake in adipose tissue due to impaired adipogenesis, the increased glucose uptake observed in skeletal muscle may account for the improved glucose homeostasis. Our results indicate that HMGA1 plays an important function in the regulation of white and brown adipogenesis in vivo and suggests that impaired adipocyte differentiation and decreased fat mass is not always associated with impaired whole-body glucose homeostasis.

Insulin-like Growth Factor I (IGF-I)-induced Chronic Gliosis and Retinal Stress Lead to Neurodegeneration in a Mouse Model of Retinopathy

Background: In the retina, insulin-like growth factor I (IGF-I) is neuroprotective and essential for vasculature homeostasis. Results: Transgenic mice overexpressing Igf-I in the retina present chronic gliosis and retinal stress leading to neurodegeneration. Conclusion: IGF-I induces deleterious processes in the retina that, chronically, may overcome its neurotrophic properties. Significance: Increased intraocular IGF-I may contribute to the pathogenesis of conditions such as ischemic or diabetic retinopathies. Insulin-like growth factor I (IGF-I) exerts multiple effects on different retinal cell types in both physiological and pathological conditions. Despite the growth factors 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.

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.

348. Correction of CNS and Somatic Pathology by Intra-Cerebrospinal Fluid Gene Therapy for Mucopolysaccharidosis Type II

Mucopolysaccharidosis type II (MPSII), or Hunter Syndrome, is a X-linked recessive Lysosomal Storage Disease (LSD) caused by deficiency in Iduronate-2-sulfatase (IDS), an enzyme involved in the stepwise degradation of the glycosaminoglycans (GAGs) heparan sulfate (HS) and dermatan sulfate (DS). GAG storage builds up in the CNS and peripheral tissues causing severe neurologic and multisystemic somatic disease. Patients usually die during the second decade of life. Periodic 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 limits the efficacy of the approach in treating neurological symptoms. Alternatively, the periodic infusion of ERT to the cerebrospinal fluid is under clinical testing, but serious adverse events associated with the use of intrathecal drug delivery devices have been reported. Here we report a gene therapy approach directly addressing the CNS pathology of MPSII. Through a minimally invasive procedure we delivered adeno-associated virus vectors of serotype 9 encoding IDS (AAV9-Ids) to the CSF of MPSII mice with already established disease. Four months after vector administration, 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 the brain transcriptomic signature and disappearance of neuroinflammation. Moreover, our approach not only resulted in widespread distribution of vector in the CNS but also in liver transduction, providing a peripheral source of therapeutic protein that corrected the storage disease in visceral organs, with evidence of cross-correction of non-transduced organs by circulating enzyme. Importantly, MPSII mice treated with AAV9-Ids also showed normalization of behavioural deficits. These results provide a strong proof of concept for the clinical translation of our approach for the treatment of Hunter patients with cognitive impairment.

248. A Mouse Model of Type VII Glycogenosis to Assay Gene Therapy Approaches

Top of pageAbstract Type VII glycogenosis, or Taruis disease, is a muscular disorder characterized by the appearance of muscular fatigue, hemolysis and myoglobinuria after exercise. It is associated with an abnormal accumulation of muscle glycogen, due to an increase in its synthesis and a blockade of its mobilization. The underlying defect is a deficiency in muscle 6-phosphofructo-1-kinase isoenzyme (PFK-1M). In order to obtain a murine model of Taruis disease, heterozygous and homozygous PFK-1M knock-out mice were generated by standard gene-targeting methods in mouse ES cells. Heterozygous mice showed a 50% decrease in muscle PFK-1M mRNA levels, which led to a 50% decrease in muscle enzyme activity. This decrease did not cause alterations in muscle glycogen levels. Neither expression of the gene nor activity of the enzyme was found in skeletal muscle extracts from homozygous mice. These animals showed a three-fold increase in muscle glycogen levels and morphological abnormalities in muscle fibers such as necrotic and regenerating fibers or altered mitochondrias. Histopathological analysis of other tissues revealed glycogen accumulations in cardiac muscle and diaphragm. Moreover, due to the loss of erythrocyte PFK-1M activity, these animals showed higher levels of circulating reticulocytes. In addition, the spleen of PFK-1M deficient animals was 5 times bigger than those in control animals, due to hemolysis and compensatory extramedullary erythropoiesis. Furthermore, these animals were intolerant to exercise as described in human patients. The results indicate that mice lacking PFK-1M show pathological alterations similar to those observed in patients with type VII glycogenosis. Thus, these animals are the first murine model of type VII glycogenosis in which the disease can be studied and gene therapy approaches can be assayed and developed.