Toward a gene therapy for neurological and somatic MPSIIIA

Mucopolysaccharidosis Type IIIA (MPSIIIA) represents an unmet medical need. MPSIIIA shares with many other lysosomal storage disorders (LSD) the characteristic of being a severe neurodegenerative disease accompanied by mild somatic involvement. Thus, the main target organ for the development of new treatments is the central nervous system (CNS), but overall clinical efficacy would be greatly enhanced by simultaneous correction of peripheral disease. We have recently developed a novel treatment for MPSIIIA based on the delivery to the cerebrospinal fluid of serotype 9 adeno-associated virus (AAV9)-derived vectors. This gene therapy strategy corrected both CNS and somatic pathology in animal models through widespread transduction of CNS, peripheral nervous system (PNS), and liver. The work set the grounds for the clinical translation of the approach to treat MPSIIIA in humans. Here we discuss some important considerations that further support the applicability of this treatment to MPSIIIA and other LSD with CNS and somatic involvement

Insulin-like growth factor I (IGF-I)-induced chronic gliosis and retinal stress lead to neurodegeneration in a mouse model of retinopathy

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

Modulation of CD8\u3csup\u3e+\u3c/sup\u3e T cell responses to AAV vectors with IgG-derived MHC class II epitopes

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen- specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible

Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy

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

Insulin-like growth factor 2 overexpression induces β-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 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

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.This work was supported by grants from Ministerio de Economía y Competi- tividad (MINECO) and FEDER, Plan Nacional I+D+I (SAF2014-54866R), andGeneralitat de Catalunya (2014SGR1669and ICREA Academia Award to F.B.), Spain, from the European Commission (MYOCURE, PHC-14-2015 667751) and the European Foundation for the Study of Diabetes (EFSD/MSD European Research Programme on Novel Therapies for Type 2 Diabetes,2013). V.J. was recipient of a post-doctoral research fellowship from EFSD/ Lilly. E.C., V.S., and C.M. received a predoctoral fellowship from Ministerio de Educación, Cultura y Deporte, and J.R. from Ministerio de Economía y Competitividad, Spain. The authors thank Marta Moya and Maria Molas for technical assistance.S

Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy

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

Long-term retinal PEDF overexpression prevents neovascularization in a murine adult model of retinopathy

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

Progressive neurologic and somatic disease in a novel mouse model of human mucopolysaccharidosis type IIIC

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. Summary: A new animal model of the severe neurodegenerative lysosomal disorder mucopolysaccharidosis IIIC recapitulates the human disease, with progressive CNS and somatic lysosomal pathology, and shortened lifespan

Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for Diabetes : 8-Year Follow-Up in Dogs

Diabetes is a complex metabolic disease that exposes patients to the deleterious effects of hyperglycemia on various organs. Achievement of normoglycemia with exogenous insulin treatment requires the use of high doses of hormone, which increases the risk of life-threatening hypoglycemic episodes. We developed a gene therapy approach to control diabetic hyperglycemia based on co-expression of the insulin and glucokinase genes in skeletal muscle. Previous studies proved the feasibility of gene delivery to large diabetic animals with adeno-associated viral (AAV) vectors. Here, we report the long-term (∼8 years) follow-up after a single administration of therapeutic vectors to diabetic dogs. Successful, multi-year control of glycemia was achieved without the need of supplementation with exogenous insulin. Metabolic correction was demonstrated through normalization of serum levels of fructosamine, triglycerides, and cholesterol and remarkable improvement in the response to an oral glucose challenge. The persistence of vector genomes and therapeutic transgene expression years after vector delivery was documented in multiple samples from treated muscles, which showed normal morphology. Thus, this study demonstrates the long-term efficacy and safety of insulin and glucokinase gene transfer in large animals and especially the ability of the system to respond to the changes in metabolic needs as animals grow older