Excretion of excess nitrogen and increased survival by loss of SLC6A19 in a mouse model of ornithine transcarbamylase deficiency.

Protein catabolism ultimately yields toxic ammonia, which must be converted to urea by the liver for renal excretion. In extrahepatic tissues, ammonia is temporarily converted primarily to glutamine for subsequent hepatic extraction. Urea cycle disorders (UCDs) are inborn errors of metabolism causing impaired ureagenesis, leading to neurotoxic accumulation of ammonia and brain glutamine. Treatment includes dietary protein restriction and oral "ammonia scavengers." These scavengers chemically combine with glutamine and glycine to yield excretable products, creating an alternate pathway of waste nitrogen disposal. The amino acid transporter SLC6A19 is responsible for >95% of absorption and reabsorption of free neutral amino acids in the small intestine and kidney, respectively. Genetic SLC6A19 deficiency causes massive neutral aminoaciduria but is typically benign. We hypothesized that inhibiting SLC6A19 would open a novel and effective alternate pathway of waste nitrogen disposal. To test this, we crossed SLC6A19 knockout (KO) mice with spfash mice, a model of ornithine transcarbamylase (OTC) deficiency. Loss of SLC6A19 in spfash mice normalized plasma ammonia and brain glutamine and increased median survival in response to a high protein diet from 7 to 97 days. While induced excretion of amino acid nitrogen is likely the primary therapeutic mechanism, reduced intestinal absorption of dietary free amino acids, and decreased muscle protein turnover due to loss of SLC6A19 may also play a role. In summary, the results suggest that SLC6A19 inhibition represents a promising approach to treating UCDs and related aminoacidopathies.

AAV8-mediated expression of N-acetylglucosamine-1-phosphate transferase attenuates bone loss in a mouse model of mucolipidosis II.

Mucolipidoses II and III (ML II and ML III) are lysosomal disorders in which the mannose 6-phosphate recognition marker is absent from lysosomal hydrolases and other glycoproteins due to mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Both disorders are caused by the same gene, but ML II represents the more severe phenotype. Bone manifestations of ML II include hip dysplasia, scoliosis, rickets and osteogenesis imperfecta. In this study, we sought to determine whether a recombinant adeno-associated viral vector (AAV2/8-GNPTAB) could confer high and prolonged gene expression of GNPTAB and thereby influence the pathology in the cartilage and bone tissue of a GNPTAB knock out (KO) mouse model. The results demonstrated significant increases in bone mineral density and content in AAV2/8-GNPTAB-treated as compared to non-treated KO mice. We also showed that IL-6 (interleukin-6) expression in articular cartilage was reduced in AAV2/8-GNPTAB treated ML II mice. Together, these data suggest that AAV-mediated expression of GNPTAB in ML II mice can attenuate bone loss via inhibition of IL-6 production. This study emphasizes the value of the MLII KO mouse to recapitulate the clinical manifestations of the disease and highlights its amenability to therapy.

Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice.

Enzyme replacement therapy is often hampered by the rapid clearance and degradation of the administered enzyme, limiting its efficacy and requiring frequent dosing. Encapsulation of therapeutic molecules into red blood cells (RBCs) is a clinically proven approach to improve the pharmacokinetics and efficacy of biologics and small molecule drugs. Here we evaluated the ability of RBCs encapsulated with phenylalanine hydroxylase (PAH) to metabolize phenylalanine (Phe) from the blood and confer sustained enzymatic activity in the circulation. Significant quantities of PAH were successfully encapsulated within murine RBCs (PAH-RBCs) with minimal loss of endogenous hemoglobin. While intravenously administered free PAH enzyme was rapidly eliminated from the blood within a few hours, PAH-RBCs persisted in the circulation for at least 10days. A single injection of PAH-RBCs was able to decrease Phe levels by nearly 80% in normal mice. These results demonstrate the ability of enzyme-loaded RBCs to metabolize circulating amino acids and highlight the potential to treat disorders of amino acid metabolism.

Inhibiting glycosphingolipid synthesis ameliorates hepatic steatosis in obese mice.

UNLABELLED: Steatosis in the liver is a common feature of obesity and type 2 diabetes and the precursor to the development of nonalcoholic steatohepatitis (NASH), cirrhosis, and liver failure. It has been shown previously that inhibiting glycosphingolipid (GSL) synthesis increases insulin sensitivity and lowers glucose levels in diabetic rodent models. Here we demonstrate that inhibiting GSL synthesis in ob/ob mice not only improved glucose homeostasis but also markedly reduced the development of hepatic steatosis. The ob/ob mice were treated for 7 weeks with a specific inhibitor of glucosylceramide synthase, the initial enzyme involved in the synthesis of GSLs. Besides lowering glucose and hemoglobin A1c (HbA1c) levels, drug treatment also significantly reduced the liver/body weight ratio, decreased the accumulation of triglycerides, and improved several markers of liver pathology. Drug treatment reduced liver glucosylceramide (GL1) levels in the ob/ob mouse. Treatment also reduced the expression of several genes associated with hepatic steatosis, including those involved in lipogenesis, gluconeogenesis, and inflammation. In addition, inhibiting GSL synthesis in diet-induced obese mice both prevented the development of steatosis and partially reversed preexisting steatosis. CONCLUSION: These data indicate that inhibiting GSL synthesis ameliorates the liver pathology associated with obesity and diabetes, and may represent a novel strategy for treating fatty liver disease and NASH.

Inhibiting neutral amino acid transport for the treatment of phenylketonuria.

The neuropathological effects of phenylketonuria (PKU) stem from the inability of the body to metabolize excess phenylalanine (Phe), resulting in accumulation of Phe in the blood and brain. Since the kidney normally reabsorbs circulating amino acids with high efficiency, we hypothesized that preventing the renal uptake of Phe might provide a disposal pathway that could lower systemic Phe levels. SLC6A19 is a neutral amino acid transporter responsible for absorption of the majority of free Phe in the small intestine and reuptake of Phe by renal proximal tubule cells. Transgenic KO mice lacking SLC6A19 have elevated levels of Phe and other amino acids in their urine but are otherwise healthy. Here, we crossed the Pahenu2 mouse model of PKU with the Slc6a19-KO mouse. These mutant/KO mice exhibited abundant excretion of Phe in the urine and an approximately 70% decrease in plasma Phe levels. Importantly, brain Phe levels were decreased by 50%, and the levels of key neurotransmitters were increased in the mutant/KO mice. In addition, a deficit in spatial working memory and markers of neuropathology were corrected. Finally, treatment of Pahenu2 mice with Slc6a19 antisense oligonucleotides lowered Phe levels. The results suggest that inhibition of SLC6A19 may represent a novel approach for the treatment of PKU and related aminoacidopathies.

Increased hepatic insulin action in diet-induced obese mice following inhibition of glucosylceramide synthase.

BACKGROUND: Obesity is characterized by the accumulation of fat in the liver and other tissues, leading to insulin resistance. We have previously shown that a specific inhibitor of glucosylceramide synthase, which inhibits the initial step in the synthesis of glycosphingolipids (GSLs), improved glucose metabolism and decreased hepatic steatosis in both ob/ob and diet-induced obese (DIO) mice. Here we have determined in the DIO mouse model the efficacy of a related small molecule compound, Genz-112638, which is currently being evaluated clinically for the treatment of Gaucher disease, a lysosomal storage disorder. METHODOLOGY/PRINCIPAL FINDINGS: DIO mice were treated with the Genz-112638 for 12 to 16 weeks by daily oral gavage. Genz-112638 lowered HbA1c levels and increased glucose tolerance. Whole body adiposity was not affected in normal mice, but decreased in drug-treated obese mice. Drug treatment also significantly lowered liver triglyceride levels and reduced the development of hepatic steatosis. We performed hyperinsulinemic-euglycemic clamps on the DIO mice treated with Genz-112638 and showed that insulin-mediated suppression of hepatic glucose production increased significantly compared to the placebo treated mice, indicating a marked improvement in hepatic insulin sensitivity. CONCLUSIONS/SIGNIFICANCE: These results indicate that GSL inhibition in obese mice primarily results in an increase in insulin action in the liver, and suggests that GSLs may have an important role in hepatic insulin resistance in conditions of obesity.

Inhibiting glycosphingolipid synthesis improves glycemic control and insulin sensitivity in animal models of type 2 diabetes.

Previous reports have shown that glycosphingolipids can modulate the activity of the insulin receptor, and studies in transgenic mice suggest a link between altered levels of various gangliosides and the development of insulin resistance. Here, we show that an inhibitor of glycosphingolipid synthesis can improve glucose control and increase insulin sensitivity in two different diabetic animal models. In the Zucker diabetic fatty rat, the glucosylceramide synthase inhibitor (1R,2R)-nonanoic acid[2-(2',3'-dihydro-benzo [1, 4] dioxin-6'-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]- amide-l-tartaric acid salt (Genz-123346) lowered glucose and A1C levels and improved glucose tolerance. Drug treatment also prevented the loss of pancreatic beta-cell function normally observed in the Zucker diabetic fatty rat and preserved the ability of the animals to secrete insulin. In the diet-induced obese mouse, treatment with Genz-123346 normalized A1C levels and improved glucose tolerance. Analysis of the phosphorylation state of the insulin receptor and downstream effectors showed increased insulin signaling in the muscles of the treated Zucker diabetic fatty rats and diet-induced obese mice. These results suggest that inhibiting glycosphingolipid synthesis can significantly improve insulin sensitivity and glucose homeostasis and may therefore represent a novel therapeutic approach for the treatment of type 2 diabetes.

Transient siRNA-mediated attenuation of liver expression from an alpha-galactosidase A plasmid reduces subsequent humoral immune responses to the transgene product in mice.

Hepatocytes are an effective depot for protein production from gene therapy vectors. However, when gene transfer vectors or their delivery induces hepatic inflammation, adaptive immune responses against the transgene product can ensue. In BALB/c mice, hydrodynamic delivery of a CMV-driven plasmid DNA (pDNA) bearing human alpha-galactosidase A (alphagal) to the liver generated antibodies against alphagal. This humoral immune response was more robust in a transgenic knockout for alphagal, the Fabry mouse. The antibody response could be attenuated in both mouse strains by using a promoter more restricted to hepatocytes. In an attempt to reduce further the humoral responses to alphagal, expression from the transgene was attenuated by using siRNA during the period of initial delivery-associated liver inflammation. In both mouse models and with both promoters, codelivering an alphagal siRNA resulted in a 2 log decrease in initial expression that then increased over the next few weeks to levels generated by the pDNA alone. This strategy led to both attenuated antibodies and an immune status approximating "tolerance" to alphagal. Importantly, in the Fabry mouse, an alphagal siRNA together with a hepatocyte-restricted promoter gave minimal anti-alphagal antibodies and profound tolerance, suggesting that such an approach might have clinical utility for genetic diseases.

CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo.

Systemic delivery of cationic lipid-plasmid DNA (pDNA) complexes induces an acute inflammatory response with adverse hematologic changes and liver damage. Immunostimulatory CpG motifs in the pDNA are known to contribute substantially to this response. Here we constructed a pDNA vector (pGZB) that has been depleted of 80% of the CpG motifs present in the original vector. Compared with the unmodified vector, systemic administration of pGZB induced considerably fewer changes in blood parameters, reduced levels of inflammatory cytokines, and decreased liver damage. Despite the extensive sequence modifications within pGZB, there were still robust levels of transgene expression. Furthermore, in contrast to the transient expression observed from the unmodified vector, we observed sustained or increasing expression for up to 49 days from pGZB in the lung and liver of immunocompetent BALB/c mice. Studies adding CpG motifs in trans or in cis indicate that the reduced CpG content of pGZB was the major determinant for its persistent expression. This combination of decreased toxicity and sustained expression suggests that CpG-depleted pDNA vectors can greatly improve the safety and efficacy of synthetic gene delivery systems.

Partial correction of the alpha-galactosidase A deficiency and reduction of glycolipid storage in Fabry mice using synthetic vectors.

BACKGROUND: Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A, leading to an accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in most tissues of the body. The goal of this study was to determine if systemic delivery of a nonviral vector could correct the enzyme deficiency and reduce the levels of GL-3 in different tissues of a transgenic knockout mouse model of the disease. METHODS: Cationic lipid was complexed with a CpG-depleted plasmid DNA vector and then injected intravenously into Fabry mice. The levels of alpha-galactosidase A and GL-3 in different tissues were assayed at various time points after injection. RESULTS: Expression of alpha-galactosidase A was detected in the different tissues of Fabry mice for up to 3 months after complex administration, but resulted in minimal reductions in GL-3 levels. However, the use of the anti-inflammatory drug dexamethasone and multiple dosing increased alpha-galactosidase A expression and resulted in significant reductions of GL-3 in all the organs with the exception of the kidney. In addition, injecting complex into young Fabry mice partially prevented the normal accumulation of GL-3 in the heart, lung, and liver. CONCLUSIONS: Systemic delivery of a cationic lipid-pDNA complex partially corrected the enzyme deficiency and reduced glycolipid storage in a mouse model of Fabry disease. The results are one of the few demonstrations of long-term efficacy in a genetic disease model using nonviral vectors. However, substantial improvements in expression, especially in critical organs such as the kidney, are required before these vectors can become a viable approach to treat Fabry disease and other lysosomal storage disorders.