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.

Comparison of Microflow and Analytical Flow Liquid Chromatography Coupled to Mass Spectrometry Global Metabolomics Methods Using a Urea Cycle Disorder Mouse Model.

Microscale-based separations are increasingly being applied in the field of metabolomics for the analysis of small-molecule metabolites. These methods have the potential to provide improved sensitivity, less solvent waste, and reduced sample-size requirements. Ion-pair free microflow-based global metabolomics methods, which we recently reported, were further compared to analytical flow ion-pairing reagent containing methods using a sample set from a urea cycle disorder (UCD) mouse model. Mouse urine and brain homogenate samples representing healthy, diseased, and disease-treated animals were analyzed by both methods. Data processing was performed using univariate and multivariate techniques followed by analyte trend analysis. The microflow methods performed comparably to the analytical flow ion-pairing methods with the ability to separate the three sample groups when analyzed by partial least-squares analysis. The number of detected metabolic features present after each data processing step was similar between the microflow-based methods and the ion-pairing methods in the negative ionization mode. The observed analyte trend and coverage of known UCD biomarkers were the same for both evaluated approaches. The 12.5-fold reduction in sample injection volume required for the microflow-based separations highlights the potential of this method to support studies with sample-size limitations.

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.

Gene therapy for lysosomal storage disorders.

Lysosomal storage diseases (LSDs) are a group of single-gene disorders that have proven to be highly informative in revealing the merits of gene transfer as a technology platform. Over the past several years considerable progress has been made in delivering therapeutic genes to peripheral tissues as well as the central nervous system. The current leading vectors for direct genetic modification of target cells in vivo are derived from adeno-associated viruses (AAV) and lentiviruses. These vectors are capable of conferring widespread, robust, and sustained expression of a given gene in several mouse models of LSDs. Here we review recent progress using recombinant AAV and lentiviruses to treat various LSDs and the remaining challenges to translate the results in mice to human patients.

Adeno-associated virus-mediated expression of acid sphingomyelinase decreases atherosclerotic lesion formation in apolipoprotein E(-/-) mice.

BACKGROUND: The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis. METHODS: Apolipoprotein E deficient (ApoE(-/-) ) mice were injected intravenously with a recombinant adeno-associated virus (AAV8-ASM) that constitutively expressed high levels of human ASM in liver and plasma. RESULTS: Plasma sphingomyelin levels were reduced at early but not later time points after the administration of AAV8-ASM despite persistently elevated circulating ASM. No change in serum lipoprotein levels was observed. Thirteen or 17 weeks after the administration of AAV8-ASM, the amount of plaque formation in the aortic sinus was comparable to that of mice treated with a control AAV. CONCLUSIONS: Unexpectedly, the lesion area of the entire aorta was reduced significantly in the AAV8-ASM virus-treated group. Hepatic expression and secretion of ASM into the circulation did not accelerate or exacerbate, but rather decreased, lesion formation in ApoE(-/-) mice. Thus, plasma ASM activity does not appear to be rate limiting for plaque formation during atherogenesis.

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.

Inhibition of osteoclastogenesis by prolyl hydroxylase inhibitor dimethyloxallyl glycine.

Studies examining the effects of hypoxia upon osteoclast biology have consistently revealed a stimulatory effect; both osteoclast differentiation and resorption activity have been shown to be enhanced in the presence of hypoxia. In the present study we examined the effects of the hypoxia mimetics dimethyloxallyl glycine (DMOG) and desferrioxamine (DFO) upon osteoclastogenesis. In contrast to hypoxia, our studies revealed a dose-dependent inhibition of osteoclast formation from macrophages treated with DMOG and DFO. Moreover, expression of a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha) did not enhance osteoclastogenesis and actually attenuated the differentiation process. DMOG did not affect cell viability or receptor activator of nuclear factor kappaB ligand (RANKL)-dependent phosphorylation of mitogen-activated protein (MAP) kinases. However, RANKL-dependent transcription of tartrate-resistant acid phosphatase (TRAP) was reduced in the presence of DMOG. Additionally, DMOG promoted transcription of the pro-apoptotic mediator B-Nip3. These studies suggest that a hypoxia-responsive factor other than HIF-1alpha is necessary for enhancing the formation of osteoclasts in hypoxic settings.

Systemic Insulin-like growth factor-1 reverses hypoalgesia and improves mobility in a mouse model of diabetic peripheral neuropathy.

Peripheral neuropathy is a particularly debilitating complication of both type 1 and type 2 diabetes characterized by sensory and motor neuron damage and decreased circulating levels of insulin-like growth factor 1 (IGF-1). Quite often, an early hyperalgesia is followed by hypoalgesia and muscle weakness. Hypoalgesia can lead to significant morbidity for which there is no current treatment. Hyperglycemic, streptozotocin (STZ)-induced rodent models reproduce these symptoms. We investigated whether increasing systemic IGF-1 could improve neuronal function in hyper- and hypoalgesic STZ-treated mice. Increased circulating levels of IGF-1 were achieved by delivering a plasmid or adeno-associated viral (AAV) vector bearing mouse IGF-1 to the liver. Treating mice in the hyperalgesia stage prevented later hypoalgesia. Treating mice in the hypoalgesia stage reversed existing hypoalgesia. This latter effect could be seen by merely restoring IGF-1 serum levels to normalcy, which was possible to achieve by IGF-1 gene therapy or insulin treatment. Sensory nerve functional correction was seen to be correlated with attenuated Schwann cell vacuolization and demyelination in peripheral sensory nerve fibers. A further increase in serum IGF-1 levels with gene therapy also improved motor function, consistent with the observed prevention of both muscle atrophy and peripheral motor nerve fiber demyelination. These results suggest that the restoration of systemic levels of IGF-1 may prove to be a highly effective therapeutic modality for treating diabetic peripheral neuropathy.

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.

Controlling the kinetics of transgene expression by plasmid design.

While a vast array of liposomes, peptides, and molecular conjugates have been evaluated for nonviral gene transfer, the entity containing the actual gene itself is almost always a plasmid. The layout of most plasmid DNA (pDNA) vectors is usually quite simple, consisting of a promoter, transgene, polyadenylation signal, and a backbone that permits propagation of the plasmid in bacteria. Additional sequence elements and modifications can be incorporated to influence the stability of gene expression and retention of the pDNA molecule in a given tissue. This review describes the different choices that can be made when designing a pDNA vector for transient, sustained, or regulated expression. The choice of promoter is a major determinant governing the kinetics of expression, but other factors, such as CpG content and the topological form of the pDNA are also influential. Vectors can also be designed to respond to the local environment of a given cell or tissue, or engineered to respond to a small molecule drug.

DNA sequences in cationic lipid:pDNA-mediated systemic toxicities.

Systemic delivery of synthetic gene transfer vectors such as cationic lipid:plasmid DNA (pDNA) complexes elicits a range of acute physiologic responses, which in the context of therapeutic gene delivery represent dose-limiting toxicities. The most prominent responses are transient leukopenia, thrombocytopenia, serum transaminase elevations, and elevations of proinflammatory cytokines such as interferon-gamma (IFN-gamma), interleukin-12 (IL-12), and tumor necrosis factor-alpha (TNF-alpha). The unmethylated CpG sequences present in plasmid DNA have been implicated as a major cause of the robust cytokine response that follows systemic administration of cationic lipid:pDNA complexes. However, the factors causing the additional significant toxicities (leukopenia, thrombocytopenia, and serum transaminase elevations) recently shown to be associated with vector administration have not been defined. We show here that DNA sequences, such as immune stimulatory CpG sequences, play a significant role in inducing the additional acute toxicities associated with cationic lipid:pDNA complex administration. Importantly, while methylating these CpG sequences results in greatly reduced cytokine levels, this modification does not eliminate their ability to generate the other systemic toxicities. Examples of non-CpG DNA sequences that induce distinct toxicity profiles when administered systemically in the form of cationic lipid:DNA complexes are also identified. Taken together, these results imply that specific DNA sequences are responsible for a significant portion of the systemic toxicities observed after administration of cationic lipid:pDNA complexes.

Correction of the nonlinear dose response improves the viability of adenoviral vectors for gene therapy of Fabry disease.

Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-alpha gal encoding human alpha-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-alpha gal by 10-fold (from 10(11) to 10(10) particles/mouse) resulted in a greater than 200-fold loss in transgene expression. In Fabry mice, the reduced expression of alpha-galactosidase A, using the lower dose of Ad2/CMVHI-alpha gal, was associated with less than optimal clearance of the accumulated glycosphingolipid (GL-3) from the affected lysosomes. It was determined that this lack of linearity in dose response was not due to an inability to deliver the recombinant viral vectors to the liver but rather to sequestration, at least in part, of the viral vectors by the Kupffer cells. This lack of correlation between dose and expression levels could be obviated by supplementing the low dose of Ad2/CMVHI-alpha gal with an unrelated adenoviral vector or by depleting the Kupffer cells before administration of Ad2/CMVHI-alpha gal. Prior removal of the Kupffer cells, using clodronate liposomes, facilitated the use of a 100-fold lower dose of Ad2/CMVHI-alpha gal (10(9) particles/mouse) to effect the nearly complete clearance of GL-3 from the affected organs of Fabry mice. These results suggest that practical strategies that minimize the interaction between the recombinant adenoviral vectors and the reticuloendothelial system (RES) may improve the therapeutic window of this vector system. In this regard, we showed that pretreatment of mice with gamma globulins also resulted in significantly enhanced adenovirus-mediated transduction and expression of alpha-galactosidase A in the liver.

Recipient intramuscular cotransfection of naked plasmid transforming growth factor beta1 and interleukin 10 ameliorates lung graft ischemia-reperfusion injury.

OBJECTIVE: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in lung graft ischemia-reperfusion injury. In this study we analyzed whether recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 would result in amelioration of lung graft ischemia-reperfusion injury. METHODS: Forty-eight hours before transplantation, 6 groups (n = 6) of F344 rats received intramuscular injection of naked plasmid encoding chloramphenicol acetyltransferase, chloramphenicol acetyltransferase plus beta-galactosidase, transforming growth factor beta(1), interleukin 10, or transforming growth factor beta(1) plus interleukin 10 or were not treated. Donor lungs were flushed and stored for 18 hours at 4 degrees C before transplantation. Twenty-four hours later, grafts were assessed immediately before the animals were killed. Arterial oxygenation, wet/dry ratio, myeloperoxidase, and proinflammatory cytokines (interleukin 1, tumor necrosis factor alpha, interferon gamma, and interleukin 2) were measured, and immunohistochemistry was performed. RESULTS: For lung graft function, the arterial oxygenation was considerably higher in the cotransfected group receiving transforming growth factor beta(1) plus interleukin 10 compared with that in all other groups (P < or =.03). The wet/dry ratio, reflecting lung edema, was reduced in the cotransfected group compared with that in control animals (nontreated, P <.02; chloramphenicol acetyltransferase, P <.03; chloramphenicol acetyltransferase plus beta-galactosidase, P <.01). Myeloperoxidase, which measures neutrophil sequestration, was also reduced with cotransfection compared with that seen in control animals (P < or =.03). All proinflammatory cytokines were decreased in the cotransfected group compared with those in all other groups (interleukin 1beta, P <.04; tumor necrosis factor alpha, P <.002; interferon gamma, P <.0001; interleukin 2, P <.03). These results indicate that cotransfection provides a synergistic benefit in graft function versus either cytokine alone, neutrophil sequestration, or inflammatory cytokine expression. Immunohistochemistry showed positive staining of transforming growth factor beta(1) plus interleukin 10 in type I and II pneumocytes and localized edema fluid. CONCLUSIONS: Recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 provides a synergistic effect in ameliorating lung reperfusion injury after prolonged ischemia.

Reducing the immunostimulatory activity of CpG-containing plasmid DNA vectors for non-viral gene therapy.

The mammalian innate immune system has the ability to recognise and direct a response against incoming foreign DNA. The primary signal that triggers this response is unmethylated CpG motifs present in the DNA sequence of various disease-causing pathogens. These motifs are rare in vertebrate DNA, but abundant in bacterial and some viral DNAs. Because gene therapy generally involves the delivery of DNA from either plasmids of bacterial origin or recombinant viruses, an acute inflammatory response of variable severity inevitably results. The response is most serious for non-viral gene delivery vectors composed of cationic lipid-DNA complexes, producing adverse effects at lower doses and lethality at higher doses of complex. This review examines the role of immunostimulatory CpG motifs in the acute inflammatory response to non-viral gene therapy vectors. Strategies to neutralise or eliminate CpG motifs within plasmid DNA vectors, and the existing limitations of CpG reduction on improving the safety profile of non-viral vectors, will be discussed.

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.

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression.

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (>or=56 d) in vivo transgene expression in the absence of lung inflammation.

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.

Toxicity of cationic lipid-DNA complexes.

As with any conventional drug, the body's response to cationic lipid-DNA complexes is highly dependent on both the dose administered and the route of delivery. At relatively low doses there is little to no effect on organ function or tissue architecture, but at higher doses, acute inflammation and tissue damage can occur that is sometimes quite profound. Of the two most common routes of delivery, intravenous (i.v.) or intrapulmonary, i.v. administration tends to cause more severe adverse effects and can be lethal at higher doses of complex. Both routes activate an innate immune response that includes the induction of proinflammatory cytokines and immune cell activation, a major portion of which has been attributed to the presence of immunostimulatory CpG motifs within the plasmid DNA vector. Removing CpGs from the plasmid vector reduces several, but not all of the acute inflammatory responses to cationic lipid-DNA complexes. Therefore, other strategies are required to improve the therapeutic potential of these vectors, such as transient immune suppression, aerosolization of the complex, and novel formulations that have increased efficiency of transduction and decreased interaction with immune cells.