Malgorzata Przybylska

Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes.

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with SssI-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible cause- and-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.

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 β-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.

Inhibiting glycosphingolipid synthesis ameliorates hepatic steatosis in obese mice

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. (HEPATOLOGY 2009.)

Partial correction of the α‐galactosidase A deficiency and reduction of glycolipid storage in Fabry mice using synthetic vectors

Fabry disease is a recessive, X‐linked disorder caused by a deficiency of the lysosomal enzyme α‐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.

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.

Increased Duration of Transgene Expression in the Lung with Plasmid DNA Vectors Harboring Adenovirus E4 Open Reading Frame 3

For gene therapy to be effective in the treatment of chronic diseases, plasmid DNA (pDNA) vectors that provide persistent expression of therapeutic levels of the transgene product are desirable. Studies in the lung with adenovirus vectors showed that products of the adenovirus E4 region can act both in cis and in trans to increase the duration of expression when transcription of the transgene was under the control of the human cytomegalovirus (CMV) promoter. To determine if these E4-encoded proteins could also effect greater persistence of expression from a nonviral vector, a complex composed of cationic lipid GL-67, a CMV promoter plasmid (pCF1-CAT), and an E4-containing adenovirus vector (Ad2/betagal-4) was instilled into the lungs of BALB/c nu/nu mice. Significant increases in the duration of transgene expression were observed for up to 10 weeks postinstillation compared with expression from mice instilled with control complexes containing an adenovirus vector deleted of most of E4 (Ad2/betagal-2). This effect could also be observed in immunodeficient NIH-rnu rats as well as in immunocompetent BALB/c mice. Studies with CMV promoter mutants indicated that a region proximal to the promoter was necessary for the E4-mediated increase in longevity of expression. In addition to the CMV promoter, a CMV enhancer-human mucin I (MUC-I) hybrid promoter also responded to these E4-encoded proteins with increased persistence of transgene expression, but a human interleukin 8 (IL-8) promoter did not. Ad2/betagal-4 could be replaced by a pDNA vector expressing only the E4 region, indicating that products of the E4 region alone were sufficient in the absence of expression from the rest of the adenovirus genome. Further analysis indicated that the protein encoded by open reading frame 3 (ORF3) alone was sufficient for conferring the increase in persistence of expression. These data indicate that expression of a single protein from the adenovirus genome can significantly improve the duration of transgene expression from pDNA vectors, and increases the feasibility of using nonviral vectors for the treatment of chronic diseases.

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.

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.

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.