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Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA−/− mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA−/− mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA−/− mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA−/− mice, phosphorylase activity is suppressed in the GAA−/− mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity. [ABSTRACT FROM AUTHOR]

Transcriptional control of transgene expression is crucial to successful gene therapy, yet few promoter/enhancer combinations have been tested in clinical trials. We created a simple, desktop computer database and populated it with promoter sequences from publicly available sources. From this database, we rapidly identified novel CpG-free promoter sequences suitable for use in non-inflammatory, non-viral in vivo gene transfer. In a simple model of lung gene transfer, five of the six promoter elements selected, chosen without prior knowledge of their transcriptional activities, directed significant transgene expression. Each of the five novel promoters directed transgene expression for at least 14 days post-delivery, greatly exceeding the duration achieved with the commonly used CpG-rich viral enhancer/promoters. Novel promoter activity was also evaluated in a more clinically relevant model of aerosol-mediated lung gene transfer and in the liver following delivery via high-pressure tail vein injection. In each case, the novel CpG-free promoters exhibited higher and/or more sustained transgene expression than commonly used CpG-rich enhancer/promoter sequences. This study demonstrates that novel CpG-free promoters can be readily identified and that they can direct significant levels of transgene expression. Furthermore, the database search criteria can be quickly adjusted to identify other novel promoter elements for a variety of transgene expression applications. [ABSTRACT FROM AUTHOR]

Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type ofthe disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases. [ABSTRACT FROM AUTHOR]

Background The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. Methods We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug). Results TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls. Conclusions Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects. [ABSTRACT FROM AUTHOR]

Fabry disease is an X-linked glycosphingolipid storage disorder caused by a deficiency in the activity of the lysosomal hydrolase α-galactosidase A (α-gal). This deficiency results in accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in lysosomes. Endothelial cell storage of GL-3 frequently leads to kidney dysfunction, cardiac and cerebrovascular disease. The current treatment for Fabry disease is through infusions of recombinant α-gal (enzyme-replacement therapy; ERT). Although ERT can markedly reduce the lysosomal burden of GL-3 in endothelial cells, variability is seen in the clearance from several other cell types. This suggests that alternative and adjuvant therapies may be desirable. Use of glucosylceramide synthase inhibitors to abate the biosynthesis of glycosphingolipids (substrate reduction therapy, SRT) has been shown to be effective at reducing substrate levels in the related glycosphingolipidosis, Gaucher disease. Here, we show that such an inhibitor (eliglustat tartrate, Genz-112638) was effective at lowering GL-3 accumulation in a mouse model of Fabry disease. Relative efficacy of SRT and ERT at reducing GL-3 levels in Fabry mouse tissues differed with SRT being more effective in the kidney, and ERT more efficacious in the heart and liver. Combination therapy with ERT and SRT provided the most complete clearance of GL-3 from all the tissues. Furthermore, treatment normalized urine volume and uromodulin levels and significantly delayed the loss of a nociceptive response. The differential efficacies of SRT and ERT in the different tissues indicate that the combination approach is both additive and complementary suggesting the possibility of an improved therapeutic paradigm in the management of Fabry disease. [ABSTRACT FROM AUTHOR]

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid β-glucosidase), with consequent cellular accumulation of glucosylceramide (GL-1). The disease is managed by intravenous administrations of recombinant glucocerebrosidase (imiglucerase), although symptomatic patients with mild to moderate type 1 Gaucher disease for whom enzyme replacement therapy (ERT) is not an option may also be treated by substrate reduction therapy (SRT) with miglustat. To determine whether the sequential use of both ERT and SRT may provide additional benefits, we compared the relative pharmacodynamic efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V/null). As expected, ERT with recombinant glucocerebrosidase was effective in reducing the burden of GL-1 storage in the liver, spleen, and lung of 3-month-old Gaucher mice. SRT using a novel inhibitor of glucosylceramide synthase (Genz-112638) was also effective, albeit to a lesser degree than ERT. Animals administered recombinant glucocerebrosidase and then Genz-112638 showed the lowest levels of GL-1 in all the visceral organs and a reduced number of Gaucher cells in the liver. This was likely because the additional deployment of SRT following enzyme therapy slowed the rate of reaccumulation of GL-1 in the affected organs. Hence, in patients whose disease has been stabilized by intravenously administered recombinant glucocerebrosidase, orally administered SRT with Genz-112638 could potentially be used as a convenient maintenance therapy. In patients naïve to treatment, ERT followed by SRT could potentially accelerate clearance of the offending substrate. [ABSTRACT FROM AUTHOR]

Background When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs). Methods We have compared laser microdissection, pronase digestion and nasal brushing for: (i) the ability to enrich RECs from the wild-type mouse nose and (ii) the length of time to perform the procedure. Using TaqMan, we subsequently assessed gene transfer in enriched RECs after nasal perfusion of GL67A/pCF1-CFTR complexes in a CF mouse model. Results Laser microdissection successfully isolated RECs; however, time-consuming sample preparation made this technique unsuitable for high-throughput studies. Pronase digestion was sufficiently rapid but only yielded 19% (range = 13%) RECs ( n = 6). The nasal brushing method was superior, yielding 92% (range = 15%) RECs ( n = 8) and was equally effective in CF knockout mice (91%, range = 14%, n = 10). Importantly, gene transfer was detectable in brushed RECs from 70% of perfused mice and the number of vector-specific transcripts was comparable to 3.5% of endogenous wild-type Cftr levels. Conclusions Isolation of RECs by brushing allows accurate assessment of GTA transfection efficiency in an experimental system that is relevant for CF gene therapy. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Surfactant protein (SP)-D plays an important role in host defense and pulmonary surfactant homeostasis. In SP-D-deficient (Sftpd-/-) mice, the abnormal large surfactant forms seen at the ultrastructural level are taken up inefficiently by type II cells, resulting in an over threefold increase in the surfactant pool size. The mechanisms by which SP-D influences surfactant ultrastructure are unknown. We hypothesized that SP-D binds to surfactant immediately after being secreted and influences surfactant ultrastructure conversion. In newborn and adult sheep lungs, immunogold-labeled SP-D was associated with both lamellated membranous lipid structures of newly secreted surfactant and with small aggregate surfactant but not with tubular myelin. Since SP-D preferentially binds to phosphatidylinositol (PI) in vitro, the postnatal changes in PI were assessed. PI content in the bronchoalveolar lavage fluid increased after birth and peaked at 2-5 days of age, a time of rapid conversion of surfactant forms that is associated with the peak of surfactant lipid pool size. SP-D selectively interacted with PI-rich liposomes in vitro, causing their lysis. Similarly, the abnormal surfactant ultrastructure in Sftpd-/- mice was corrected by the addition of SP-D or melittin, and both peptides caused lysis of lipid vesicles. The normal conversion of surfactant ultrastructure requires SP-D that preferentially interacts with PI-rich, newly secreted surfactant, causing lysis of surfactant lipid membranes, converting the lipid forms into smaller surfactant lamellated structures that are critical for surfactant uptake by type II cells and normal surfactant homeostasis. SP-D regulates the dramatic decreases in the surfactant pool size that occurs in the newborn period. [ABSTRACT FROM AUTHOR]

Background: Surfactant protein D (SP-D) plays an important role in innate defense against influenza A viruses (IAVs) and other pathogens. Methods: We tested antiviral activities of recombinant human SP-D against a panel of IAV strains that vary in glycosylation sites on their hemagglutinin (HA). For these experiments a recombinant version of human SP-D of the Met11, Ala160 genotype was used after it was characterized biochemically and structurally. Results: Oligosaccharides at amino acid 165 on the HA in the H3N2 subtype and 104 in the H1N1 subtype are absent in collectin-resistant strains developed in vitro and are important for mediating antiviral activity of SP-D; however, other glycans on the HA of these viral subtypes also are involved in inhibition by SP-D. H3N2 strains obtained shortly after introduction into the human population were largely resistant to SP-D, despite having the glycan at 165. H3N2 strains have become steadily more sensitive to SP-D over time in the human population, in association with addition of other glycans to the head region of the HA. In contrast, H1N1 strains were most sensitive in the 1970s-1980s and more recent strains have become less sensitive, despite retaining the glycan at 104. Two H5N1 strains were also resistant to inhibition by SP-D. By comparing sites of glycan attachment on sensitive vs. resistant strains, specific glycan sites on the head domain of the HA are implicated as important for inhibition by SP-D. Molecular modeling of the glycan attachment sites on HA and the carbohydrate recognition domain of SPD are consistent with these observations. Conclusion: Inhibition by SP-D correlates with presence of several glycan attachment sites on the HA. Pandemic and avian strains appear to lack susceptibility to SP-D and this could be a contributory factor to their virulence. [ABSTRACT FROM AUTHOR]

The article reports on the results of a study of the effect of exogenous recombinant human surfactant protein-D (rhSP-D) on protection of the adult mouse lung from lipopolysaccharide (LPS)-induced and lipoteichoic acid (LTA)-induced injury. A description of study methods is presented. The study concluded that intratracheal rhSP-D inhibited inflammation induced by intratracheal LPS and LTA instillation in the lung.

Rationale: The susceptibility of neonates to pulmonary and systemic infection has been associated with the immaturity of both lung structure and the immune system. Surfactant protein (SP) D is a member of the collectin family of innate immune molecules that plays an important role in innate host defense of the lung.Objectives: We tested whether treatment with recombinant human SP-D influenced the response of the lung and systemic circulation to intratracheally administered Escherichia coli lipopolysaccharides.Methods: After intratracheal lipopolysaccharide instillation, preterm newborn lambs were treated with surfactant and ventilated for 5 h.Measurement: Survival rate, physiologic lung function, lung and systemic inflammation, and endotoxin level in plasma were evaluated.Main Results: In control lambs, intratracheal lipopolysaccharides caused septic shock and death associated with increased endotoxin in plasma. In contrast, all lambs treated with recombinant human SP-D were physiologically stable and survived. Leakage of lipopolysaccharides from the lungs to the systemic circulation was prevented by intratracheal recombinant human SP-D. Recombinant human SP-D prevented systemic inflammation and decreased the expression of IL-1beta, IL-8, and IL-6 in the spleen and liver. Likewise, recombinant human SP-D decreased IL-1beta and IL-6 in the lung and IL-8 in the plasma. Recombinant human SP-D did not alter pulmonary mechanics following endotoxin exposure. Recombinant human SP-D was readily detected in the lung 5 h after intratracheal instillation.Conclusions: Intratracheal recombinant human SP-D prevented shock caused by endotoxin released from the lung during ventilation in the premature newborn. [ABSTRACT FROM AUTHOR]

Background: The cationic lipid Genzyme lipid (GL) 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. Methods: Anti-lacZ and ENaC (epithelial sodium channel) siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. Results: In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to β-galactosidase reduced βgal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. Conclusion: This study suggests that although siRNAs and asODNs can be developed to inhibit gene expression in culture systems and certain organs in vivo, barriers to nucleic acid transfer in airway epithelial cells seen with large DNA molecules may also affect the efficiency of in vivo uptake of small nucleic acid molecules. [ABSTRACT FROM AUTHOR]

Intravenous endostatin gene transfection results in tumor suppression in a murine pulmonary metastasis model. We transfected the endostatin gene at different times, in order to achieve an optimal protective effect. pST2-Endo encoding murine endostatin was injected in a complex with cationic lipid. Pulmonary metastases were caused by intravenous injection of murine fibrosarcoma cells. Mice were observed for 14 days following fibrosarcoma cell inoculation (FSI). In the study groups, the animals were transfected with pST2-Endo at three different times: 2 days before and 3 and 7 days after FSI. In the group transfected with pST2-Endo 2 days before FSI, the weights of the lungs and tumor-occupied area ratio were significantly less than in the other groups. Significant inhibition of tumor neovascularization was documented by means of CD31 immunohistochemistry. The effect of repeated endostatin transfection on survival after FSI was determined. Animals repeatedly transfected with the endostatin gene survived significantly longer than the groups treated with a single endostatin gene transfection. A stable endostatin-expressing fibrosarcoma transfectant was created and tested for migration and invasion. Compared with controls, endostatin expression reduced migration and invasion by 15%. It is concluded that endostation gene transfection before FSI and repeated transfection thereafter results in significant tumor suppression. [ABSTRACT FROM AUTHOR]

Background 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. Methods Cationic lipid was complexed with a CpG-depleted plasmid DNA vector and then injected intravenously into Fabry mice. The levels of α-galactosidase A and GL-3 in different tissues were assayed at various time points after injection. Results Expression of α-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 α-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. Copyright © 2003 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Editorial. Investigates the feasibility of treating lung cancer with alternative gene therapies, such as the cationic lipid-mediated gene delivery system. Leading cause of cancer in the United States; Increase in the incidence of lung cancer in males; Use of clinical gene therapy trials with viral vectors containing p53 in the treatment of localized tumors; Advantages and disadvantages of using cationic lipid.