Neuronopathic glycosphingolipidoses are a sub-group of lysosomal storage disorders for which there are presently no effective therapies. Here, we evaluated the potential of substrate reduction therapy (SRT) using an inhibitor of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide (GL1) and related glycosphingolipids. The substrates that accumulate in Sandhoff disease (e.g., ganglioside GM2 and its nonacylated derivative, lyso-GM2) are distal to the drug target, GCS. Treatment of Sandhoff mice with a GCS inhibitor that has demonstrated CNS access (Genz-682452) reduced the accumulation of GL1 and GM2, as well as a variety of disease-associated substrates in the liver and brain. Concomitant with these effects was a significant decrease in the expression of CD68 and glycoprotein non-metastatic melanoma B protein (Gpnmb) in the brain, indicating a reduction in microgliosis in the treated mice. Moreover, using in vivo imaging, we showed that the monocytic biomarker translocator protein (TSPO), which was elevated in Sandhoff mice, was normalized following Genz-682452 treatment. These positive effects translated in turn into a delay (similar to 28 days) in loss of motor function and coordination, as measured by rotarod latency, and a significant increase in longevity (similar to 17.5%). Together, these results support the development of SRT for the treatment of gangliosidoses, particularly in patients with residual enzyme activity.

Obstructive Sleep Apnoea (OSA) is frequent in patients with type 2 diabetes. The aim of this study is to evaluate prevalence of OSA in patients with type 1 diabetes.In a cross-sectional design, all patients with type 1 diabetes attending the outpatient clinic were offered screening for OSA for one night with the ApneaLink + home-monitoring device. OSA was classified by the Apnoea–Hypopnea index (AHI; apnoeas/hypopneas per hour sleep). Symptoms of OSA were scored using the Epworth Sleepiness Score. Presence of autonomic neuropathy was evaluated by the Vagus® device.A total of 200 of 518 eligible patients with type 1 diabetes (39%) participated (68% men; age 52 ± 15 years (mean ± SD), diabetes duration 24 ± 14 years and BMI 25.3 ± 3.3 kg/m2). OSA was diagnosed in 92 patients (46% (95% CI: 40–53)). Five patients had known OSA, and OSA was newly diagnosed in 87 patients, predominantly mild OSA (60 patients (69%)).OSA was present in 32% of the patients with normal BMI, in 60% of overweight patients, and in 61% of obese patients. Patients with type 1 diabetes and OSA were largely asymptomatic and did not report more sleepiness than patients without OSA. At multivariate analysis, age, BMI and presence of nephropathy were positively associated with risk of OSA.The prevalence of asymptomatic OSA is high in a cohort of patients with type 1 diabetes. Older age, overweight, and presence of nephropathy are associated with OSA.

Certain recessively inherited diseases result from an enzyme deficiency within lysosomes. In mucopolysaccharidoses (MPS), a defect in glycosaminoglycan (GAG) degradation leads to GAG accumulation followed by progressive organ and multiple system dysfunctions. Current methods of GAG analysis used to diagnose and monitor the diseases lack sensitivity and throughput. Here we report a LC-MS method with accurate metabolite mass analysis for identifying and quantifying biomarkers for MPS type I without the need for extensive sample preparation. The method revealed 225 LC-MS features that were >1000-fold enriched in urine, plasma and tissue extracts from untreated MPS I mice compared to MPS I mice treated with iduronidase to correct the disorder. Levels of several trisaccharides were elevated >10000-fold. To validate the clinical relevance of our method, we confirmed the presence of these biomarkers in urine, plasma and cerebrospinal fluid from MPS I patients and assessed changes in their levels after treatment.

Gaucher disease (GD) is caused by a deficiency of glucocerebrosidase and the consequent lysosomal accumulation of unmetabolized glycolipid substrates. Enzyme-replacement therapy adequately manages the visceral manifestations of nonneuronopathic type-1 Gaucher patients, but not the brain disease in neuronopathic types 2 and 3 GD. Substrate reduction therapy through inhibition of glucosylceramide synthase (GCS) has also been shown to effectively treat the visceral disease. Here, we evaluated the efficacy of a novel small molecule inhibitor of GCS with central nervous system (CNS) access (Genz-682452) to treat the brain disease. Treatment of the conduritol β epoxide-induced mouse model of neuronopathic GD with Genz-682452 reduced the accumulation of liver and brain glycolipids (>70% and >20% respectively), extent of gliosis, and severity of ataxia. In the genetic 4L;C* mouse model, Genz-682452 reduced the levels of substrate in the brain by >40%, the extent of gliosis, and paresis. Importantly, Genz-682452-treated 4L;C* mice also exhibited an ~30% increase in lifespan. Together, these data indicate that an orally available antagonist of GCS that has CNS access is effective at attenuating several of the neuropathologic and behavioral manifestations associated with mouse models of neuronopathic GD. Therefore, Genz-682452 holds promise as a potential therapeutic approach for patients with type-3 GD.

Fabry disease, an X-linked glycosphingolipid storage disorder, is caused by the deficient activity of α-galactosidase A (α-Gal A). This results in the lysosomal accumulation in various cell types of its glycolipid substrates, including globotriaosylceramide (GL-3) and lysoglobotriaosylceramide (globotriaosyl lysosphingolipid, lyso-GL-3), leading to kidney, heart, and cerebrovascular disease. To complement and potentially augment the current standard of care, biweekly infusions of recombinant α-Gal A, the merits of substrate reduction therapy (SRT) by selectively inhibiting glucosylceramide synthase (GCS) were examined. Here, we report the development of a novel, orally available GCS inhibitor (Genz-682452) with pharmacological and safety profiles that have potential for treating Fabry disease. Treating Fabry mice with Genz-682452 resulted in reduced tissue levels of GL-3 and lyso-GL-3 and a delayed loss of the thermal nociceptive response. Greatest improvements were realized when the therapeutic intervention was administered to younger mice before they developed overt pathology. Importantly, as the pharmacologic profiles of α-Gal A and Genz-682452 are different, treating animals with both drugs conferred the greatest efficacy. For example, because Genz-682452, but not α-Gal A, can traverse the blood-brain barrier, levels of accumulated glycosphingolipids were reduced in the brain of Genz-682452-treated but not α-Gal A-treated mice. These results suggest that combining substrate reduction and enzyme replacement may confer both complementary and additive therapeutic benefits in Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder that is caused by a deficiency of α-galactosidase A. The disease ultimately manifests as multiple organ dysfunctions owing to excessive accumulation of globotriaosylceramide (Gb3). Among the several complications of Fabry disease, ascending thoracic aortic aneurysm is relatively common, which is classically associated with connective tissue disorders characterized by abnormal defects or deficiencies in structural proteins such as collagen and elastin. Although an elevated Gb3 level is regarded as a prerequisite for the manifestations of Fabry disease, only this excess accumulation cannot explain the pathophysiology of these complications. Recently, an increased plasma level of lyso-Gb3 was suggested as a new biomarker in Fabry disease. Therefore, the aim of this study was to assess the effects of lyso-Gb3 on the pathogenesis of thoracic ascending aortic aneurysms in Fabry disease, with a particular focus on the responses related to aortic remodeling by fibroblasts. We found that lyso-Gb3 inhibited the growth of fibroblasts, as well as their differentiation into myofibroblasts, and collagen expression. Moreover, all of these compromised responses could be attributed to the effects of lyso-Gb3 on downregulation of KCa3.1 channel expression, and these impairments could be rescued when activating the KCa3.1 channel or increasing intracellular Ca2+ concentration. This study provides new evidence that lyso-Gb3 inhibits the differentiation into myofibroblasts and collagen synthesis of fibroblasts owing to decreased Ca2+ levels by KCa3.1 channel dysfunction. These findings suggest that the KCa3.1 channel can serve as a new target to attenuate and prevent development of ascending thoracic aortic aneurysm in Fabry disease.

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disease caused by mutations in NPC1 or NPC2, the gene products of which are involved in cholesterol transport in late endosomes. NPC is characterized by an accumulation of cholesterol, sphingomyelin and glycosphingolipids in the visceral organs, primarily the liver and spleen. In the brain, there is a redistribution of unesterified cholesterol and a concomitant accumulation of glycosphingolipids. It has been suggested that reducing the aberrant lysosomal storage of glycosphingolipids in the brain by a substrate reduction therapy (SRT) approach may prove beneficial. Inhibiting glucosylceramide synthase (GCS) using the iminosugar-based inhibitor miglustat (NB-DNJ) has been reported to increase the survival of NPC mice. Here, we tested the effects of Genz-529468, a more potent iminosugar-based inhibitor of GCS, in the NPC mouse. Oral administration of Genz-529468 or NB-DNJ to NPC mice improved their motor function, reduced CNS inflammation, and increased their longevity. However, Genz-529468 offered a wider therapeutic window and better therapeutic index than NB-DNJ. Analysis of the glycolipids in the CNS of the iminosugar-treated NPC mouse revealed that the glucosylceramide (GL1) but not the ganglioside levels were highly elevated. This increase in GL1 was likely caused by the off-target inhibition of the murine non-lysosomal glucosylceramidase, Gba2. Hence, the basis for the observed effects of these inhibitors in NPC mice might be related to their inhibition of Gba2 or another unintended target rather than a result of substrate reduction.

In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding a-galactosidase A (alpha-gal). As in mice, sustained expression in monkeys attenuated antibody responses to alpha-gal. However, this effect was not robust, and sustained alpha-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.

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.

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.

In murine models of lysosomal storage diseases, sustained hepatocyte-restricted expression and secretion of the therapeutic enzyme is capable of correcting the peripheral pathology associated with the storage product. For example, systemic delivery of a recombinant AAV8 vector encoding human alpha-galactosidase A to Fabry mice resulted in complete clearance of the glycosphingolipid storage product in peripherally affected tissues when sustained serum alpha-galactosidase A levels were in excess of 1 ug/ml. In humans, transient blood levels of human FIX have been obtained after hepatocyte transduction with an AAV2 vector. The abbreviatedexpression was attributed to cytotoxic lymphocyte (CTL) recognition of viral capsid epitopes on the transduced cells. In view of these potential immune-based limitations on expression, we have asked whether we could generate prolonged circulating levels of human alpha-galactosidase A following delivery of an AAV2/8 vector encoding human alpha-galactosidase A under control of a hepatocyte-restricted promoter to immune suppressed rhesus macaques. We have compared systemic delivery using a peripheral vein approach and local delivery using a hepatic vein approach that was developed in a rabbit model. The hepatic vein approach uses a balloon catheter to isolate a lobe of the liver and block blood flow into the vena cava. We then flush the vasculature of the lobe retrograde to blood flow to reduce the concentration of any resident anti-viral antibodies and to allow the viral vector to “dwell” within the lobe for a defined period after delivery. A three month immune suppression regime was used to blunt any neutralizing immune responses to virally-transduced cells or human alpha-galactosidase A. After removing immune suppression, animals will be challenged with purified human alpha- galactosidase A to assess their degree of tolerance to this foreign protein. Although this experiment is not yet complete, we will present up-to-date expression and toxicity data comparing the two delivery techniques.

Gaucher disease is the most common of the >40 described lysosomal storage disorders. The primary manifestation of Gaucher disease is the accumulation of glucosylceramide (GL-1), predominantly in the macrophages of liver and spleen, due to a genetic defect in the lysosomal hydrolase glucocerebrosidase (GC). A mouse model of Type I Gaucher disease (D409V/null) was used to assess the efficacy of AAV8-mediated gene therapy. This mouse model exhibits only 5% of normal GC activity in visceral tissues resulting in elevated GL-1 levels and the appearance of enlarged macrophages (Gaucher cells) in liver, lung, and spleen at 3 months of age. The AAV vector (AAV8/DC172-hGC) used for these studies contained a hepatocyte-restricted promoter, DC172 (human 1-microglobulin enhancer, human 1-anti-trypsin promoter), to drive the expression of human GC. Intravenous administration of 31011 drp of AAV8/DC172-hGC into 4-week old D409V/null mice (prior to development of disease pathology) generated high levels of GC in the liver and serum, and subsequent uptake by the spleen and lung. Expression was sustained for the duration of the study (6 months) and was effective at preventing both the accumulation of GL-1 in the tissues and the consequent onset of Gaucher disease symptoms. A subsequent study in older mice (4-month old) with established disease pathology showed that administration of a similar dose of AAV8/DC172-hGC was also effective at reversing GL-1 storage in the affected tissues. Furthermore, AAV-treated mice were largely devoid of the lipid-engorged macrophages that were frequently observed in vehicle-treated D409V/null mice. A dose-response study, in which 5-month old mice received 31011, 11011 or 31010 drp of AAV8/DC172-hGC, yielded high and sustained expression of GC in all treatment groups. Three months post-injection, both the 31011 and 11011 drp dosed mice demonstrated a dramatic reduction in GL-1 levels and the number of Gaucher storage cells in liver, lung and spleen. Treatment also corrected the abnormal pathology, cell differentials and MIP-1 levels in the lungs of the D409V/null mice in a dose-dependent manner. Additionally, no antibodies to GC were detected, in accordance with previous AAV gene therapy studies using hepatocyte-restricted promoters. These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in the mouse model and thus support its continued development in the treatment of Gaucher Disease.

Gaucher disease is the most common of the more than 40 currently described lysosomal storage diseases. It is caused by mutations in the gene encoding the lysosomal hydrolase, glucocerebrosidase (GC). The loss or deficiency of this enzyme results in the accumulation of the substrate, glucosylceramide (GL-1), in tissue macrophages primarily of the liver and spleen. A mouse model of the disease (D409V/null) was recently generated. Characterization of this murine model indicated that it displayed several of the biochemical and phenotypic abnormalities shown associated with the human disease. The D409V/null mice exhibited approximately only 5% of normal levels of the enzyme in the visceral tissues and consequently, elevated levels of GL-1 in the liver, spleen, lung and also the bone marrow of these animals. Associated with the abnormal storage of GL-1 in the tissues were the appearance of characteristically enlarged macrophages (Gaucher cells) similar to those observed in Gaucher patients, particularly in the liver, spleen and lung of the mice. To evaluate the potential of gene therapy for treating this disease, an AAV 2/2 and 2/8-pseudotyped vector encoding the human GC were constructed. Since secretion of GC from transduced cells occurs only in cells highly overexpressing the enzyme, efforts were made to optimize its expression. A codon optimized and CpG-reduced synthetic cDNA for human GC was placed under the transcriptional control of an 1-antitrypsin promoter to which was appended two copies of the 1-microglobulin enhancer (DC172). Studies showed that expression from the DC172 promoter was hepatic-restricted and was significantly higher than that attained with either a CMV or the previously described DC190 promoter (human serum albumin promoter linked to two copies of the human prothrombin enhancer). Intravenous administration of 31011 particles of either AAV2/2-DC172-shGC or AAV2/8-DC172-shGC into 4 weeks old D409V/null mice resulted in hepatic transduction and subsequent secretion of supraphysiological levels of GC into the systemic circulation. Expression of GC in animals administered AAV2/8-DC172-hGC were 50 to 100-fold higher than from the corresponding AAV2/2 vector and remained undiminished at 4 months. In contrast to the untreated mice, which displayed characteristic Gaucher cells in the lungs, liver and spleen, treated animals (at 2 months post-treatment) were devoid of these cells and harbored normal tissue levels of GL-1. The absence of elevated GL-1 levels in the tissues of treated animals confirmed that the hepatically-produced enzyme was in a form that was conducive to recapture by the affected cells and that importantly, the levels attained were sufficient to prevent the development of disease manifestations. These data support the continued development of AAV vectors for gene therapy of Gaucher disease.

Objective: Gene therapy is a promising strategy to modify ischemia-reperfusion injury and rejection after transplantation. We evaluated variables that may affect ex vivo gene transfer to rat lung isografts. Methods: Left lungs were harvested and perfused via the pulmonary vein with chloramphenicol acetyltransferase complementary deoxyribonucleic acid complexed with cationic liposomes. Several variables were examined: (1) Influence of temperature: In group I (n = 4), grafts were stored for 4 hours at 23 degrees C and transplanted. Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. In groups II and III (n = 4), grafts were stored at 10 degrees and 4 degrees C, respectively. Arterial oxygen tension and inflammatory infiltrate were also determined, (2) Influence of storage time: Grafts were preserved at 10 degrees C for 1, 2, 3, 4 (n = 4), and 10 hours (n = 5), Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. (3) Rapidity and duration of transgene expression: Grafts were preserved at 10 degrees C for 1 hour and then transplanted, Chloramphenicol acetyltransferase activity was assessed 2, 4, 6, 12, and 24 hours and 2, 7, 14, 21, and 28 days after implantation. Results: Chloramphenicol acetyltransferase expression was apparently less in lungs transfected at 4 degrees C than in those transfected at 10 degrees and 23 degrees C. Storage for 1 hour at 10 degrees C was sufficient to yield significant expression. Increasing the exposure time to 10 hours did not increase toxicity. There were no differences in arterial oxygen tension between transfected and nontransfected lungs. Chloramphenicol acetyltransferase expression was detected for at least 28 days, Conclusion: Ex vivo liposome-mediated transfection of lung isografts can he achieved after a short time of cold storage, with minimal toxicity.

In an effort to improve the efficiency of cationic lipid mediated gene transfer, over 90 novel cationic lipids of diverse structural types were synthesized and evaluated in vitro. Four cationic lipids derived from phospholipids were examined. The most promising cationic lipid formulations were tested in vivo by intranasal or transtracheal instillation into the lungs of BALB/c mice. The most active formulations gave CAT reporter gene expression levels which are greater than 500 fold over that which could be attained using free DNA alone. Certain cationic lipid formulations have been shown to facilitate substantial expression of the CFTR (cystic fibrosis transmembrane conductance regulator) gene in vitro as determined by the SPQ and 'Ussing Chamber assays.