Efficacy of Enzyme and Substrate Reduction Therapy with a Novel Antagonist of Glucosylceramide Synthase for Fabry Disease.

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.

Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.

The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep.

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.

Dysregulation of multiple facets of glycogen metabolism in a murine model of Pompe disease.

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.

Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease.

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 of the 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.

Rapid identification of novel functional promoters for gene therapy.

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.

Assessment of the nuclear pore dilating agent trans-cyclohexane-1,2-diol in differentiated airway epithelium.

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.

Iminosugar-based inhibitors of glucosylceramide synthase prolong survival but paradoxically increase brain glucosylceramide levels in Niemann-Pick C mice.

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.

Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease.

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.

Distribution of acid sphingomyelinase in rodent and non-human primate brain after intracerebroventricular infusion.

One treatment approach for lysosomal storage diseases (LSDs) is the systemic infusion of recombinant enzyme. Although this enzyme replacement is therapeutic for the viscera, many LSDs have central nervous system (CNS) components that are not adequately treated by systemic enzyme infusion. Direct intracerebroventricular (ICV) infusion of a high concentration of recombinant human acid sphingomyelinase (rhASM) into the CNS over a prolonged time frame (hours) has shown therapeutic efficacy in a mouse model of Niemann-Pick A (NP/A) disease. To evaluate whether such an approach would translate to a larger brain, rhASM was infused into the lateral ventricles of both rats and Rhesus macaques, and the resulting distribution of enzyme characterized qualitatively and quantitatively. In both species, ICV infusion of rhASM resulted in parenchymal distribution of enzyme at levels that were therapeutic in the NP/A mouse model. Enzyme distribution was global in nature and exhibited a relatively steep gradient from the cerebrospinal fluid compartment to the inner parenchyma. Additional optimization of an ICV delivery approach may provide a therapeutic option for LSDs with neurologic involvement.

Systemic administration of AAV8-α-galactosidase A induces humoral tolerance in nonhuman primates despite low hepatic expression.

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 α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal. However, this effect was not robust, and sustained α-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.

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer.

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.

Substrate reduction augments the efficacy of enzyme therapy in a mouse model of Fabry disease.

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.

Preexisting immunity and low expression in primates highlight translational challenges for liver-directed AAV8-mediated gene therapy.

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.

Induction of immune tolerance to a therapeutic protein by intrathymic gene delivery.

The efficacy of recombinant enzyme therapy for genetic diseases is limited in some patients by the generation of a humoral immune response to the therapeutic protein. Inducing immune tolerance to the protein prior to treatment has the potential to increase therapeutic efficacy. Using an AAV8 vector encoding human acid α-glucosidase (hGAA), we have evaluated direct intrathymic injection for inducing tolerance. We have also compared the final tolerogenic states achieved by intrathymic and intravenous injection. Intrathymic vector delivery induced tolerance equivalent to that generated by intravenous delivery, but at a 25-fold lower dose, the thymic hGAA expression level was 10,000-fold lower than the liver expression necessary for systemic tolerance induction. Splenic regulatory T cells (Tregs) were apparent after delivery by both routes, but with different phenotypes. Intrathymic delivery resulted in Tregs with higher FoxP3, TGFß, and IL-10 mRNA levels. These differences may account for the differences noted in splenic T cells, where only intravenous delivery appeared to inhibit their activation. Our results imply that different mechanisms may be operating to generate immune tolerance by intrathymic and intravenous delivery of an AAV vector, and suggest that the intrathymic route may hold promise for decreasing the humoral immune response to therapeutic proteins in genetic disease indications.

Evaluation of systemic follistatin as an adjuvant to stimulate muscle repair and improve motor function in Pompe mice.

Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.

Inhibition of glycogen biosynthesis via mTORC1 suppression as an adjunct therapy for Pompe disease.

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1-2years of age to a more slowly progressive course that causes significant morbidity and early mortality in children and adults. Recombinant human GAA (rhGAA) improves clinical outcomes with variable results. Adjunct therapy that increases the effectiveness of rhGAA may benefit some Pompe patients. Co-administration of the mTORC1 inhibitor rapamycin with rhGAA in a GAA knockout mouse reduced muscle glycogen content more than rhGAA or rapamycin alone. These results suggest mTORC1 inhibition may benefit GSDs that involve glycogen accumulation in muscle.

Improved management of lysosomal glucosylceramide levels in a mouse model of type 1 Gaucher disease using enzyme and substrate reduction therapy.

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid beta-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.

Surfactant protein-d inhibits lung inflammation caused by ventilation in premature newborn lambs.

RATIONALE: Premature newborns frequently require manual ventilation for resuscitation during which lung injury occurs. Although surfactant protein (SP)-D regulates pulmonary inflammation, SP-D levels are low in the preterm lung. Commercial surfactants for treatment of respiratory distress syndrome do not contain SP-D. OBJECTIVES: To determine whether addition of recombinant human SP-D (rhSP-D) to commercial surfactant influences lung inflammation in ventilated premature newborn lambs. METHODS: Prematurely delivered lambs (130 d gestation age) were resuscitated with 100% O(2) and peak inspiratory pressure 40 cm H(2)O for 20 minutes and then treated with Survanta or Survanta containing rhSP-D. Ventilation was then changed to regulate tidal volume at 8 to 9 ml/kg. At 5 hours of age lambs were killed for sample collection. MEASUREMENTS AND MAIN RESULTS: Sequential blood gas and tidal volume were similar in lambs treated with or without rhSP-D, indicating that lung immaturity and ventilatory stress used to support premature lambs were comparable between the two groups. Ventilation caused pulmonary inflammation in lambs treated with surfactant alone. In contrast, surfactant containing rhSP-D decreased neutrophil numbers in bronchoalveolar lavage fluid and decreased neutrophil elastase activity in lung tissue. IL-8 mRNA and IL-8 protein were significantly decreased in the +rhSP-D group lamb lungs, to 20% of those in controls. The addition of rhSP-D also rendered Survanta more resistant to plasma protein inhibition of surfactant function. CONCLUSIONS: Treatment with rhSP-D-containing surfactant inhibited lung inflammation and enhanced the resistance of surfactant to inhibition, supporting its potential usefulness for prevention of lung injury in the preterm newborn.

Low-frequency ultrasound increases non-viral gene transfer to the mouse lung.

The aim of the study was to assess if low-frequency ultrasound (US), in the range of 30-35 kHz, increases non-viral gene transfer to the mouse lung. US is greatly attenuated in the lung due to large energy losses at the air/tissue interfaces. The advantages of low-frequency US, compared with high-frequency US are: (i) increased cavitation (responsible for the formation of transient pores in the cell membrane) and (ii) reduced energy losses during lung penetration. Cationic lipid GL67/plasmid DNA (pDNA), polyethylenimine (PEI)/pDNA and naked pDNA were delivered via intranasal instillation and the animals were then exposed to US (sonoporation) at 0.07 or 0.1 MPa for 10 min. Under these conditions, US did not enhance GL67 or PEI-mediated transfection. It did, however, increase naked pDNA gene transfer by approximately 4 folds. Importantly, this was achieved in the absence of microbubbles, which are crucial for the commonly used high-frequency (1 MHz) sonoporation but may not be able to withstand nebulization in a clinically relevant setup. Lung hemorrhage was also assessed and shown to increase with US pressure in a dose-dependent manner. We have thus, established that low-frequency US can enhance lung gene transfer with naked pDNA and this enhancement is more effective than the previously reported 1 MHz US.