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Background: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis., Methods: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0.9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867., Findings: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3.7%, 95% CI 0.1-7.3; p=0.046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups., Interpretation: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials., Funding: Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme., (Copyright © 2015 Alton et al. Open Access article distributed under the terms of CC BY. Published by Elsevier Ltd.. All rights reserved.)

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.

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.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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.

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.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

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.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

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, TGFβ, 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.

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.

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.

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.

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.

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.

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.

We have assessed whether viscoelastic gels known to inhibit mucociliary clearance can increase lipid-mediated gene transfer. Methylcellulose or carboxymethylcellulose (0.25-1.5%) was mixed with complexes of the cationic lipid GL67A and plasmids encoding luciferase and perfused onto the nasal epithelium of mice. Survival after perfusion with 1% CMC or 1% MC was 90 and 100%, respectively. In contrast 1.5% CMC was uniformly lethal likely due to the viscous solution blocking the airways. Perfusion with 0.5% CMC containing lipid/DNA complexes reproducibly increased gene expression by approximately 3-fold (n=16, p<0.05). Given this benefit, likely related to increased duration of contact, we also assessed the effect of prolonging contact time of the liposome/DNA complexes by delivering our standard 80 microg DNA dose over either approximately 22 or 60 min of perfusion. This independently increased gene transfer by 6-fold (n=8, p<0.05) and could be further enhanced by the addition of 0.5% CMC, leading to an overall 25-fold enhancement (n=8, p<0.001) in gene expression. As a result of these interventions CFTR transgene mRNA transgene levels were increased several logs above background. Interestingly, this did not lead to correction of the ion transport defects in the nasal epithelium of cystic fibrosis mice nor for immunohistochemical quantification of CFTR expression. To assess if 0.5% CMC also increased gene transfer in the mouse lung, we used whole body nebulisation chambers. CMC was nebulised for 1h immediately before, or simultaneously with GL67A/pCIKLux. The former did not increase gene transfer, whereas co-administration significantly increased gene transfer by 4-fold (p<0.0001, n=18). This study suggests that contact time of non-viral gene transfer agents is a key factor for gene delivery, and suggests two methods which may be translatable for use in man., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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.

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.)

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.

Proteasome inhibitors have been shown to increase adeno-associated virus (AAV)-mediated transduction in vitro and in vivo. To assess if proteasome inhibitors also increase lipid-mediated gene transfer with relevance to cystic fibrosis (CF), we first assessed the effects of doxorubicin and N-acetyl-l-leucinyl-l-leucinal-l-norleucinal in non-CF (A549) and CF (CFTE29o-) airway epithelial cell lines. CFTE29o- cells did not show a response to Dox or LLnL; however, gene transfer in A549 cells increased in a dose-related fashion (p < 0.05), up to approximately 20-fold respectively at the optimal dose (no treatment: 9.3 x 10(4) +/- 1.5 x 10(3), Dox: 1.6 x 10(6)+/-2.6 x 10(5), LLnL: 1.9 x 10(6) +/- 3.2 x 10(5)RLU/mg protein). As Dox is used clinically in cancer chemotherapy we next assessed the effect of this drug on non-viral lung gene transfer in vivo. CF knockout mice were injected intraperitoneally (IP) with Dox (25-100 mg/kg) immediately before nebulisation with plasmid DNA carrying a luciferase reporter gene under the control of a CMV promoter/enhancer (pCIKLux) complexed to the cationic lipid GL67A. Dox also significantly (p < 0.05) increased expression of a plasmid regulated by an elongation factor 1alpha promoter (hCEFI) approximately 8-fold. Although administration of Dox before lung gene transfer may not be a clinically viable option, understanding how Dox increases lung gene expression may help to shed light on intracellular bottle-necks to gene transfer, and may help to identify other adjuncts that may be more appropriate for use in man.

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.

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

The availability of a murine model of Pompe disease has enabled an evaluation of the relative merits of various therapeutic paradigms, including gene therapy. We report here that administration of a recombinant adeno-associated virus serotype 8 (AAV8) vector (AAV8/DC190-GAA) encoding human acid alpha-glucosidase (GAA) into presymptomatic Pompe mice resulted in nearly complete correction of the lysosomal storage of glycogen in all the affected muscles. A relatively high dose of AAV8/DC190-GAA was necessary to attain a threshold level of GAA for inducing immunotolerance to the expressed enzyme and for correction of muscle function, coordination, and strength. Administration of AAV8/DC190-GAA into older Pompe mice with overt disease manifestations was also effective at correcting the lysosomal storage abnormality. However, these older mice exhibited only marginal improvements in motor function and no improvement in muscle strength. Examination of histologic sections showed evidence of skeletal muscle degeneration and fibrosis in aged Pompe mice whose symptoms were abated or rescued by early but not late treatment with AAV8/DC190-GAA. These results suggest that AAV8-mediated hepatic expression of GAA was effective at addressing the biochemical and functional deficits in Pompe mice. However, early therapeutic intervention is required to maintain significant muscle function and should be an important consideration in the management and treatment of Pompe disease.

Imaging of in vivo gene expression using luciferase expression in various organs has been used for several years. In contrast to other organs, in vivo imaging of the lung, particularly after non-viral gene transfer has not been extensively studied. The aim of this study was to address several questions: (1) Does in vivo light emission correlate with standard tissue homogenate-based luciferase detection in a dose-dependent manner? Recombinant Sendai virus (SeV) transduces airway epithelial cells very efficiently and was used to address this question, (2) Is the sensitivity of the assay sufficient to detect non-viral gene transfer? We treated mice with SeV-Lux vector using our standard "sniffing" protocol, a method that predominantly results in lung deposition. Dose-related in vivo light emission was visible in all animals. Importantly, there was a significant correlation (r>0.90, p<0.0001) between the in vivo and ex vivo assays in both the left and right lung. We next transfected the nasal epithelium via nasal perfusion or the lungs ("sniffing") of mice with a luciferase plasmid (pCIKLux) complexed to the cationic lipid GL67 (n=25-27/group) and imaged luciferase expression in vivo 24h after transfection. Gene expression was detectable in both organs. Correlation between the in vivo and ex vivo assays was significant (r=0.52, p<0.005) in the left, but not the right lung. The correlation in the nose was weaker (r=0.45, p<0.05). To our knowledge these studies show for the first time that this non-invasive method of assessing pulmonary gene transfer is viable for evaluating non-viral gene transfer agents.

Background: Acute lung injury is a common cause of morbidity and mortality following pulmonary or systemic infections. Surfactant protein-D is a member of the collectin family of proteins, which play important roles in innate host defense of the lung. In this study, the effect of exogenous recombinant human SP-D (rhSP-D) on protection of the adult mouse lung from lipopolysaccharide (LPS)-induced and lipoteichoic acid (LTA)-induced injury was assessed., Methods: The effect of rhSP-D on LPS-induced and LTA-induced lung inflammation and injury was assessed with and without exogenous pulmonary surfactant in Sftpd+/+ and Sftpd-/- mice. A total of 204 mice (6 mice per group) were used for the present study., Results: Sftpd-/- mice were more susceptible to intratracheal LPS than were Sftpd+/+ mice. rhSP-D decreased neutrophilic infiltrates induced by LPS and LTA in the lungs of both Sftpd+/+ and Sftpd-/- mice. The addition of exogenous pulmonary surfactant to rhSP-D further decreased LPS-induced and LTA-induced pulmonary inflammation in Sftpd-/- and Sftpd+/+ mice., Conclusions: Intratracheal rhSP-D inhibited inflammation induced by intratracheal LPS and LTA instillation in the lung. The antiinflammatory effects of rhSP-D were enhanced by the addition of pulmonary surfactant, providing a potential therapy for the treatment of lung inflammation.

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a monogenic disorder caused by the loss of tripeptidyl peptidase 1 (TPP1) activity as a result of mutations in CLN2. Absence of TPP1 results in lysosomal storage with an accompanying axonal degeneration throughout the central nervous system (CNS), which leads to progressive neurodegeneration and early death. In this study, we compared the efficacies of pre- and post-symptomatic injections of recombinant adeno-associated virus (AAV) for treating the cellular and functional abnormalities of CLN2 mutant mice. Intracranial injection of AAV1-hCLN2 resulted in widespread human TPP1 (hTPP1) activity in the brain that was 10-100-fold above wild-type levels. Injections before disease onset prevented storage and spared neurons from axonal degeneration, reflected by the preservation of motor function. Furthermore, the majority of CLN2 mutant mice treated pre-symptomatically lived for at least 330 days, compared with a median survival of 151 days in untreated CLN2 mutant controls. In contrast, although injection after disease onset ameliorated lysosomal storage, there was evidence of axonal degeneration, motor function showed limited recovery, and the animals had a median lifespan of 216 days. These data illustrate the importance of early intervention for enhanced therapeutic benefit, which may provide guidance in designing novel treatment strategies for cLINCL patients.

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.

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 beta-galactosidase reduced betagal 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.

Antiviral antibodies within the human population remain a barrier to the effective clinical use of viral gene transfer vectors. We have asked whether local, balloon catheter-mediated delivery of a viral vector to the rabbit liver using a hepatic vein might mitigate the neutralizing effects of antiviral antibodies. We have compared directly the ability of adenovirus (Ad2) encoding nuclear-localized beta-galactosidase to infect the rabbit liver after local and systemic delivery in both the presence and the absence of defined anti-Ad2 antibody titers. In naive rabbits, local delivery resulted in higher beta-galactosidase expression compared to systemic delivery. In the presence of passively administered anti-Ad2 antibodies, local delivery resulted in expression levels that were comparable to those obtained in naive rabbits by systemic delivery. Local delivery also resulted in the majority of expression originating from hepatocytes, even in passively immunized animals, a result that could not be duplicated using the systemic approach. Since systemic delivery of adenovirus in naive animal models results in transgene expression levels often regarded as therapeutic, these results predict that local hepatic vein delivery of a viral vector is a clinically practical approach to mitigate neutralizing antiviral antibodies and generate therapeutic levels of transgene expression.

Hepatocytes are an effective depot for protein production from gene therapy vectors. However, when gene transfer vectors or their delivery induces hepatic inflammation, adaptive immune responses against the transgene product can ensue. In BALB/c mice, hydrodynamic delivery of a CMV-driven plasmid DNA (pDNA) bearing human alpha-galactosidase A (alphagal) to the liver generated antibodies against alphagal. This humoral immune response was more robust in a transgenic knockout for alphagal, the Fabry mouse. The antibody response could be attenuated in both mouse strains by using a promoter more restricted to hepatocytes. In an attempt to reduce further the humoral responses to alphagal, expression from the transgene was attenuated by using siRNA during the period of initial delivery-associated liver inflammation. In both mouse models and with both promoters, codelivering an alphagal siRNA resulted in a 2 log decrease in initial expression that then increased over the next few weeks to levels generated by the pDNA alone. This strategy led to both attenuated antibodies and an immune status approximating "tolerance" to alphagal. Importantly, in the Fabry mouse, an alphagal siRNA together with a hepatocyte-restricted promoter gave minimal anti-alphagal antibodies and profound tolerance, suggesting that such an approach might have clinical utility for genetic diseases.

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

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

CpG-reduced, CMV-based plasmid DNA constructs encoding human alpha-galactosidase A and factor IX were injected into C57Bl/6, BALB/c, and CD1 mice using hydrodynamics-based delivery of plasmid DNA (pDNA), and gene expression was monitored for 6 months. Linearized and supercoiled pDNAs were compared for their abilities to support long-term expression and to generate immune responses to the transgene product. In all mouse strains supercoiled CpG-reduced pDNA encoding alpha-galactosidase A and factor IX generated higher and more sustained levels of circulating gene product than their supercoiled CpG-replete analogs. Linearizing supercoiled CpG-reduced pDNA did not significantly increase levels of circulating gene product beyond levels supercoiled CpG-reduced pDNA could achieve. Linearizing supercoiled CpG-replete pDNA vectors significantly increased expression compared to their supercoiled CpG-replete analogs, but the increase was short-lived or subtherapeutic. Regardless of vector, liver depot expression did not elicit significant antibody responses to human alpha-galactosidase A or factor IX. Taken together, these data suggest that a clinically acceptable hydrodynamics-based approach targeting the liver combined with CpG-reduced pDNA vectors may represent a viable option for individuals with hemophilia, a lysosomal storage disease, or other disease in which prolonged depot expression of a therapeutic protein from the liver is desirable., (Copyright The American Society of Gene Therapy)

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.

Immunostimulatory CpG motifs have been implicated as a major contributor to the acute inflammatory response associated with nonviral vectors, most prominently seen after systemic delivery of cationic lipid-plasmid DNA (pDNA) complexes. We have shown previously that complexes containing pDNA vectors that have been largely depleted of CpG motifs have significantly reduced acute toxicity when delivered systemically. However, several CpGs remain in these vectors and the toxicity is not negligible, especially at higher doses of complex. To determine the maximal reduction in the acute toxic response that could be achieved by eliminating CpG signaling, we injected cationic lipid-pDNA complexes into transgenic mice that are deficient in Toll-like receptor 9 (TLR9), which is the receptor that recognizes immunostimulatory CpG motifs. We observed significantly decreased adverse hematological changes and liver damage in TLR9(-/-) mice compared to normal mice and increased survival at higher doses of complex. However, a pronounced loss of lymphocytes and platelets was still observed in the TLR9(-/-) mice at higher doses. We also measured the toxicity in normal mice of systemically delivered complexes containing non-CpG oligonucleotides. Although serum transaminase levels were reduced, a loss of lymphocytes and platelets akin to that seen in the TLR9(-/-) mice was observed. Taken together, these findings suggest that signaling through TLR9 contributes to the majority but not all of the toxic responses associated with systemic delivery of cationic lipid-pDNA complexes.

Background: Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal enzyme alpha-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 alpha-galactosidase A and GL-3 in different tissues were assayed at various time points after injection., Results: Expression of alpha-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 alpha-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.)

Hydrodynamic delivery is an efficient and inexpensive procedure to deliver a wide range of nucleic acids to hepatic tissues and other organs in vivo. The successful application of hydrodynamic delivery is dependent on the rapid injection of a large aqueous volume containing DNA, RNA or other molecules into the vasculature of the liver. In this review, the development of the procedures for hydrodynamic delivery will be described and the parameters necessary for attaining maximal gene expression will be highlighted. A review of the mechanisms for transfecting hepatocytes, as well as potential uses of this approach in various research and clinical applications, will also be discussed.

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

Rapid systemic injection of naked plasmid DNA (pDNA) in a large volume into a mouse tail vein has been shown to result in a high level of gene expression in the liver. However, the potential therapeutic benefit to humans embodied in hydrodynamic transfection of the liver cannot be realized until a clinically viable method for gene delivery is developed. In light of this fact, we have devised and evaluated several methods for delivering pDNA to the isolated rabbit liver using minimally invasive catheter-based techniques. Using a lobar technique, pDNA was delivered hydrodynamically to an isolated hepatic lobe using a balloon occlusion balloon catheter to occlude a selected hepatic vein. A whole organ technique was used wherein the entire hepatic venous system was isolated and the pDNA solution injected hydrodynamically into the vena cava between two balloons used to block hepatic venous outflow. Lobar delivery of a plasmid encoding a secreted alkaline phosphatase (SEAP) reporter gene resulted in significant levels of transgene product in the serum. A nonsecreted transgene product, chloramphenicol acetyltransferase (CAT), showed the highest levels of expression in the injected lobe distal to the injection site. Compared to lobar delivery, whole organ delivery yielded much higher serum levels of SEAP expression and a significantly broader hepatic parenchymal distribution of CAT expression. These preliminary studies suggest that catheter-mediated hydrodynamic delivery of pDNA to the isolated liver may provide a method for human gene therapy that is both therapeutically significant and clinically practicable.

We have demonstrated recently that treatment of established peritoneal mesothelial tumors with complexes composed of cationic lipid and noncoding plasmid DNA (pNull) results in the inhibition of tumor growth accompanied by the induction of a tumor-specific cellular immune response. In this study, treatment of mice bearing intraperitoneal (i.p.) M3 melanoma tumors with i.p. injections of lipid/pNull complex was found to inhibit tumor growth and induce the development of a cytolytic response against several M3 melanoma-associated antigens. Depletion of CD8(+) T cells, as opposed to natural killer (NK) or CD4(+) T cells, essentially abrogated the therapeutic effect of lipid+pNull complex, thus supporting the involvement of cytotoxic CD8(+) T cells in the antitumor response. The antitumor effect of lipid/pNull complex was maximal following delivery into a tumor-bearing compartment. For example, i.p. delivery of complex was more effective than intravenous (i.v.) or subcutaneous (s.c.) treatment of i.p. M3 tumors. In addition, i.v. injection of complex displayed therapeutic activity against lung metastases caused by i.v. injection of tumor cells, and intratumoral injection of complex into solid s.c. tumors caused regression in most animals. Importantly, the immune response induced by local treatment of tumors with complex also offered systemic protection against tumor cells at distal sites, as illustrated by the eradication of both peritoneal tumors and lung metastases in mice treated with complex delivered i.p. Treatment with lipid/pNull complex, therefore, represents an attractive immune-based treatment modality that could potentially be applied to many tumor types.

Systemic delivery of cationic lipid-plasmid DNA (pDNA) complexes induces an acute inflammatory response with adverse hematologic changes and liver damage. Immunostimulatory CpG motifs in the pDNA are known to contribute substantially to this response. Here we constructed a pDNA vector (pGZB) that has been depleted of 80% of the CpG motifs present in the original vector. Compared with the unmodified vector, systemic administration of pGZB induced considerably fewer changes in blood parameters, reduced levels of inflammatory cytokines, and decreased liver damage. Despite the extensive sequence modifications within pGZB, there were still robust levels of transgene expression. Furthermore, in contrast to the transient expression observed from the unmodified vector, we observed sustained or increasing expression for up to 49 days from pGZB in the lung and liver of immunocompetent BALB/c mice. Studies adding CpG motifs in trans or in cis indicate that the reduced CpG content of pGZB was the major determinant for its persistent expression. This combination of decreased toxicity and sustained expression suggests that CpG-depleted pDNA vectors can greatly improve the safety and efficacy of synthetic gene delivery systems., ((c)2002 Elsevier Science (USA).)