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

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

Abstract: 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 &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Due to the lack of acid α-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. [ABSTRACT FROM AUTHOR]

Abstract: 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 80μg DNA dose over either approximately 22 or 60min 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 &y& Elsevier]

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.Molecular Therapy (2008) 16 8, 1400–1408 doi:10.1038/mt.2008.115 [ABSTRACT FROM AUTHOR]

Abstract: 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 α-galactosidase A (αgal) to the liver generated antibodies against αgal. This humoral immune response was more robust in a transgenic knockout for αgal, 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 αgal, 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 αgal 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 αgal. Importantly, in the Fabry mouse, an αgal siRNA together with a hepatocyte-restricted promoter gave minimal anti-αgal antibodies and profound tolerance, suggesting that such an approach might have clinical utility for genetic diseases. [Copyright &y& Elsevier]

CpG-reduced, CMV-based plasmid DNA constructs encoding human α-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 α-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 α-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 &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a neurodegenerative disorder that results from the loss of tripeptidyl peptidase I (TPP1) enzyme activity. Patients with cLINCL possess seizures that are frequently associated with blindness, exhibit cognitive and behavioral deficits that are progressive, and death by 7–20 years of age. Accumulation of autofluorescent storage material is a cardinal feature of the disease, as well as axonal degeneration and loss of cortical neurons. A recent study by our group in a knockout mouse model of cLINCL showed that recombinant AAV2 or AAV5 significantly reduced autofluorescent storage and curvilinear bodies in cells of the brain. Reversal of pathology was restricted to injected and nearby structures at a dose of 2.0 e11 genome copies (gc)/ml. To further evaluate AAV gene therapy, a codon-optimized synthetic human CLN2 cDNA was engineered, packaged into AAV1 capsids (AAV1-hCLN2), concentrated to a high titer (3.3 e12 gc/ml), and injected into the CLN2 (−/−) thalamus of one hemisphere. At 1 month post-injection, brain homogenates of the injection site contained TTP1 enzyme activity that was 15-fold higher than corresponding regions of untreated CLN2 (+/+) mice. TPP1 activity was also observed in the rostral and caudal regions of injected hemisphere, and in the contralateral hemisphere at levels 2-fold higher than CLN2 (+/+) controls. These data demonstrate that the AAV1 vector is capable of producing widespread and high levels of TPP1 activity in vivo. A cohort of 4-week old CLN2 (−/−) mice were then injected with AAV1-hCLN2 into multiple structures of the right (striatum, hippocampus, cerebellum) and left (motor cortex, thalamus, medulla) hemispheres to investigate global reversal of pathology, functional recovery, and extension of lifespan. The results of this experiment including behavioral testing and survival data will be presented.Molecular Therapy (2006) 13, S344–S344; doi: 10.1016/j.ymthe.2006.08.982 [ABSTRACT FROM AUTHOR]