Origin, synchronization, and propagation of sleep slow waves in children.

STUDY OBJECTIVES: Sleep slow wave activity, as measured using EEG delta power (<4 Hz), undergoes significant changes throughout development, mirroring changes in brain function and anatomy. Yet, age-dependent variations in the characteristics of individual slow waves have not been thoroughly investigated. Here we aimed at characterizing individual slow wave properties such as origin, synchronization, and cortical propagation at the transition between childhood and adulthood. METHODS: We analyzed overnight high-density (256 electrodes) EEG recordings of healthy typically developing children (N = 21, 10.3 ± 1.5 years old) and young healthy adults (N = 18, 31.1 ± 4.4 years old). All recordings were preprocessed to reduce artifacts, and NREM slow waves were detected and characterized using validated algorithms. The threshold for statistical significance was set at p = 0.05. RESULTS: The slow waves of children were larger and steeper, but less widespread than those of adults. Moreover, they tended to mainly originate from and spread over more posterior brain areas. Relative to those of adults, the slow waves of children also displayed a tendency to more strongly involve and originate from the right than the left hemisphere. The separate analysis of slow waves characterized by high and low synchronization efficiency showed that these waves undergo partially distinct maturation patterns, consistent with their possible dependence on different generation and synchronization mechanisms. CONCLUSIONS: Changes in slow wave origin, synchronization, and propagation at the transition between childhood and adulthood are consistent with known modifications in cortico-cortical and subcortico-cortical brain connectivity. In this light, changes in slow-wave properties may provide a valuable yardstick to assess, track, and interpret physiological and pathological development.

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis.

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.

Gulf War veterans exhibit broadband sleep EEG power reductions in regions overlying the frontal lobe.

AIMS: Nearly a third of U.S. veterans who deployed in support of the 1990-1991 Persian Gulf War are affected by Gulf War illness (GWI). Here we aimed to characterize whether subjective sleep complaints in GWI veterans are associated with objective sleep EEG disturbances relative to healthy veterans and controls; and whether Gulf War veterans show alterations in neural activity during sleep that differentiate them from healthy subjects. MAIN METHODS: We used high-density EEG (HDEEG) to assess regional patterns of rapid eye movement (REM) sleep and non-REM (NREM) sleep between three groups: Gulf War male veterans with fatigue and GWI, Gulf War male veterans without fatigue or GWI, and control males. The groups were matched relative to age, sex and obstructive sleep apnea. Topographic comparisons of nocturnal NREM and REM sleep were made between groups for all frequency bands. KEY FINDINGS: Topographic analysis revealed a broadband reduction in EEG power in a circumscribed region overlying the frontal lobe in both groups of Gulf War veterans, regardless of GWI and fatigue. This frontal reduction in neural activity was present, to some extent, across all frequency bands in NREM and REM sleep. SIGNIFICANCE: Given that our findings were observed in all Gulf War veterans, it appears unlikely that frontal sleep HDEEG power reductions prove wholly responsible for fatigue symptoms. These results provide avenues for research which may someday contribute to improved clinical care of formerly deployed veterans of the Persian Gulf War.

A high-density electroencephalography study reveals abnormal sleep homeostasis in patients with rapid eye movement sleep behavior disorder.

Rapid eye movement (REM) sleep behavior disorder (RBD) is characterized by disrupting motor enactments during REM sleep, but also cognitive impairments across several domains. In addition to REM sleep abnormalities, we hypothesized that RBD patients may also display EEG abnormalities during NREM sleep. We collected all-night recordings with 256-channel high-density EEG in nine RBD patients, predominantly early-onset medicated individuals, nine sex- and age- matched healthy controls, and nine additional controls with matched medications and comorbidities. Power spectra in delta to gamma frequency bands were compared during both REM and NREM sleep, between phasic and tonic REM sleep, and between the first versus last cycle of NREM sleep. Controls, but not RBD patients, displayed a decrease in beta power during phasic compared to tonic REM sleep. Compared to controls, RBD patients displayed a reduced decline in SWA from early to late NREM sleep. Overnight changes in the distribution of the amplitude of slow waves were also reduced in RBD patients. Without suppression of beta rhythms during phasic REM sleep, RBD patients might demonstrate heightened cortical arousal, favoring the emergence of behavioral episodes. A blunted difference between REM sleep sub-stages may constitute a sensitive biomarker for RBD. Moreover, reduced overnight decline in SWA suggests a reduced capacity for synaptic plasticity in RBD patients, which may favor progression towards neurodegenerative diseases.

Limitations of the murine nose in the development of nonviral airway gene transfer.

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.

Seasonal influences on sleep and executive function in the migratory White-crowned Sparrow (Zonotrichia leucophrys gambelii).

BACKGROUND: We have previously shown that the White-crowned Sparrow (WCS) decreases sleep by 60% during a period of migratory restlessness relative to a non-migratory period when housed in a 12 h light: 12 h dark cycle. Despite this sleep reduction, accuracy of operant performance was not impaired, and in fact rates of responding were elevated during the migratory period, effects opposite to those routinely observed following enforced sleep deprivation. To determine whether the previously observed increases in operant responding were due to improved performance or to the effects of migration on activity level, here we assessed operant performance using a task in which optimal performance depends on the bird's ability to withhold a response for a fixed interval of time (differential-reinforcement-of-low-rate-behavior, or DRL); elevated response rates ultimately impair performance by decreasing access to food reward. To determine the influence of seasonal changes in day length on sleep and behavioral patterns, we recorded sleep and assessed operant performance across 4 distinct seasons (winter, spring, summer and fall) under a changing photoperiod. RESULTS: Sleep amount changed in response to photoperiod in winter and summer, with longest sleep duration in the winter. Sleep duration in the spring and fall migratory periods were similar to what we previously reported, and were comparable to sleep duration observed in summer. The most striking difference in sleep during the migratory periods compared to non-migratory periods was the change from discrete day-night temporal organization to an almost complete temporal fragmentation of sleep. The birds' ability to perform on the DRL task was significantly impaired during both migratory periods, but optimal performance was sustained during the two non-migratory periods. CONCLUSIONS: Birds showed dramatic changes in sleep duration across seasons, related to day length and migratory status. Migration was associated with changes in sleep amount and diurnal distribution pattern, whereas duration of sleep in the non-migratory periods was largely influenced by the light-dark cycle. Elevated response rates on the DRL task were observed during migration but not during the short sleep duration of summer, suggesting that the migratory periods may be associated with decreased inhibition/increased impulsivity. Although their daily sleep amounts and patterns may vary by season, birds are susceptible to sleep loss throughout the year, as evidenced by decreased responding rates following enforced sleep deprivation.

An immunocytochemical assay to detect human CFTR expression following gene transfer.

BACKGROUND: To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery. METHODS: We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR. RESULTS: The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells. CONCLUSION: We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.

Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.

Gene therapy is being investigated in the treatment of lung-related aspects of the genetic disease, Cystic fibrosis (CF). Clinical studies have demonstrated CF transmembrane conductance regulator (CFTR) expression in the airways of adults with CF using a variety of gene transfer agents. In utero gene therapy is an alternative approach that facilitates vector transduction of rapidly expanding populations of target cells while avoiding immune recognition of the vector. In CF, in utero gene transfer could potentially delay the onset of disease symptoms in childhood and compensate for the role, if any, that CFTR plays in the developing organs. Previously published studies have suggested that transient expression of CFTR in utero was sufficient to rescue the fatal intestinal defect in S489X Cftr(tm1Unc)/Cftr(tm1Unc) knockout mice. We replicated these studies using an identical CFTR-expressing adenoviral vector and CF mouse strain in sufficiently large numbers to provide robust Kaplan-Meier survival data. Although each step of the procedure was carefully controlled and vector-specific CFTR expression was confirmed in the fetal organs after treatment, there was statistically no significant improvement in the survival of mice treated in utero with AdCFTR, compared with contemporaneous control animals.

Enhanced lung gene expression after aerosol delivery of concentrated pDNA/PEI complexes.

A major limitation of many self-assembling nonviral gene transfer formulations is that they are commonly prepared at relatively low component concentrations. While this typically has little impact on their use in cell culture, it can severely limit the progress of in vivo studies. In order to overcome this, we have developed a simple, scalable, pharmaceutically acceptable concentration method that has allowed us to increase the concentration of a commonly used pDNA/PEI formulation from 0.2 to >8 mg/ml plasmid DNA (pDNA). Crucially, the concentration method was found to have only minimal impact on the electrostatic properties or size of the pDNA/PEI particles. When delivered as an aerosol to the mouse lung, the concentrated pDNA/PEI formulations resulted in a 15-fold increase in lung reporter gene expression, with minimal impact in terms of inflammation or toxicity. Importantly, this performance advantage was replicated after aerosol administration to sheep lungs, with reporter gene expression being similarly approximately 15-fold higher than with the conventional pDNA/PEI formulation, and lung inflammation falling to background levels. These findings demonstrate that concentrated pDNA/PEI formulations offer increased aerosol gene transfer with decreased inflammatory sequelae, and represent a promising advance in the field of nonviral lung gene transfer. It seems likely that similar benefits might be achievable with alternative delivery routes and with other nonviral formulations.

Transgene sequences free of CG dinucleotides lead to high level, long-term expression in the lung independent of plasmid backbone design.

Non-viral aerosol gene therapy offers great potential for treating chronic lung diseases of the airways such as cystic fibrosis (CF). Early clinical trials showed that transgene expression in the airways was transient whereas maximal duration of transgene expression is essential in order to minimise the frequency of aerosol treatments. Improved vector design, such as careful selection of the promoter/enhancer, can lead to more persistent levels of transgene expression, but multiple factors affect expression in vivo. Following aerosol delivery to the lungs of mice, we measured reporter gene expression from a CpG-free luciferase transgene cassette in the context of both a plasmid and minicircle vector configuration and showed that the vector backbone had no effect on expression. Transgene activity was affected by the vector backbone however, when a similar, but sub-optimal CpG-containing transgene was used, suggesting that aspects of the plasmid backbone had a negative impact on transgene expression. Similar studies were performed in Toll-like receptor-9 (TLR9) knockout mice to investigate a potential role for the TLR9 signalling pathway in detecting CpGs in the vector sequence. Even in the absence of TLR9, persistent expression could only be achieved with a CpG-free transgene. Together, these data indicate that in order to achieve high levels of persistent expression in vivo, a CpG-free transgene cassette is required.

Circadian Disruption in Psychiatric Disorders.

Evidence suggests that abnormalities in circadian rhythms might prove causally or pathophysiologically significant in psychiatric illness. The circadian regulation of hormonal and behavioral timekeeping processes is often altered in patients with major depression, bipolar disorder, and schizophrenia, and a susceptibility to rhythm instability may contribute to the functional impairment. For some patients, interventions that stabilize or resynchronize circadian rhythms prove therapeutically effective. Circadian disruption in the clinical profiles of most psychiatric illnesses and the treatment efficacy of chronobiological interventions suggest that attention to circadian phenotypes in a range of psychiatric disorders may help to uncover shared pathophysiologic mechanisms.

Regional reductions in sleep electroencephalography power in obstructive sleep apnea: a high-density EEG study.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is associated with significant alterations in neuronal integrity resulting from either hypoxemia and/or sleep loss. A large body of imaging research supports reductions in gray matter volume, alterations in white matter integrity and resting state activity, and functional abnormalities in response to cognitive challenge in various brain regions in patients with OSA. In this study, we used high-density electroencephalography (hdEEG), a functional imaging tool that could potentially be used during routine clinical care, to examine the regional distribution of neural activity in a non-clinical sample of untreated men and women with moderate/severe OSA. DESIGN: Sleep was recorded with 256-channel EEG in relatively healthy subjects with apnea-hypopnea index (AHI) > 10, as well as age-, sex-, and body mass index-matched controls selected from a research population initially recruited for a study on sleep and meditation. SETTING: Sleep laboratory. PATIENTS OR PARTICIPANTS: Nine subjects with AHI > 10 and nine matched controls. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Topographic analysis of hdEEG data revealed a broadband reduction in EEG power in a circumscribed region overlying the parietal cortex in OSA subjects. This parietal reduction in neural activity was present, to some extent, across all frequency bands in all stages and episodes of nonrapid eye movement sleep. CONCLUSION: This investigation suggests that regional deficits in electroencephalography (EEG) power generation may be a useful clinical marker for neural disruption in obstructive sleep apnea, and that high-density EEG may have the sensitivity to detect pathological cortical changes early in the disease process.

Pharmacological Characterization of a Novel ENaCα siRNA (GSK2225745) With Potential for the Treatment of Cystic Fibrosis.

Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC(50) (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5' RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC(50) = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.Molecular Therapy - Nucleic Acids (2013) 2, e65; doi:10.1038/mtna.2012.57; published online 15 January 2013.

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.

CpG-free plasmid expression cassettes for cystic fibrosis gene therapy.

Clinical studies are underway for the aerosol delivery of plasmid DNA complexed with Genzyme Lipid GL67A to the lungs of patients with cystic fibrosis (CF). Plasmid vectors contain several functional elements all of which play a role in determining the efficacy of the final clinical product. To optimise the final plasmid, variations of CpG-free 5' enhancer elements and 3'UTR regions were inserted into a common CpG-free, plasmid backbone containing Luciferase or CFTR transgenes. Plasmids were compared in immortalised cell culture, human airway liquid interface primary cell cultures, and mouse lung models to determine which design directed optimal transgene expression. Following aerosol delivery to mouse lung, plasmids containing the murine CMV enhancer showed higher peak Luciferase activity than the human CMV enhancer, but the human version resulted in persistent expression. In cell culture, the SV40 3'UTR and a novel BGH2 3'UTR exhibited up to 20-fold higher Luciferase activity than the commonly used BGH 3'UTR, but in mouse lung aerosol studies the activity and duration was greater for BGH 3'UTR. Systematic evaluation of each functional component of the plasmid has resulted in an improved design, exhibiting superior levels and duration of lung gene expression.

The use of CpG-free plasmids to mediate persistent gene expression following repeated aerosol delivery of pDNA/PEI complexes.

Aerosol gene therapy offers great potential for treating acquired and inherited lung diseases. For treatment of chronic lung diseases such as cystic fibrosis, asthma and emphysema, non-viral gene therapy will likely require repeated administration to maintain transgene expression in slowly dividing, or terminally differentiated, lung epithelial cells. When complexed with plasmid DNA (pDNA), the synthetic polymer, 25 kDa branched Polyethylenimine (PEI), can be formulated for aerosol delivery to the lungs. We show that pDNA/PEI aerosol formulations can be repeatedly administered to airways of mice on at least 10 occasions with no detectable toxicity. Interestingly, peak reporter gene activity upon repeated delivery was significantly reduced by up to 75% compared with a single administration, despite similar pDNA lung deposition at each subsequent aerosol exposure. Although the precise mechanism of inhibition is unknown, it is independent of mouse strain, does not involve an immune response, and is mediated by PEI. Importantly, using a dosing interval of 56 days, delivery of a fourth-generation, CpG-free plasmid generated high-level, sustained transgene expression, which was further boosted at subsequent administrations. Together these data indicate that pDNA/PEI aerosol formulations offer a versatile platform for gene delivery to the lung resulting in sustained transgene expression suitable for treatment of chronic lung diseases.

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.

The use of carboxymethylcellulose gel to increase non-viral gene transfer in mouse airways.

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.

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression.

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (>or=56 d) in vivo transgene expression in the absence of lung inflammation.

Lack of repeat transduction by recombinant adeno-associated virus type 5/5 vectors in the mouse airway.

While recombinant adeno-associated virus (rAAV) vectors promote long-term transgene expression in the lungs and other organs, the goal of correcting chronic inherited lung diseases such as cystic fibrosis with this type of viral gene transfer vector is limited by the requirement of achieving stable potent transgene expression, potentially requiring vector readministration. Here we evaluated the abilities of rAAV type 5/5 (rAAV5/5) vectors based on the genome and capsid of AAV5 to efficiently transduce the lungs and nasal epithelium of mice after repeated administration. Transduction efficiency as judged by reporter gene expression was markedly reduced on a second rAAV5/5 administration and effectively abolished on a third. Varying the period between administrations from 8 to 36 weeks did not allow efficient repeated administration. A rapid rise in anti-AAV5 antibodies was noted after rAAV5/5 vector administration that was sustained for the entire period of investigation (in some cases exceeding 9 months). Furthermore, this antibody response and subsequent failure to repeatedly administer the vector were not rescued by the in vivo expression of CTLA4Ig from an rAAV5/5 vector. These results suggest that without the development of an effective and clinically acceptable immunosuppression strategy, treatments for chronic diseases that require repeated administration of rAAV5/5 vectors will be unsuccessful.