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.
Respiratory epithelium is the target of therapies, such as gene therapy, for cystic fibrosis (CF) lung disease. To determine the usefulness of the nasal epithelium as a pre-screen for lung-directed ...therapies, we profiled gene expression in CF and non-CF nasal and bronchial epithelium samples using Illumina HumanRef-8 Expression BeadChips. 863 genes were differentially expressed between CF and non-CF bronchial epithelium but only 15 were differentially expressed between CF and non-CF nasal epithelium (
≥
1.5-fold,
P
≤
0.05). The most enriched pathway in CF bronchial epithelium was inflammatory response, whereas in CF nasal epithelium it was amino acid metabolism. We also compared nasal and bronchial epithelium in each group and identified differential expression of cellular movement genes in CF patients and cellular growth genes in non-CF subjects. We conclude that CF and non-CF nasal and bronchial epithelium are transcriptionally distinct and CF nasal epithelium is not a good surrogate for the lung respiratory epithelium.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The protein transduction domain (PTD) of the HIV-1 Tat protein can facilitate the cellular and nuclear uptake of macromolecular particles. Here, we demonstrate that incorporation without covalent ...linkage of a 17-amino acid PTD peptide into gene delivery lipoplexes improves gene transfer. Tat/Liposome/DNA (TLD) transfection, as evaluated by Fluorescence Activated Cell Scan analysis of a Green Fluorescence Protein expression plasmid, enabled transfection of highly recalcitrant primary cells in the form of air/liquid interface cultures of sheep tracheal epithelium. Treatment with chloroquine increased, and incubation at low temperature decreased, TLD transfection, suggesting that the endocytosis uptake pathway is involved.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Efficient gene transfer to the airways by nonviral vectors is a function of different parameters, among which the size and the charge of the transfecting particles. The aim of this study was to ...determine the transfection efficiency of polyethylenimine (PEI)/albumin polyplexes in ex vivo and in vivo models of respiratory epithelium and to correlate it with biophysical characteristics of the particles. Complexes were obtained by adding different amounts of human serum albumin (HSA) to PEI polyplexes preformed in saline. The presence of HSA caused the formation of bigger and more negative polyplexes and increased PEI transfection efficiency in primary respiratory epithelial cells by 4–6-fold. For in vivo administration to the lung, PEI polyplexes were formed in water and optimized with respect to the N/P ratio. PEI/pC-Luc complexes gave the highest luciferase expression at N/P 15 when administered through the trachea. At this N/P ratio, the size and the surface charge of albumin-containing polyplexes were not different as compared with plain PEI polyplexes. Formulation of PEI polyplexes in the presence of HSA or murine serum albumin (MSA) resulted in a 2-fold increase in luciferase expression. In mice treated with PEI or PEI/MSA polyplexes containing the nuclear β-gal gene, X-gal staining revealed that transfected cells localized at the bronchiolar epithelium and that PEI/MSA transfected four times as many cells as PEI (p < 0.05). Finally, double administration of PEI/MSA polyplexes resulted in a further enhancement of transfection of the lung. Our data show that serum albumin enhances PEI-mediated gene transfer to airway epithelial cells in vivo, likely facilitating the uptake of polyplexes, and indicate that this formulation would fulfill the requirement of repeated administration, as necessary in chronic lung diseases like cystic fibrosis.
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IJS, KILJ, NUK, PNG, UL, UM
Transcription activity of chromatin is associated with histone acetylation which is regulated by recruitment of histone acetyltransferases and deacetylases (HDAC) to specific chromatin regions. We ...have tested how expression of a transfected or stably introduced gene correlates with histone acetylation. Our results demonstrate that expression of transiently transfected green fluorescence protein (GFP) genes is significantly enhanced by HDAC inhibitors. Although HDAC treatment did not induce noticeable changes in the chromatin structure of genomic DNA, chromatin immunoprecipitation showed that more transiently transfected DNA is assembled into chromatin containing acetylated histones in HDAC inhibitor treated cells when compared to untreated cells. For stably integrated GFP, the expression response to HDAC inhibitors varies between independent stable cell lines. However, there was no difference in histone acetylation associated with the integrated transgene between HDAC inhibitor responsive and non-responsive cells. Furthermore, the overall enhancement of transgene expression by HDAC inhibitors was not as pronounced as in transiently transfected cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background
Cystic fibrosis (CF) is a chronic, life-limiting disease caused by mutations in the CF transmembrane conductance regulator (
CFTR
) gene leading to abnormal airway surface ion transport, ...chronic lung infections, inflammation and eventual respiratory failure. With the exception of the small-molecule potentiator, ivacaftor (Kalydeco
®
, Vertex Pharmaceuticals, Boston, MA, USA), which is suitable for a small proportion of patients, there are no licensed therapies targeting the basic defect. The UK Cystic Fibrosis Gene Therapy Consortium has taken a cationic lipid-mediated
CFTR
gene therapy formulation through preclinical and clinical development.
Objective
To determine clinical efficacy of the formulation delivered to the airways over a period of 1 year in patients with CF.
Design
This was a randomised, double-blind, placebo-controlled Phase IIb trial of the
CFTR
gene–liposome complex pGM169/GL67A. Randomisation was performed via InForm™ version 4.6 (Phase Forward Incorporated, Oracle, CA, USA) and was 1 : 1, except for patients in the mechanistic subgroups (2 : 1). Allocation was blinded by masking nebuliser chambers.
Settings
Data were collected in the clinical and scientific sites and entered onto a trial-specific InForm, version 4.6 database.
Participants
Patients with CF aged ≥ 12 years with forced expiratory volume in the first second (FEV
1
) between 50% and 90% predicted and any combination of
CFTR
mutations. The per-protocol group (≥ 9 doses) consisted of 54 patients receiving placebo (62 randomised) and 62 patients receiving gene therapy (78 randomised).
Interventions
Subjects received 5 ml of nebulised pGM169/G67A (active) or 0.9% saline (placebo) at 28 (±5)-day intervals over 1 year.
Main outcome measures
The primary end point was the relative change in percentage predicted FEV
1
over the 12-month period. A number of secondary clinical outcomes were assessed alongside safety measures: other spirometric values; lung clearance index (LCI) assessed by multibreath washout; structural disease on computed tomography (CT) scan; the Cystic Fibrosis Questionnaire – Revised (CFQ-R), a validated quality-of-life questionnaire; exercise capacity and monitoring; systemic and sputum inflammatory markers; and adverse events (AEs). A mechanistic study was performed in a subgroup in whom transgene deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) was measured alongside nasal and lower airway potential difference.
Results
There was a significant (
p
= 0.046) treatment effect (TE) of 3.7% 95% confidence interval (CI) 0.1% to 7.3% in the primary end point at 12 months and in secondary end points, including forced vital capacity (FVC) (
p
= 0.031) and CT gas trapping (
p
= 0.048). Other outcomes, although not reaching statistical significance, favoured active treatment. Effects were noted by 1 month and were irrespective of sex, age or
CFTR
mutation class. Subjects with a more severe baseline FEV
1
had a FEV
1
TE of 6.4% (95% CI 0.8% to 12.1%) and greater changes in many other secondary outcomes. However, the more mildly affected group also demonstrated benefits, particularly in small airway disease markers such as LCI. The active group showed a significantly (
p
= 0.032) greater bronchial chloride secretory response. No difference in treatment-attributable AEs was seen between the placebo and active groups.
Conclusions
Monthly application of the pGM169/GL67A gene therapy formulation was associated with an improvement in lung function, other clinically relevant parameters and bronchial CFTR function, compared with placebo.
Limitations
Although encouraging, the improvement in FEV
1
was modest and was not accompanied by detectable improvement in patients’ quality of life.
Future work
Future work will focus on attempts to increase efficacy by increasing dose or frequency, the coadministration of a CFTR potentiator, or the use of modified viral vectors capable of repeated administration.
Trial registration
ClinicalTrials.gov NCT01621867.
Funding
This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research partnership.
Purpose
In this proof-of-concept pilot study, our purpose was to determine correlations between gut microbiota composition and alterations in cardiorespiratory fitness and psychosocial outcomes among ...post-primary treatment breast cancer survivors (BCS).
Methods
Composition of the gut microbiota in BCS (
n
= 12) was assessed at baseline (M0) and at the end of 3 months (M3) using Illumina MiSeq DNA Sequencing of the 16S rRNA gene. Gut microbiota composition was analyzed using the QIIME bioinformatics software and represented through diversity metrics and taxa analyses. Cardiorespiratory fitness, fatigue, anxiety, depression, and sleep dysfunction were assessed at M0 and M3 via the submaximal treadmill test, Fatigue Symptom Inventory, Hospital Anxiety and Depression Scale, and Pittsburgh Sleep Quality Index, respectively.
Results
Increased fatigue interference in BCS was associated with increased mean within-sample Shannon diversity (organism richness and evenness) (
p
= 0.009). Weighted UniFrac analysis (shifts in taxa relative abundance) revealed significant differences in between-sample (beta) diversity for changes in fatigue interference (
p
= 0.01) and anxiety (
p
= 0.022), with a trend observed for fatigue intensity and sleep dysfunction (
p
< 0.1). Unweighted UniFrac analysis (shifts in taxa types) found significant beta diversity differences for cardiorespiratory fitness (
p
= 0.026). Prior to false discovery correction (FDR), changes in fitness, fatigue, anxiety, and sleep dysfunction were associated with the frequency of certain gut bacteria genera (e.g.,
Faecalibacterium
,
Prevotella
,
Bacteroides
) (
p
< 0.05).
Conclusions
Correlations may exist between alterations in gut microbiota composition and longitudinal changes in cardiorespiratory fitness, fatigue, and anxiety in BCS. Further research examining the role of the microbiota-gut-brain axis in exercise-induced effects on psychosocial outcomes in BCS is warranted.
Geophysical and geochemical data indicate there is abundant fluid expulsion in the Nootka fault zone (NFZ) between the Juan de Fuca and Explorer plates and the Nootka continental slope. Here we ...combine observations from >20 years of investigations to demonstrate the nature of fluid‐flow along the NFZ, which is the seismically most active region off Vancouver Island. Seismicity reaching down to the upper mantle is linked to near‐seafloor manifestation of fluid flow through a network of faults. Along the two main fault traces, seismic reflection data imaged bright spots 100–300 m below seafloor that lie above changes in basement topography. The bright spots are conformable to sediment layering, show opposite‐to‐seafloor reflection polarity, and are associated with frequency reduction and velocity push‐down indicating the presence of gas in the sediments. Two seafloor mounds ~15 km seaward of the Nootka slope are underlain by deep, nonconformable high‐amplitude reflective zones. Measurements in the water column above one mound revealed a plume of warm water, and bottom‐video observations imaged hydrothermal vent system biota. Pore fluids from a core at this mound contain predominately microbial methane (C1) with a high proportion of ethane (C2) yielding C1/C2 ratios <500 indicating a possible slight contribution from a deep source. We infer the reflective zones beneath the two mounds are basaltic intrusions that create hydrothermal circulation within the overlying sediments. Across the Nootka continental slope, gas hydrate‐related bottom‐simulating reflectors are widespread and occur at depths indicating heat flow values of 80–90 mW/m2.
Key Points
Fluid flow is focused along Nootka Fault traces resulting in shallow bright spots
Two seafloor mounds are the result of basaltic intrusions in the Nootka fault zone
Gas hydrates occur at the Nootka Slope and are imaged seismically as bottom‐simulating reflectors suggesting a regional heat flow of ~80 mW/m2 along the slope
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
This paper presents a meta-algorithm for approximating the Pareto optimal set of costly black-box multiobjective optimization problems given a limited number of objective function evaluations. The ...key idea is to switch among different algorithms during the optimization search based on the predicted performance of each algorithm at the time. Algorithm performance is modeled using a machine learning technique based on the available information. The predicted best algorithm is then selected to run for a limited number of evaluations. The proposed approach is tested on several benchmark problems and the results are compared against those obtained using any one of the candidate algorithms alone.
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