SummaryBackgroundLung 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.MethodsWe 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.FindingsBetween 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.InterpretationMonthly 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.FundingMedical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme.

Loss-of-function (LOF) variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including developmental and epileptic encephalopathy and cardiac arrhythmia. β1 Subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming α subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Here, we investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase and show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA sequencing, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b -null cardiac tissue, which lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. We propose that human disease variants resulting in SCN1B LOF cause transcriptional dysregulation that contributes to altered excitability. Moreover, these results provide important insights into the mechanism of SCN1B -linked channelopathies, adding RIP-excitation coupling to the multifunctionality of sodium channel β1 subunits.

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BackgroundCystic 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-mediatedCFTRgene therapy formulation through preclinical and clinical development.ObjectiveTo determine clinical efficacy of the formulation delivered to the airways over a period of 1 year in patients with CF.DesignThis was a randomised, double-blind, placebo-controlled Phase IIb trial of theCFTRgene–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.SettingsData were collected in the clinical and scientific sites and entered onto a trial-specific InForm, version 4.6 database.ParticipantsPatients with CF aged ≥ 12 years with forced expiratory volume in the first second (FEV1) between 50% and 90% predicted and any combination ofCFTRmutations. The per-protocol group (≥ 9 doses) consisted of 54 patients receiving placebo (62 randomised) and 62 patients receiving gene therapy (78 randomised).InterventionsSubjects received 5 ml of nebulised pGM169/G67A (active) or 0.9% saline (placebo) at 28 (±5)-day intervals over 1 year.Main outcome measuresThe primary end point was the relative change in percentage predicted FEV1over 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.ResultsThere 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 orCFTRmutation class. Subjects with a more severe baseline FEV1had a FEV1TE 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.ConclusionsMonthly 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.LimitationsAlthough encouraging, the improvement in FEV1was modest and was not accompanied by detectable improvement in patients’ quality of life.Future workFuture 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 registrationClinicalTrials.gov NCT01621867.FundingThis project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research partnership.

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There have been several reports that individuals with Fragile X syndrome (FXS) and animal models of FXS have communication deficits. The present study utilized two different call classification taxonomies to examine the sex-specificity of ultrasonic vocalization (USV) production on postnatal day (PD8) in the FVB strain of Fmr1 knockout (KO) mice. One classification protocol requires the investigator to score each call by hand, while the other protocol uses an automated algorithm. Results using the hand-scoring protocol indicated that male Fmr1 KO mice exhibited longer calls (P = .03) than wild types on PD8. Male KOs also produced fewer complex, composite, downward, short and two-syllable call-types, as well as more frequency steps and chevron call-types. Female heterozygotes exhibited no significant changes in acoustic or temporal aspects of calls, yet showed significant changes in call-type production proportions across two different classification taxonomies (P < .001). They

The Fmr1 knockout (KO) mouse has commonly been used to investigate communication impairments, one of the key diagnostic symptoms observed in Fragile X syndrome (FXS) and Autism spectrum disorder (ASD). Many studies have found alterations in ultrasonic vocalizations (USVs) in neonatal Fmr1 KO mice, however, there is limited research investigating whether these deficits continue into adulthood. In the present study, we examine differences in female urine-induced ultrasonic vocalizations, scent marking behavior, odor discrimination, and open field activity in adult male Fmr1 KO and wildtype (WT) mice. Overall, we found extensive alterations between genotypes in both spectral and temporal properties of ultrasonic vocalizations. There was no difference in the average number of calls emitted by both genotypes, however, Fmr1 KO mice emitted calls of a higher frequency, decreased amplitude, and shorter duration than WT mice. Spectrographic analyses revealed statistically significant differences between genotypes in the types of calls emitted. Contrastingly, we found no differences in scent marking behavior, a form of social communication, or in odor discrimination and activity levels of the mice. The results corroborate previous studies emphasizing the importance of qualitative differences observed in vocalization behavior of Fmr1 KO mice, rather than quantitative measurements such as number of calls emitted. Overall, the study confirms the presence of abnormalities in vocalization behavior in adult Fmr1 KO mice that we believe are consistent with communication deficits seen in the syndrome.

Dysregulation of the PI3K/Akt/mTOR signaling cascade has been associated with the pathology of neurodegenerative disorders, specifically Alzheimer's disease (AD). Both in-vivo models and post-mortem brain samples of individuals with AD have commonly shown hyperactivation of the pathway. In the present study, we examine how neuron subset-specific deletion of Pten (NS-Pten) in mice, which presents with hyperactive mammalian target of rapamycin (mTOR) activity, affects the hippocampal protein levels of key neuropathological hallmarks of AD. We found NS-Pten knockout (KO) mice to have elevated levels of amyloid-β, α-synuclein, neurofilament-L, and pGSK3α in the hippocampal synaptosome compared with NS-Pten wild type mice. In contrast, there was a decreased expression of amyloid precursor protein, tau, GSK3α, and GSK3β in NS-Pten KO hippocampi. Overall, there were significant alterations in levels of proteins associated with AD pathology in NS-Pten KO mice. This study provides novel insight into how altered mTOR signaling is linked to AD pathology, without the use of an in-vivo AD model that already displays neuropathological hallmarks of the disease.