Cystic fibrosis (CF) results from mutations within the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a transmembrane chloride channel found on the apical surface of epithelial cells. The most common CF-causing mutation results in a deletion of phenylalanine 508 (ΔF508-CFTR), a residue normally found within the NBD1 domain. Loss of F508 causes NBD1 to be less thermodynamically stable and prevents proper tertiary folding of CFTR. As a result, CFTR is not properly trafficked to the cell surface. Recently, progress has been made towards the development of small molecule “correctors” that can restore CFTR tertiary structure and stabilize the channel to overcome the instability inherent in ΔF508-CFTR. However, the resultant improvement in channel activity has been modest, and the need for potent correctors remains. To fully inform such efforts, a better understanding of the molecular pathology associated with ΔF508-CFTR is required. Here we present a comprehensive study of the impact of F508 deletion on both purified NBD1 and full-length CFTR. Through the use of homology modeling, molecular dynamics simulations, mutational analysis, biochemical, biophysical and functional characterization studies, we obtained insight into how the ΔF508 mutation may lead to helical unraveling of transmembrane domains 10 and 11 (TM10, TM11), and how the known suppressor mutations V510D and R1070W, as well as novel second site suppressor mutations (SSSMs) identified in this work, may act to rescue ΔF508-CFTR maturation and trafficking.

Liposomal delivery systems for nucleic acids fall within the larger class of non-viral delivery vehicles. Chronologically, liposomal systems designed to encapsulate pDNA were among the first non-viral systems to demonstrate gene delivery. Plasmid DNA by itself has transfection activity in some systems. The enhancement in transfection efficiencies of these delivery systems over those of liposomes is imparted by the fusion properties of viral envelopes, such as the F protein of Sendai virus. The haemagglutinating virus of Japan system has also been used successfully to deliver antisense oligonucleotides in vivo to inhibit neointimal hyperplasia in a model of restenosis. The immune advantage of purely cationic lipid systems is offset somewhat by the lower potency of these delivery systems compared with viral systems. DC-Choi was used to deliver a plasmid containing the B7 co-stimulatory molecule as part of an immune system stimulation protocol designed to attack melanoma cells.

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.

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.

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.

The mammalian target of rapamycin complex 1 (mTORC1) regulates insulin-mediated glucose metabolism, cell proliferation, the oxidative branch of the pentose phosphate pathway, de novo lipogenesis, and autophagy. Ribosomal S6 kinase 1 (S6K1) and 2 (S6K2) are downstream effectors of mTORC1. To characterize the role of S6K2 in insulin-mediated metabolism, the response of S6K2 deficient mice (S6K2-/-) to a glucose challenge was compared to that of wild-type (C57BL/6) and diabetes resistant strains (BALB/c and A/J) after 35 weeks on a high fat diet (HFD). Although S6K2-/- mice fed a HFD gained as much weight as the wild-type C57BL/6 control mice, unlike the wild-type mice they remained glucose tolerant, insulin sensitive, and had lower basal blood glucose levels. Moreover, unlike S6K1 deficient mice, S6K2-/- mice have increased basal plasma insulin levels and increased beta-cell mass compared to C57BL/6, BALB/c, and A/J mice. Administration of insulin to S6K2-/-and C57BL/6 mice fed a Standard Diet (SD) resulted in phosphorylation of Ser307 on skeletal muscle Insulin Receptor Substrate 1 (IRS-1); however, when both strains were fed a HFD, phosphorylation of IRS-1 Ser307 was maintained in S6K2-/- mice but inhibited in C57BL/6 mice. Taken together, these results suggest that S6K2 inhibition may represent a strategy for treating type 2 diabetes.

Although most CF patients express CFTR protein (albeit mutant) and should therefore not recognise the wild-type CFTR protein as foreign, there is an inherent risk of activation of T-cells against the recombinant wild-type protein after gene therapy. In addition, we have previously shown that approximately 10% of CF and non-CF subjects carry self-reactive CFTR-specific T-cells (Calcedo et al, Hum Gene Ther Clin Dev 2013). The reason for this is unknown and it is also unclear whether being positive for self-reactive T-cells affects disease severity or increases the risk of further T-cell activation after gene therapy. As part of the UKCFGTC Phase I/IIa Pilot study [in which patients received a single dose (5, 10 or 20 mls) of the non-viral formulation pGM169/GL67A] peripheral blood mononuclear cells (PBMC) were collected prior to dosing and approximately 4 weeks after nebulisation of 5 ml (n = 2), 10 ml (n = 6) or 20 ml (n = 17) of pGM169/GL67A. IFN-g ELISPOT to detect CFTR-specific T-cells in PBMC was performed. CFTR-specific T-cells were detectable in one patient pre- and post-dosing. In the remaining 18 patients we did not detect CFTR-specific T-cells. In addition we quantified anti-DNA antibodies (anti-nuclear and anti-cytoplasmic) in blood samples taken pre- and approximately 4 weeks post-dosing (n = 7 (5 ml), n = 10 (10 ml) and n = 17 (20 ml). We did not observe any evidence for induction of anti-DNA antibodies after a single dose of pGM169/GL67A. The UKCFGTC has now completed a Phase IIb multi-dose clinical trial in May 2014 (ClinicalTrials.gov identification number – NCT01621867). CF patients received 12 monthly doses of pGM169/GL67A (115 completed nine or more doses), or placebo by aerosol. PBMC were collected on two occasions prior to dose 1 to establish baseline levels for CFTR-specific T-cells, approximately 4 weeks after Dose 4 or 5, and 2 to 4 weeks after Dose 12 and the ELISPOT was performed. In addition anti-DNA antibodies were quantified. The Phase IIb trial will be unblinded in Summer 2014 to allow data analysis and all data will be presented at the conference. Funded by the NIHR/EME Programme and the Cystic Fibrosis Trust.