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.

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of novel vaccines and therapeutics. Here, we report on diverse camelid single-domain antibodies to influenza hemagglutinin from which we generated multi-domain antibodies with unprecedented breadth and potency. Multi-domain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle EM structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multi-domain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide a groundbreaking new strategy to prevent infection with influenza virus and other highly variable pathogens.

Airway-directed gene transfer has emerged as a promising approach for the treatment of the two genetic diseases of the lung, namely cystic fibrosis and α-1-antitrypsin deficiency. Herein we describe the transduction efficiency of a novel adeno-associated virus (AAV) vector, AAV2/9, across murine nasal and lung airway epithelia. At the peak of gene expression AAV2/9-mediated human α-1-antitrypsin gene expression in serum was ≈60-fold better than that of AAV2/5. We found that AAV2/9-mediated n LacZ gene transfer in nasal and lung airways was relatively stable for 9 months, suggesting that a progenitor airway cell population was transduced. Most interestingly, we show that AAV2/9 can be readministered in the presence of high levels of serum-circulating neutralizing antibodies as early as 1 month after initial exposure, with minimal effect on overall reporter gene expression, rendering it a promising gene transfer vector candidate for use in humans.

The potential for gene therapy to be an effective treatment for cystic fibrosis (CF) airway disease has been limited by inefficient gene transfer vector particle delivery and lack of persistent gene expression. We have developed an airway conditioning process that, when combined with a human immunodeficiency virus (HIV)-derived lentivirus (LV) vector, resulted in persistent in vivo expression of transgenes in airway epithelium. Pretreatment of mouse nasal epithelium with the detergent lysophosphatidylcholine (LPC) prior to instillation of a single dose of an LV vector carrying the LacZ marker gene produced significant LacZ gene expression in nasal airway epithelium for at least 92 days. Transduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using the same LV vector system resulted in partial recovery of electrophysiologic function in the nasal airway epithelium of CF mice (cftr(tm1Unc) knockout) for at least 110 days. This first demonstration of LV-mediated in vivo recovery of CFTR function in CF airway epithelium illustrates the potential of combining a preconditioning of the airway surface with a simple and brief LV vector exposure to produce therapeutic gene expression in airway.

Vectors based on adeno-associated virus (AAV) serotype 9 are candidates for in vivo gene delivery to many organs, but the receptor(s) mediating these tropisms have yet to be defined. We evaluated AAV9 uptake by glycans with terminal sialic acids (SAs), a common mode of cellular entry for viruses. We found, however, that AAV9 binding increased when terminal SA was enzymatically removed, suggesting that galactose, which is the most commonly observed penultimate monosaccharide to SA, may mediate AAV9 transduction.

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We have characterized the ability of adeno-associated virus (AAV) serotypes 1-9 in addition to nineteen novel vectors isolated from various tissues, to transduce mouse and human ciliated airway epithelium (HAE). Vectors expressing alpha-1-antitrypsin (AAT) and beta-galactosidase were co-instilled into the mouse lung. Of all the vectors tested rh.64R1, AAV5 and AAV6 were the most efficient. The high transduction observed in mouse was reproduced in HAE cell cultures for both rh.64R1 and AAV6 but not for AAV5. Since AAV6 was the most efficient vector in mouse and HAE we also tested the transduction efficiencies of the AAV6 singleton vectors (i.e., AAV6 variants with targeted mutations) in these models. Of these, AAV6.2 transduced mouse airway epithelium and HAE with greater efficiency than all other AAV vectors tested. We demonstrated that AAV6.2 exhibits improved transduction efficiency compared to previously reported AAVs in mouse airways and in culture models of human airway epithelium and that this vector requires further development for preclinical and clinical testing.

CRB1 mutations lead to early-onset severe loss of vision with thickened, disorganized, nonseeing retina. Impaired peripheral vision can persist in late disease stages. Rd8 mice also have a disorganized retina, but there is sufficient photoreceptor integrity to produce largely normal retinal function. Differences between human and mouse diseases will complicate proof-of-concept studies intended to advance treatment initiatives.