Pharmacological and pre-clinical safety profile of rSIV.F/HN, a hybrid lentiviral vector for cystic fibrosis gene therapy.

RATIONALE AND OBJECTIVE: Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators offer significant improvements, but approximately 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimized for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. METHODS: Air-liquid interface cultures of primary human bronchial epithelial cells (HBEC) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalized human lung epithelial cell line mimicking Class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay (PLA) for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. RESULTS: rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a Class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence ≤10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalization assay. CONCLUSIONS: The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

Genetic therapies in cystic fibrosis.

PURPOSE OF REVIEW: Advances in cystic fibrosis (CF) therapies over the past decade pivotally changed the morbidity and mortality of CF with the advent of cystic fibrosis transmembrane conductance regulator (CFTR) modulators that rescue dysfunctional CFTR protein in individuals with eligible genotypes. However, a significant proportion of the CF population is in need of alternative treatment strategies to address CFTR variants that are ineligible for therapeutic protein correction and/or potentiation. Current drug development efforts of nucleic-acid based therapies (i.e., DNA and RNA based therapies) in CF are informed by historic challenges of CF gene therapy trials, recent FDA guidance informed by non-CF gene therapy trials, and advances in therapeutic applications related to severe acute respiratory syndrome coronavirus 2 vaccine development. These historic and timely developments are of significant relevance for advancing genetic therapies in CF. RECENT FINDINGS: This article reviews the main themes of semi-permanent genetic therapy strategies covering recent literature focused on: adenovirus and adeno-associated virus vector delivery, advances in lentivirus vector use and safety considerations, mRNA delivery and antisense oligonucleotide drug development. SUMMARY: Currently, drug development and clinical trials for genetic therapies in CF are rapidly progressing. This review aims to increase the foundational knowledge of CF genetic therapies.

Identification and validation of a novel pathogenic variant in GDF2 (BMP9) responsible for hereditary hemorrhagic telangiectasia and pulmonary arteriovenous malformations.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant multisystemic vascular dysplasia, characterized by arteriovenous malformations (AVMs), mucocutaneous telangiectasia and nosebleeds. HHT is caused by a heterozygous null allele in ACVRL1, ENG, or SMAD4, which encode proteins mediating bone morphogenetic protein (BMP) signaling. Several missense and stop-gain variants identified in GDF2 (encoding BMP9) have been reported to cause a vascular anomaly syndrome similar to HHT, however none of these patients met diagnostic criteria for HHT. HHT families from UK NHS Genomic Medicine Centres were recruited to the Genomics England 100,000 Genomes Project. Whole genome sequencing and tiering protocols identified a novel, heterozygous GDF2 sequence variant in all three affected members of one HHT family who had previously screened negative for ACVRL1, ENG, and SMAD4. All three had nosebleeds and typical HHT telangiectasia, and the proband also had severe pulmonary AVMs from childhood. In vitro studies showed the mutant construct expressed the proprotein but lacked active mature BMP9 dimer, suggesting the mutation disrupts correct cleavage of the protein. Plasma BMP9 levels in the patients were significantly lower than controls. In conclusion, we propose that this heterozygous GDF2 variant is a rare cause of HHT associated with pulmonary AVMs.

Transepithelial nasal potential difference in patients with, and at risk of acute respiratory distress syndrome.

BACKGROUND: Impaired alveolar fluid clearance, determined in part by alveolar sodium transport, is associated with acute respiratory distress syndrome (ARDS). Nasal sodium transport may reflect alveolar transport. The primary objective of this prospective, observational study was to determine if reduced nasal sodium transport, as measured by nasal potential difference (NPD), was predictive of the development of and outcome from ARDS. METHODS: NPD was measured in 15 healthy controls and in 88 patients: 40 mechanically ventilated patients defined as 'at-risk' for ARDS, 61 mechanically ventilated patients with ARDS (13 who were previously included in the 'at-risk' group) and 8 ARDS survivors on the ward. RESULTS: In at-risk subjects, maximum NPD (mNPD) was greater in those who developed ARDS (difference -8.4 mV; 95% CI -13.8 to -3.7; p=0.005) and increased mNPD predicted the development of ARDS before its onset (area under the curve (AUC) 0.75; 95% CI 0.59 to 0.89). In the ARDS group, mNPD was not significantly different for survivors and non-survivors (p=0.076), and mNPD was a modest predictor of death (AUC 0.60; 95% CI 0.45 to 0.75). mNPD was greater in subjects with ARDS (-30.8 mV) than in at-risk subjects (-24.2 mV) and controls (-19.9 mV) (p<0.001). NPD values were not significantly different for survivors and controls (p=0.18). CONCLUSIONS: Increased NPD predicts the development of ARDS in at-risk subjects but does not predict mortality. NPD increases before ARDS develops, is greater during ARDS, but is not significantly different for controls and survivors. These results may reflect the upregulated sodium transport necessary for alveolar fluid clearance in ARDS. NPD may be useful as a biomarker of endogenous mechanisms to stimulate sodium transport. Larger studies are now needed to confirm these associations and predictive performance.

The murine lung as a factory to produce secreted intrapulmonary and circulatory proteins.

We have shown that a lentiviral vector (rSIV.F/HN) pseudotyped with the F and HN proteins from Sendai virus generates high levels of intracellular proteins after lung transduction. Here, we evaluate the use of rSIV.F/HN for production of secreted proteins. We assessed whether rSIV.F/HN transduction of the lung generates therapeutically relevant levels of secreted proteins in the lung and systemic circulation using human α1-anti-trypsin (hAAT) and factor VIII (hFVIII) as exemplars. Sedated mice were transduced with rSIV.F/HN carrying either the secreted reporter gene Gaussia luciferase or the hAAT or hFVIII cDNAs by nasal sniffing. rSIV.F/HN-hAAT transduction lead to therapeutically relevant hAAT levels (70 µg/ml) in epithelial lining fluid, with stable expression persisting for at least 19 months from a single application. Secreted proteins produced in the lung were released into the circulation and stable expression was detectable in blood. The levels of hFVIII in murine blood approached therapeutically relevant targets. rSIV.F/HN was also able to produce secreted hAAT and hFVIII in transduced human primary airway cells. rSIV.F/HN transduction of the murine lungs leads to long-lasting and therapeutically relevant levels of secreted proteins in the lung and systemic circulation. These data broaden the use of this vector platform for a large range of disease indications.

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.

Antipseudomonal Bacteriophage Reduces Infective Burden and Inflammatory Response in Murine Lung.

As antibiotic resistance increases, there is a need for new therapies to treat infection, particularly in cystic fibrosis (CF), where Pseudomonas aeruginosa is a ubiquitous pathogen associated with increased morbidity and mortality. Bacteriophages are an attractive alternative treatment, as they are specific to the target bacteria and have no documented side effects. The efficacy of phage cocktails was established in vitro. Two P. aeruginosa strains were taken forward into an acute murine infection model with bacteriophage administered either prophylactically, simultaneously, or postinfection. The infective burden and inflammation in bronchoalveolar lavage fluid (BALF) were assessed at various times. With low infective doses, both control mice and those undergoing simultaneous phage treatment cleared P. aeruginosa infection at 48 h, but there were fewer neutrophils in BALF of phage-treated mice (median, 73.2 × 10(4)/ml [range, 35.2 to 102.1 × 10(4)/ml] versus 174 × 10(4)/ml [112.1 to 266.8 × 10(4)/ml], P < 0.01 for the clinical strain; median, 122.1 × 10(4)/ml [105.4 to 187.4 × 10(4)/ml] versus 206 × 10(4)/ml [160.1 to 331.6 × 10(4)/ml], P < 0.01 for PAO1). With higher infective doses of PAO1, all phage-treated mice cleared P. aeruginosa infection at 24 h, whereas infection persisted in all control mice (median, 1,305 CFU/ml [range, 190 to 4,700 CFU/ml], P < 0.01). Bacteriophage also reduced CFU/ml in BALF when administered postinfection (24 h) and both CFU/ml and inflammatory cells in BALF when administered prophylactically. A reduction in soluble inflammatory cytokine levels in BALF was also demonstrated under different conditions. Bacteriophages are efficacious in reducing both the bacterial load and inflammation in a murine model of P. aeruginosa lung infection. This study provides proof of concept for future clinical trials in patients with CF.

Cystic fibrosis gene therapy: a mutation-independent treatment.

PURPOSE OF REVIEW: Since cloning of the disease-causing gene 27 years ago, the development of cystic fibrosis (CF) gene therapy has been pursued. Here, we will summarize key findings with a particular focus on recent developments. RECENT FINDINGS: Almost 3 decades of research have highlighted the complexity of lung gene transfer and have generated a body of data that has recently led to the completion of a large phase IIB study. This trial has, for the first time, shown that nonviral gene transfer can, albeit modestly, stabilize lung function in CF and provides the impetus for further development of more potent gene transfer agents. Lentiviral vectors, specifically pseudotyped to enable entry into airway epithelial cells have most recently been developed. Persistent expression after a single dose and the ability to be administered repeatedly suggest that these viral vectors hold promise for the treatment of CF; a first-in-man clinical trial will shortly be initiated. SUMMARY: Although the development of CF gene therapy has been slower than initially anticipated, recent progress has been encouraging and has renewed the interest of academics and industry to pursue lung gene therapy.

Moving forward: cystic fibrosis gene therapy.

Since cloning of the CFTR gene more than 20 years ago a large number of pre-clinical and clinical CF gene therapy studies have been performed and a vast amount of information and know-how has been generated. Here, we will review key studies with a particular emphasis on clinical findings. We have learnt that the lung is a more difficult target than originally anticipated, and we describe the strength and weaknesses of the most commonly used airway gene transfer agents (GTAs). In our view, one of the most significant developments in recent years is the generation of lentiviral vectors, which efficiently transduce lung tissue. However, focused and co-ordinated efforts assessing lentiviral vector safety and scaling up of production will be required to move this vector into clinical lung gene therapy studies.

Cyanide levels found in infected cystic fibrosis sputum inhibit airway ciliary function.

We have previously reported cyanide at concentrations of up to 150 µM in the sputum of cystic fibrosis patients infected with Pseudomonas aeruginosa and a negative correlation with lung function. Our aim was to investigate possible mechanisms for this association, focusing on the effect of pathophysiologically relevant cyanide levels on human respiratory cell function. Ciliary beat frequency measurements were performed on nasal brushings and nasal air-liquid interface (ALI) cultures obtained from healthy volunteers and cystic fibrosis patients. Potassium cyanide decreased ciliary beat frequency in healthy nasal brushings (n = 6) after 60 min (150 µM: 47% fall, p<0.0012; 75 µM: 32% fall, p<0.0001). Samples from cystic fibrosis patients (n = 3) showed similar results (150 µM: 55% fall, p = 0.001). Ciliary beat frequency inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited ciliary beat frequency in ALI cultures, albeit to a lesser extent. Ciliary beat frequency measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants defective in virulence factor production implicated cyanide as a key component inhibiting the ciliary beat frequency. If cyanide production similarly impairs mucocilliary clearance in vivo, it could explain the link with increased disease severity observed in cystic fibrosis patients with detectable cyanide in their airway.

Lung clearance index and high-resolution computed tomography scores in primary ciliary dyskinesia.

RATIONALE: Lung clearance index (LCI) is a more sensitive measure of lung function than spirometry in cystic fibrosis (CF) and correlates well with abnormalities in high-resolution computed tomography (HRCT) scanning. We hypothesized LCI would be equally sensitive to lung disease in primary ciliary dyskinesia (PCD). OBJECTIVES: To test the relationships between LCI, spirometry, and HRCT in PCD and to compare them to the established relationships in CF. METHODS: Cross-sectional study of 127 patients with CF and 33 patients with PCD, all of whom had spirometry and LCI, of which a subset of 21 of each had HRCT performed. HRCT was scored for individual features and these features compared with physiological parameters. MEASUREMENTS AND MAIN RESULTS: Unlike in CF, and contrary to our hypothesis, there was no correlation between spirometry and LCI in PCD and no correlation between HRCT features and LCI or spirometry in PCD. CONCLUSIONS: We show for the first time that HRCT, spirometry, and LCI have different relationships in different airway diseases and that LCI does not appear to be a sensitive test of airway disease in advanced PCD. We hypothesize that this results from dissimilarities between the components of large and small airway disease in CF and PCD. These differences may in part lead to the different prognosis in these two neutrophilic airway diseases.

A randomised, double-blind, placebo-controlled phase IIB clinical trial of repeated application of gene therapy in patients with cystic fibrosis.

The UK Cystic Fibrosis Gene Therapy Consortium has been working towards clinical gene therapy for patients with cystic fibrosis for several years. We have recently embarked on a large, multi-dose clinical trial of a non-viral, liposome-based formulation powered for the first time to detect clinical benefit. The article describes the details of the protocol.

Changes in physiological, functional and structural markers of cystic fibrosis lung disease with treatment of a pulmonary exacerbation.

BACKGROUND: Clinical trials in cystic fibrosis (CF) have been hindered by the paucity of well characterised and clinically relevant outcome measures. AIM: To evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for a clinical trial of CFTR gene therapy. METHODS: A multicentre observational study of adult and paediatric patients with CF (>10 years) treated for a physician-defined exacerbation of CF pulmonary symptoms. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers. RESULTS: Statistically significant improvements were seen in forced expiratory volume in 1 s (p<0.001, n=32), lung clearance index (p<0.01, n=32), symptoms (p<0.0001, n=37), CT scores for airway wall thickness (p<0.01, n=31), air trapping (p<0.01, n=30) and large mucus plugs (p=0.0001, n=31), serum C-reactive protein (p<0.0001, n=34), serum interleukin-6 (p<0.0001, n=33) and serum calprotectin (p<0.0001, n=31). DISCUSSION: We identify the key biomarkers of inflammation, imaging and physiology that alter alongside symptomatic improvement following treatment of an acute CF exacerbation. These data, in parallel with our study of biomarkers in patients with stable CF, provide important guidance in choosing optimal biomarkers for novel therapies. Further, they highlight that such acute therapy predominantly improves large airway parameters and systemic inflammation, but has less effect on airway inflammation.

Oral contraceptives do not appear to affect cystic fibrosis disease severity.

Several studies suggest that sex may affect cystic fibrosis (CF) disease severity, with females with CF being more severely affected. In this context, it has been suggested that sex hormones may influence the CF phenotype. A large proportion of females with CF regularly use oral contraceptives (OCs), but the effect of their use on disease severity is unclear. Here, we retrospectively assessed the effects of OCs on clinical outcomes in females with CF. Data from 681 females were available, of whom 42% had taken OCs for varying periods of time. We first performed an inter-patient analysis comparing annual change in % predicted forced expiratory volume in 1 s, body mass index and total days of intravenous antibiotic use over a 5-yr study period in 57 females exposed to and 57 females not exposed to OCs. There were no differences between the two groups. We next performed an intra-patient analysis of the same outcomes over a 3-yr period of OC exposure and a 3-yr period of no OC exposure in the same patient (exposure followed by non-exposure, n=27; non-exposure followed by exposure, n=23), but again did not detect any differences in any of the clinical outcomes. Our data suggests that the use of OCs does not affect CF disease severity.

Assessment of F/HN-pseudotyped lentivirus as a clinically relevant vector for lung gene therapy.

RATIONALE: Ongoing efforts to improve pulmonary gene transfer thereby enabling gene therapy for the treatment of lung diseases, such as cystic fibrosis (CF), has led to the assessment of a lentiviral vector (simian immunodeficiency virus [SIV]) pseudotyped with the Sendai virus envelope proteins F and HN. OBJECTIVES: To place this vector onto a translational pathway to the clinic by addressing some key milestones that have to be achieved. METHODS: F/HN-SIV transduction efficiency, duration of expression, and toxicity were assessed in mice. In addition, F/HN-SIV was assessed in differentiated human air-liquid interface cultures, primary human nasal epithelial cells, and human and sheep lung slices. MEASUREMENTS AND MAIN RESULTS: A single dose produces lung expression for the lifetime of the mouse (~2 yr). Only brief contact time is needed to achieve transduction. Repeated daily administration leads to a dose-related increase in gene expression. Repeated monthly administration to mouse lower airways is feasible without loss of gene expression. There is no evidence of chronic toxicity during a 2-year study period. F/HN-SIV leads to persistent gene expression in human differentiated airway cultures and human lung slices and transduces freshly obtained primary human airway epithelial cells. CONCLUSIONS: The data support F/HN-pseudotyped SIV as a promising vector for pulmonary gene therapy for several diseases including CF. We are now undertaking the necessary refinements to progress this vector into clinical trials.

Lytic Bacteriophage Is a Promising Adjunct to Common Antibiotics across Cystic Fibrosis Clinical Strains and Culture Models of Pseudomonas aeruginosa Infection.

Bacteriophages (phages) are antimicrobials with resurgent interest that are being investigated for the treatment of antibiotic refractory infection, including for Pseudomonas aeruginosa (Pa) lung infection in cystic fibrosis (CF). In vitro work supports the use of this therapy in planktonic and biofilm culture models; however, consistent data are lacking for efficacy across different clinical Pa strains, culture models, and in combination with antibiotics in clinical use. We first examined the efficacy of a 4-phage cocktail as an adjunct to our CF centre's first-line systemic combination antibiotic therapy (ceftazidime + tobramycin) for 16 different clinical Pa strains and then determined subinhibitory interactions for a subset of these strains with each antibiotic in planktonic and biofilm culture. When a 4-phage cocktail (4 × 108 PFU/mL) was added to a ceftazidime-tobramycin combination (ceftazidime 16 mg/mL + tobramycin 8 mg/mL), we observed a 1.7-fold and 1.3-fold reduction in biofilm biomass and cell viability, respectively. The four most antibiotic resistant strains in biofilm were very susceptible to phage treatment. When subinhibitory concentrations of antibiotics and phages were investigated, we observed additivity/synergy as well as antagonism/inhibition of effect that varied across the clinical strains and culture model. In general, more additivity was seen with the phage-ceftazidime combination than with phage-tobramycin, particularly in biofilm culture, where no instances of additivity were seen when phages were combined with tobramycin. The fact that different bacterial strains were susceptible to phage treatment when compared to standard antibiotics is promising and these results may be relevant to ongoing clinical trials exploring the use of phages, in particular in the selection of subjects for clinical trials.