T Higgins

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.

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.

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.

Toxicology study assessing efficacy and safety of repeated administration of lipid/DNA complexes to mouse lung

For gene therapy to improve lung function in cystic fibrosis (CF) subjects, repeated administration of the gene transfer agent over the lifetime of patients is likely to be necessary. This requirement limits the utility of adenoviral and adeno-associated viral vectors (both previously evaluated in CF gene therapy trials) because of induced adaptive immune responses that render repeated dosing ineffective. For CF gene therapy trials, non-viral vectors are currently the only viable option. We previously showed that the cationic lipid formulation GL67A is the most efficient of several non-viral vectors analysed for airway gene transfer. Here, we assessed the efficacy and safety of administering 12 inhaled doses of GL67A complexed with pGM169, a CpG-free plasmid encoding human CFTR complementary DNA, into mice. We show that repeated administration of pGM169/GL67A to murine lungs is feasible, safe and achieves reproducible, dose-related and persistent gene expression (>140 days after each dose) using an aerosol generated by a clinically relevant nebuliser. This study supports progression into the first non-viral multidose lung trial in CF patients.

A Phase I/IIa Safety and Efficacy Study of Nebulized Liposome-mediated Gene Therapy for Cystic Fibrosis Supports a Multidose Trial

To the Editor: The vast majority of treatments for cystic fibrosis (CF) target the downstream consequences of the disease and are incompletely effective. The success of the CF transmembrane conductance regulator (CFTR) potentiator ivacaftor has illustrated the clinical benefits arising from restoration of CFTR protein function (1). This agent is applicable as a monotherapy for a minority of patients with specific, rare mutations. CFTR gene therapy, a mutation-independent alternative, has demonstrated proof of principle for gene transfer in animal models and human trials, but only one study (using a viral vector) has unsuccessfully assessed whether clinical outcomes can be improved (2). In preparation for a phase IIb clinical trial of repeatedly administered, nonviral, liposome-mediated CFTR gene transfer assessing clinically meaningful outcomes (3), the UK CF Gene Therapy Consortium (www.cfgenetherapy.org.uk) undertook a single-application safety and dose-ranging study (NCT00789867). Some of the results of these studies have been previously reported in the form of abstracts (4, 5). The chosen plasmid DNA expresses CFTR under the control of the human cytomegalovirus enhancer/elongation factor 1α sequence (6), a modified EF1a promoter aiming for extended duration of expression (7), and was rendered CpG-free to minimize a host inflammatory response (6). The cationic lipid, GL67A, was chosen on the basis of extensive preclinical testing (8). After informed consent, adult patients with CF received a single nebulized +/− nasal dose of pGM169/GL67A. Reconstitution and preparation of pGM169/GL67A was undertaken on the day, and doses were delivered in sealed negative-pressure cubicles after pretreatment with inhaled salbutamol (albuterol). Preplanned adjunctive therapies including ibuprofen, prednisolone, or paracetamol were administered to some patients. The primary outcome of the clinical study was safety; assessment included examination, standard hematology/biochemistry, adverse events, spirometry, lung clearance index, chest computed tomography scan, gas transfer, bronchial biopsy histology, and immune markers. pGM169-specific DNA and mRNA were measured on nasal and lower airway brushings, with potential difference also measured bronchoscopically and nasally. For the latter, “responders” were defined as demonstrating chloride secretion 5 mV or more greater than their mean predose value, and greater than any of their predose responses. A total of 35 subjects (Tables 1 and ​and2)2) received a nebulized dose (5 ml, n = 8; 10 ml, n = 10; 20 ml, n = 17) via an AeroEclipse II (Trudell Medical International, London, ON, Canada) breath-actuated nebulizer (9). Three subjects undertook slow delivery (∼75 vs. 25 min for each 5 ml). Standard spray devices were used for nasal delivery (2 ml, n = 21). According to pre-/post-device weighing, a mean (SD) of 88.7% (2.9%) of expected nebulized dose and 94.5% (15.0%) of expected nasal dose was delivered. There were two serious adverse events: one occurred after the predosing bronchoscopy (swelling of the uvula related to intubation) and led to observation overnight in hospital, and the other was an episode of pancreatitis occurring around Day 10 after dosing (10 ml nebulized cohort). The subject was exocrine pancreatic sufficient and had likely experienced previous similar, but undiagnosed, episodes. Table 1. Baseline Demographics Table 2. Postdosing Responses Overall, in the trial, 94.3% of subjects experienced at least one adverse event, the majority of which were mild to moderate in severity and resolved spontaneously or with standard antipyretics. The most common occurred on the day of dosing and largely resolved within 24–48 hours (Tables 1 and ​and2):2): Typically, within the first few hours after dosing, a mild, self-limiting influenza-like systemic response was seen, most frequently in the 20-ml patients. This was not affected by slow delivery or coadministration of ibuprofen or prednisolone but was clearly dose-related and reduced by paracetamol. Symptoms of headache and/or tiredness were reported by 82, 70, and 13%, and raised serum inflammatory markers were recorded in 100, 60, and 63% of the 20-, 10-, and 5-ml groups respectively, with dose-related trends in maximal values. No patient dosed with 5 ml had a temperature higher than 38°C (Table 2). A relatively asymptomatic, dose-related, restrictive drop in spirometry was also observed, with no change in respiratory rate or oxygen saturation. No patient dosed with 5 ml showed a more than 20% relative fall in FEV1 (Table 2). The 20-ml group showed a small, significant (P < 0.05) mean (SD) drop in gas transfer (transfer factor for carbon monoxide corrected for alveolar volume and hemoglobin concentration) on Day 2 of 4.5% (6.0%), which returned to baseline values by Day 14. No changes were seen in the other cohorts. Two of the 20-ml patients had small areas of ground glass opacity reported on their Day 2 chest computed tomography scans, which resolved by Day 14. No significant changes were seen in endobronchial histology (20 ml; n = 10). Consistent with the proposed excretion route for lipids, small but significant serum creatinine rises within the normal range could be detected 8 hours after dosing in the 20- and 10-ml groups, but not the 5-ml cohort; there were no other biochemical changes. Bilirubin rose on Day 1 in all dosing groups, as with creatinine, remaining within the normal range, and normalized by Day 2. There was no evidence of immune responses based on double-stranded DNA antibodies or human CFTR-specific T cells. Lung clearance index, a sensitive marker of pulmonary dysfunction (10), was included as a safety assay. Fourteen 20-ml patients with paired predosing and 28-day postdosing values showed a small but significant increase (i.e., a deterioration; Figure 1A). In contrast, and unexpectedly, on post hoc analysis, 11 of 14 patients in the lower-dosing groups (5 and 10 ml) showed a small but significant improvement (Figure 1A). Figure 1. (A) Lung clearance index (LCI) increased (worsened) by a mean (SEM) of 0.75 (0.3) units in the 20-ml group (P = 0.03), whereas it decreased (improved) in the 5-/10-ml patients by 0.32 (0.1) (P = 0.04). The difference in ... With respect to bronchial samples, 10 patients (all 20 ml) had paired pre- and postdosing bronchoscopies. pGM169-specific DNA was detected in all bronchial brushing samples at levels ∼1000-fold higher than in the nasal samples. pGM169-specific mRNA was detected in 2 of 10 postdosing samples. Paired bronchoscopic potential difference measurements were interpretable for 8 of 10 patients. There was a trend toward an increase in chloride secretion (Figure 1B) but no changes in sodium-related parameters. With respect to nasal samples, pGM169-specific DNA was detected in all 15 brushing samples taken between Day 2 and Day 14 after dosing and in two of six samples at Day 28. pGM169-specific mRNA was detected in 3 of 21 postdosing samples, with all positive samples being observed at either Day 14 (n = 2) or Day 28 (n = 1). In keeping with previous published data, there were no changes in sodium parameters on nasal potential difference. In contrast, 6 of 16 subjects (37.5%) demonstrated a “response” in terms of chloride secretory capacity. Responses were seen most commonly in the zero chloride perfusion phase and at the 14-day point; they were of sustained duration in one subject (Figure 1C). These data were important in informing the design of the phase IIb trial. Thus, based on these findings, 5 ml was selected as the optimal dose, with paracetamol being used as an adjuvant to minimize the risk of unblinding. Although well-tolerated, the adverse effects of the 20-ml doses were considered prohibitive for use in a repeated administration trial. We consider that the efficient delivery of large volumes of viscous fluid into the airways led acutely to both the influenza-like and restrictive responses, analogous to those seen after bronchoalveolar lavage, and masking the effect of plasmid DNA CpG depletion. At lower volumes, the latter effect was “revealed,” allowing safe dosing of 5 ml. The unexpected improvement in lung clearance index after only one administration at the lower doses was intriguing; larger numbers and longer follow-up are needed to confirm or refute this finding. The variable responses both in molecular and CFTR functional terms underscore the technical challenges inherent in these assays and the limited sensitivity to low levels of gene expression (11). The clean safety profile and encouraging improvements in a sensitive measure of airway health lend support to progression to a phase IIb multidose trial designed to detect clinical improvements after prolonged administration.

The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep.

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.

Genetic medicines for CF: Hype versus reality

Since identification of the CFTR gene over 25 years ago, gene therapy for cystic fibrosis (CF) has been actively developed. More recently gene therapy has been joined by other forms of “genetic medicines” including mRNA delivery, as well as genome editing and mRNA repair‐based strategies. Proof‐of‐concept that gene therapy can stabilize the progression of CF lung disease has recently been established in a Phase IIb trial. An early phase study to assess the safety and explore efficacy of CFTR mRNA repair is ongoing, while mRNA delivery and genome editing‐based strategies are currently at the pre‐clinical phase of development. This review has been written jointly by some of those involved in the various CF “genetic medicine” fields and will summarize the current state‐of‐the‐art, as well as discuss future developments. Where applicable, it highlights common problems faced by each of the strategies, and also tries to highlight where a specific strategy may have an advantage on the pathway to clinical translation. We hope that this review will contribute to the ongoing discussion about the hype versus reality of genetic medicine‐based treatment approaches in CF. Pediatr Pulmonol. 2016;51:S5–S17.

T4 Safety and expression of a single dose of lipid-mediated CFTR gene therapy to the upper and lower airways of patients with Cystic Fibrosis

Introduction and Objectives We undertook a clinical trial of non-viral CFTR gene therapy assessing safety, dose and transgene expression in preparation for a Multi-dose trial (MDT) designed to assess clinical efficacy. Methods A single nebulised and/or nasal dose of plasmid CFTR (pGM169)/GL67A was delivered to patients aged =16 years with a baseline FEV1 >60% predicted. Clinical and laboratory parameters were measured at intervals until day 28. A cohort of patients also underwent pre- and post-dosing (day 6 or 14) bronchoscopies for functional (airway potential difference (PD)) and molecular (QRT-PCR) evidence of vector-specific CFTR expression. Patients receiving a nasal dose underwent brushings for QRT-PCR and serial nasal PD measurements. Results 35 patients received a nebulised dose of 20 ml (n=17), 10 ml (n=10) or 5 ml (n=8). A short-lived, dose-related drop in FEV1 was observed over the next 6 h (mean [SD]: 20 ml 25.7 [10.2]%; 10 ml 17.7 [9.9]%; 5 ml 13.0 [4.4]% of baseline). Subjects also experienced a systemic inflammatory response which was similarly dose-related and generally limited to the first 24–48 h post-dosing. A cohort of 6 patients (4@10 ml; 2@5 ml) received 4 g paracetamol over an 18-h period post-dosing; none of these patients developed a fever. Intriguingly, these subjects also appeared to have reduced systemic inflammatory responses. Molecular (mRNA) evidence of gene transfer was observed in some individuals from upper or lower airway brushings. On lower airway PD measurement, the majority of patients showed an increase towards non-CF values after nebulised gene therapy. 19 patients received a 2 ml nasal dose and 11 (58%) had some response in chloride secretion on nasal PD. In the two most positive individuals, responses were within the normal (non-CF) range and persisted to days 63 and 91, respectively. Conclusions We consider the side effects after 20 ml nebulised dose excessive for repeated application. Those at 10 and 5 ml were more acceptable. Gene expression was confirmed in some patients, and restoration of CFTR function to the non-CF range has been observed out to 13 weeks following a single nasal dose. These data support progression of this agent to MDT.

Self-reactive CFTR T cells in humans: implications for gene therapy.

Cystic fibrosis (CF) is one of the most common autosomal recessive lethal disorders affecting white populations of northern European ancestry. To date there is no cure for CF. Life-long treatments for CF are being developed and include gene therapy and the use of small-molecule drugs designed to target specific cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. Irrespective of the type of molecular therapy for CF, which may include gene replacement, exon skipping, nonsense suppression, or molecular correctors, because all of these modulate gene expression there is an inherent risk of activation of T cells against the wild-type version of CFTR. Here we report the validation of the human interferon-γ enzyme-linked immunospot assay and its application for the analysis of CFTR-specific T cell responses in patients with CF and in non-CF subjects. We found non-CF subjects with low levels of self-reactive CFTR-specific T cells in the United States and several patients with CF with low to high levels of self-reactive CFTR-specific T cells in both the United States and the United Kingdom.

534. Preparation for a First-in-Man Lentivirus Trial in Cystic Fibrosis Patients

Background: We have recently shown that non-viral gene therapy can stabilise the decline of lung function in cystic fibrosis (CF) patients. However, the effect was modest, and it is important to develop more potent gene transfer agents in parallel. F/HN-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in pre-clinical models. In preparation for a first-in-man CF trial using the lentiviral vector we have undertaken key translational pre-clinical studies. Methods: Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air liquid interface cultures to select the lead candidate; CFTR expression and function were assessed in CF models (knockout mice and human intestinal organoids) 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. Results: A hybrid promoter consisting of the elongation factor 1α promoter and the CMV enhancer was most efficacious in both murine lungs and human air liquid interface cultures. The efficacy, 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 is stable in clinically relevant delivery devices. Conclusions: The data support progression of the F/HN pseudotyped lentiviral vector into a first-in-man CF trial due to start in Q2 2017. Regulatory-compliant toxicology studies are currently being performed.