Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis

Abstract

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.

Keywords: Cystic Fibrosis.

Figure 1

Selection of lead candidate vector. Mice and human air–liquid interface (ALI) cultures were transduced with five different lentiviral vector configurations by nasal instillation: integrase-competent (IC) vectors carrying the human elongation factor 1α (short) promoter (EF1α), a ubiquitous regulatory element which has previously been used in the context of lentivirus-mediated gene transfer, an in-house, synthetic chimeric promoter/enhancer consisting of CpG-depleted versions of the human EF1α (short) promoter and the human cytomegalovirus (CMV) enhancer (hCEF) and the original CMV-based construct, as well as integrase-defective (ID) vectors carrying the CMV promoter and hCEF promoter/enhancers (6–30E7 TU/mouse or ALI, n=6–10 mice/group, n=4 ALI/group). All vectors carried a luciferase reporter gene for quantification of gene expression by bioluminescence imaging. Negative control mice and ALIs remained untreated (UT). Gene expression was quantified in the lungs and nose of mice and in ALIs. Photon emission adjusted for differences in vector titre. (A) Representative images of transduced and UT mice and ALIs, (B) quantification of photon emission in murine lungs, (C) quantification of photoemission in murine nose and (D) in human ALIs. (B–D) Reference UT control values are shown as a dotted line (lung control: 182±6 p/s/cm²/sr, nose control: 200±10 p/s/cm²/sr, ALI control 598±1080 p/s/cm²/sr). For each group, the mean±SEM are shown. ****p<0.001 in lung and nose comparing hCEF-ID with all other vectors in mice (ANOVA followed by Tukey post hoc test), ***p<0.005 comparing hCEF-IC with UT ALI controls (Mann–Whitney).

Figure 2

Gene expression in relevant airway epithelial cells. Mice were transduced with rSIV.F/HN-hCEF-enhanced green fluorescent protein (EGFP) (8E8 TU/mouse) or remained untreated (UT) (n=3/group). Seven days after transduction, mice were culled and the lungs processed for quantification of airway cells expressing EGFP by immunohistochemistry. (A) Representative image of a lentivirus transduced mouse. (B) Representative image of an UT mouse. Scale bar=50 µm. AW, airway; P, parenchyma. (C) Quantification of EGFP in mouse airways. Each dot represents a randomly selected airway (n=10/mouse). For convenience, the data from the UT control mice were pooled. The horizontal bar shows the median. The dotted horizontal line represents the consensus therapeutic threshold of 5% airway cells. ****p<0.0001 comparing all treated mice with controls (ANOVA followed by Dunnett's multiple comparison test). GFP, green fluorescent protein.

Figure 3

Characterisation of transduced cells by immunohistochemistry. Mice were transduced with rSIV.F/HN-hCEF-enhanced green fluorescent protein (EGFP) (8E8 TU/mouse) or remained untreated (n=3/group). Seven days after transduction, mice were culled and the lungs processed for characterisation of EGFP-expressing cells by immunohistochemistry. Tissue sections were double-stained with anti-EGFP and cell-type-specific antibodies and DAPI to visualise nuclei (blue). The left panel shows EGFP-expressing cells in green, the middle panel shows cell-type-specific staining in red and the right panel shows a merged image. Arrows highlight double-labelled cells. The merged images do not in all cases show a yellow/orange signal when green and red signals are overlaid because the proteins stained are localised to different cellular compartments, for example, in type 1 pneumocytes, the EGFP is present in the cytoplasm, whereas podoplanin is a membrane protein. Scale bar=10 µm. (A) Anti-β tubulin antibody identifies ciliated airway epithelial cells, (B) anti-uteroglobin antibody identifies club cells in the airways, (C) anti-mucin 5AC antibody identifies goblet cells in the airways, (D) anti-cytokeratin 5 antibody identifies basal cells in the airways, (E) anti-podoplanin antibody identifies type 1 pneumocytes and (F) anti-surfactant protein C antibody identifies type 2 pneumocytes.

Figure 4

Assessment of acute pulmonary toxicity after lentivirus transduction. Mice were transduced with one or four doses (1E8 TU/dose at monthly intervals, n=5/group) of rSIV.F/HN-cytomegalovirus vectors carrying luciferase or enhanced green fluorescent protein reporter genes and histological analysis was performed 24 hours after the last dose. Control groups included UT and D-PBS-treated mice and mice treated with conventional (CpG containing) luciferase plasmid DNA/GL67A complexes or CpG-free CFTR plasmid pGM169/GL67A (n=3–5 mice/group). (A) Representative image of a lentivirus-treated mouse. AW, airway, P, parenchyma, arrow indicates mild cellular infiltrate, (B) semiquantitative scoring of lung inflammation. UT, untreated, one dose of rSIV (rSIV1x) and four monthly doses of rSIV (rSIV4x). Each symbol represents an individual mouse. The horizontal bar indicates the group median.

Figure 5

Transduction efficiency in mouse lung and nose in the presence of anti-human human parainfluenza virus 1 (hPIV1) antibodies. Mice were treated with human Ig (IVIg) intraperitoneally (IP, 400 μL) or by nasal instillation (IN, 100 μL). Controls did not receive IVIg (n=6/group). Twenty-four hours after passive immunisation, mice were transduced with rSIV.F/HN-hCEF-EGFPLux (1E8 TU/mouse). Control mice remained untreated (UT). Luciferase expression was quantified in lung and nose using bioluminescent imaging 24 hours after virus transduction. (A) Representative images for each cohort of mice, (B) luciferase expression nose and (C) lung. Each symbol represents one animal. The horizontal bar indicates the group median. Two independent experiments were performed (n=6/group/experiment) and a representative figure is shown. EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein.

Figure 6

Confirmation of CFTR expression and function. (A) HEK293T cells were transfected with rSIV.F/HN-hCEF-CFTR or an irrelevant control virus (negative control) at MOIs of 10 and 100. The iodide efflux assay was performed 2 days after transduction. Data are presented as mean±SEM. **p<0.05 compared with negative control (ANOVA followed by Dunnett's multiple comparison test). (B) Cystic fibrosis knockout mice were transduced with rSIV.F/HN-hCEF-CFTR (1.6E8 TU/mouse) by nasal instillation. Negative controls were treated with PBS (n=7–8/group). Mice were culled 7 and 28 days after vector administration and vector-specific mRNA was quantified in the lungs. Each symbol represents one animal. The horizontal bar shows the group median. The dotted line indicates the detection limit of the assay. ***p<0.005 and **p<0.01 compared with the negative control (Kruskal-Wallis followed by Dunn's multiple comparison test).

Figure 7

Functional confirmation of CFTR production in cystic fibrosis (CF) intestinal organoids. (A–D) CF intestinal organoids carrying two class I mutations (E60X/4015delATTT) were transduced with rSIV.F/HN-hCEF-CFTR or an irrelevant control virus (negative control). The doses in experiment 1 ranged from 0.45 to 3.6E7 TU/well and in experiment 2 from 0.06 to 0.45E7 TU/well (n=4/dose/experiment). Doses greater than 1.8E7 TU/well resulted in cell toxicity and reduced chloride transport (as measured by reduced organoid swelling upon forskolin addition) (data not shown). Analysis of chloride transport in the CF organoids 4 days after transduction therefore focused on samples treated with 0.23–0.9E7 TU/well (n=15–16 wells/group in two independent experiments). Four days post-transduction, organoid swelling upon addition of forskolin was assessed (measured as area under curve (AUC) over 120 min, baseline set at t=0). Representative organoid images are shown. Data are presented as mean±SEM. ****p<0.0001 compared with negative control (non-paired Student's t-test). (E) At the end of the experiment, organoids were harvested for protein extraction and western blot analysis. Lane 1: non-CF organoids transduced with negative control virus, Lanes 2–4: CF organoid untransduced or transduced with a negative control virus and Lane 5: CF organoids transduced with rSIV.F/HN-hCEF-CFTR.