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

Abstract

Rationale and objective: Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CFTR modulators offer significant improvements, but ∼10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimised 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 (HBECs) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalised 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 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 immortalisation assay.

Conclusions: The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

Summary of the main study findings. CFTR: cystic fibrosis transmembrane conductance regulator. Created with BioRender.com.

FIGURE 1

Transduction efficiency and transduced cell types in human bronchial epithelial cells (HBEC) air–liquid interface (ALI). For the presented experiments samples from three cystic fibrosis donors were used; n indicates the number of samples assessed, derived from these donors. a) Experimental setup. b) Quantification of transduced green fluorescent protein-positive (GFP⁺) cells at day 21 post-transduction (n=9–20). c) Immunofluorescence for ciliated (ACTUB), basal (KRT5), club (SCGB1A1) and goblet (MUC5AC) cells and co-localisation with transduced GFP⁺ cells (arrows indicate co-localisation). Scale bar=20 µm. d) Flow cytometry quantification of percentage of transduced cells in specific epithelial cell populations (n=6–9). e) Vector copy number quantitative PCR analysis in bulk samples transduced either with GFP- or cystic fibrosis transmembrane conductance regulator (CFTR)-expressing rSIV.F/HN (n=7–11). MOI: multiplicity of infection; DAPI: 4′,6-diamidino-2-phenylindole. Data were analysed using b) one-way ANOVA followed by Holm–Šídák's multiple comparison test and d, e) Kruskal–Wallis test followed by Dunn's multiple comparison tests. *: p<0.05, **: p<0.005, ***: p<0.001, ****: p<0.0001.

FIGURE 2

Transduction with rSIV.F/HN-CFTR (cystic fibrosis transmembrane conductance regulator) results in expression of codon-optimised CFTR (coCFTR) in primary cystic fibrosis human bronchial epithelial cells (HBECs). For the presented experiments samples from three cystic fibrosis donors were used; n indicates the number of samples assessed, derived from these donors. a) CoCFTR transgene gene expression showing high expression of transgene in CFTR-transduced cells, but not in the negative control green fluorescent protein (GFP)-transduced cells (n=4–13). b) Endogenous human (h) CFTR gene expression (n=4–13). c) Gene expression ratio between transgene coCFTR and hCFTR (n=11–13). Data were analysed using Kruskal–Wallis test followed by Dunn's multiple comparison tests. *: p<0.05, ***: p<0.001, ****: p<0.0001.

FIGURE 3

Functional data demonstrating that rSIV.F/HN restores cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in primary cystic fibrosis (CF) human bronchial epithelial cells (HBECs). For the presented experiments, samples from three non-CF and three CF donors were used; n indicates the number of samples assessed, derived from these donors. a) Schematic drawing of Ussing chamber measurements. ^#: forskolin peak and forskolin plateau. b) Ussing chamber data represented as the percentage of wild-type (WT) CFTR current; difference between maximum forskolin peak current and current after CFTR-inhibition is calculated (n=14–26 for all conditions except for multiplicity of infection (MOI) 30 and 90 (n=4)). c) Ussing chamber data represented as percentage of WT CFTR current; difference between forskolin plateau and CFTR-inhibited current is calculated (n=14–26 for all conditions except for MOI 30 and 90 (n=4)). d) Mean ciliary beat frequency analysis in primary non-CF, CF and transduced CF HBECs (n=36). ΔI_sc: short-circuit current change; Amil: amiloride; Fsk: forskolin; Iva: ivacaftor; GFP: green fluorescent protein; Luma: lumacaftor; Teza: tezacaftor; Elexa: elexacaftor. Data were analysed using b, c) Mann–Whitney test or d) Kruskal–Wallis test followed by Dunn's multiple comparison tests. *: p<0.05, ***: p<0.001, ****: p<0.0001.

FIGURE 4

Assessment of transduction with rSIV.F/HN-GFP (green fluorescent protein) and rSIV.F/HN-CFTR (cystic fibrosis transmembrane conductance regulator) in CFTR-knockout (KO) (CFTR-null, model of class I mutation) immortalised human small airway basal cell line (hSABCi). a) Flow cytometry quantification of transduced GFP⁺ CFTR-KO cells at day 21 post-transduction (n=6). b) Vector copy number quantitative PCR analysis in bulk CFTR-KO samples transduced with GFP- and CFTR-expressing vectors (n=6). c) Immunofluorescence for ciliated (ACTUB), basal (KRT5), club (SCGB1A1) and goblet (MUC5AC) cells and co-localisation with transduced GFP⁺ cells. Arrow indicates co-localisation. Scale bar=20 µm. d) Flow cytometry quantification of percentage of transduced cells in specific epithelial cell populations (n=6). MOI: multiplicity of infection. Data were analysed using Kruskal–Wallis test followed by Dunn's multiple comparison tests. **: p<0.01, ***: p<0.001.

FIGURE 5

Cystic fibrosis transmembrane conductance regulator (CFTR) protein detection and quantification of percentage of positive cells following lentiviral vector transduction in CFTR-knockout (KO) immortalised human small airway basal cell line (hSABCi). a) Representative micrographs of cytospun CFTR- (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue: nuclei)-stained hSABCi cells, showing untransduced CFTR-KO cells along with CFTR-KO cells transduced at multiplicities of infection (MOI) 10 and 30. Scale bar=50 μm. b) Quantification of CFTR percentage-positive CFTR-KO cells with and without rSIV.F/HN transduction, showing an average of 42.25% and 52.86% CFTR-positive cells in the MOI 10 and MOI 30 samples, respectively (n=5). Data were analysed using the Brown–Forsythe and Welch multiple comparison test. **: p<0.01.

FIGURE 6

Functional data demonstrating that rSIV.F/HN restores cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in CFTR-knockout (KO) cells in contrast to modulators. a) Codon-optimised CFTR (coCFTR) transgene gene expression showing high expression of transgene in CFTR-transduced cells, but not in green fluorescent protein (GFP)-transduced cells (n=6). b) Schematic drawing of Ussing chamber measurements. ^#: forskolin peak and forskolin plateau. c) Ussing chamber data represented as percentage of wild-type (WT) CFTR current; difference between maximum forskolin peak current and current after CFTR-inhibition is calculated (n=6–11). Of note, there was no CFTR chloride current activation after treatment with modulators (Luma+Iva, Teza+Iva, Elexa+Teza+Iva). d) Ussing chamber data represented as percentage of WT CFTR current; difference between forskolin plateau and CFTR-inhibited current is calculated (n=6–11). Of note, there was no CFTR chloride current activation after treatment with modulators (Luma+Iva, Teza+Iva, Elexa+Teza+Iva). ΔI_sc: short-circuit current change; Iva: ivacaftor; Luma: lumacaftor; Teza: tezacaftor; Elexa: elexacaftor. Data were analysed using a) Kruskal–Wallis test followed by Dunn's multiple comparison tests or c, d) Mann–Whitney test. *: p<0.05, **: p<0.01.

FIGURE 7

Insertion site (IS) analysis in primary human bronchial epithelial cells (HBECs). For the presented experiments, samples from three cystic fibrosis donors were used; n indicates the number of samples assessed, that were derived from these three donors. a) Study design. b) Number of retrieved unique IS from each sample and maximum frequency (%) of top 10 IS among all mappable IS (n=8–27). c) Frequencies of the 10 most prominent IS detected in samples transduced with multiplicity of infection (MOI) 1 and 10 and analysed at days 7 and 28. IS location and RefSeq names of genes located closest to the respective IS are given. Relative sequence count contributions of the 10 most prominent IS and all remaining mappable IS are shown. d) Top 10 common integration sites (CIS) detected in all samples transduced with MOI 1. Nearest genes for all IS within the CIS are listed. GFP: green fluorescent protein.