Cell culture

Human BMI1 transduced non-CF (NHNE, S Hart, UCL, UK) and hTERT Bmi-1 transduced G542X CF nasal epithelial cells (CFNE G542X, kind gift from S Randell, UNC, USA, [21]) p2, 8 were seeded on collagen (PureCol®) coated plasticware at a density of 3 × 105 into 75 cm2 flasks with 15 ml PneumaCult™ Ex-Basal Culture media. Media was changed every 48 h. When flasks were 80–90% confluent, cells were detached with Trypsin/EDTA (0.25% solution) and cells were pelleted in 5 ml of PneumaCult™ Ex-Basal media at 500 rpm for 5 min. The cell pellet was re-suspended in 2 ml PneumaCult™ Ex-Basal media for counting before re-seeding.

Air liquid interface (ALI) culture

NHNE, CFNE G542X and edited CFNE G542X were seeded onto collagen coated Snapwell culture inserts (cat no. 3801, Corning®) at a density of 0.5 × 106 per 1.2 cm2 in 250 μl of PneumaCult™ Ex-Basal media, 1 ml of PneumaCult™ Ex-Basal media was added to the basolateral side. After 48 h the media was aspirated from both apical and basolateral compartments and basolateral side replaced with 1 ml PneumaCult™-ALI Medium which was refreshed every 48 h. Cells were cultured for a minimum of 21 days at ALI. Treatment with ETI was performed 24 h prior to functional assessment with 1 μM VX-445, 3 μM VX-661, and 1 μM VX-770.

Transfection using RTN

Liposomes were a mixture of the cationic lipid 1,2-di-((Z)-tetradec-11-enyloxy)-N,N,N trimethylammonium propane chloride (DTDTMA or C14), and the neutral lipid Dioleoyl L-α phosphatidyl ethanolamine (DOPE), combined in a 1:1 molar ratio (Avanti Polar Lipids). Liposomes were prepared by mixing the lipids in ethanol and injecting into a NanoAssemblr microfluidics system at a flow rate of 12 ml/min (Precision Nanosystems), followed by sonication and dialysis against nuclease-free water overnight using Maxi GeBaFlex-tubing with an 8 kDa MWCO (Generon) and stored at 4 °C. Peptide E, K16GACSERSMNFCG (produced by AMS Bio) contained the SERSMNF receptor targeting motif identified by phage display biopanning on the human airway epithelial cell line (1HAEo-).

Plasmids were obtained from Addgene http://n2t.net/addgene. NG ABE8e Cas9 plasmid, Addgene plasmid #138491 [22] and EGFP gRNA_Cloning Vector BbsI, Addgene plasmid #128433. The sgRNA sequence was developed, optimised and kindly provided by L Nicosia and Prof P Harrison, University of Cork, Ireland [17]. Plasmids were prepared for transfection at a 1:1 molar ratio of ABE8e:gRNA vector. Receptor-targeted nanocomplexes (RTNs) (Fig. 1a) were prepared in water at a concentration of 4 μg / ml with respect to pDNA and in a 1:4:1 weight ratio of liposome: peptide: DNA and incubated at room temperature for 30 min to stabilise the RTNs prior to biophysical analysis. Size and zeta potential were measured in a Nano ZS Zetasizer (Malvern).

Fig. 1

Adenine base editing. a Structure of the receptor targeted nanoparticle (RTN) complex showing formulation of the liposome complex (purple), targeting peptide structure (pink) and nucleic acid cargo (purple helices), b illustration showing the genomic changes in sequence from non-CF (glycine) to the G542X (stop) variant which causes premature termination of translation, to the edited G542R (arginine) sequence Created in BioRender.com

RTNs were prepared as above but in OptiMEM instead of water for transfections of ~ 1.4 × 106 CFNE-G542X basal cells for 4 h. Plasmid uptake was visualised using confocal microscopy (Nikon A1R confocal microscope) and EGFP fluorescing cells quantified using ImageJ.

Flow-cytometric cell sorting

24 h after transfection cells were washed with phosphate buffered saline (PBS). Trypsinisation (0.25% trypsin–EDTA) of CFNE expressing EGFP in cells which had taken up editing machinery yielded ~ 1.2 × 106 cells from a single well (6-well plate). Cells were resuspended in 500 ml FACS buffer (Supplemental Table S1) and sorted by Fluorescence Activated Cell Sorting (FACS), Melody Cell Sorter, BD Biosciences. Singlets were discriminated from doublets using forward scatter (FSC) height vs FSC pulse area [23], and debris were excluded. Unsorted/sorted edited cells were seeded onto plastic (analysis of editing/scratch assay) or grown at ALI for subsequent functional analyses.

Analysis of editing

Online tools (NCBI Blast, CRISPOR) were used to analyse genomic sequences with similar homology to the guide sequence. The top 5 exon spanning and top 5 intronic sites were selected based on the sequence similarity. Genomic DNA was extracted using QIAGEN DNeasy Blood & Tissue kit according to the manufacturer’s instructions. DNA concentration was determined using a NanoDrop 2000. Genomic sites of interest were amplified by PCR using appropriate primer pairs (Supplemental Table S2). PCR was carried out using high-fidelity Phusion DNA polymerase with thermocycling: 98 °C for 2 min, then 35 cycles of 98 °C for 10 s, 53 °C for 20s, and 72 °C for 20s, followed by a final extension of 72 °C for 1 min. PCR products were verified by comparison with DNA standards on a 1% agarose gel supplemented with SYBR-safe gel stain. PCR products were treated using ExoSAP-IT (ThermoFisher) for product purification. Samples were sent for next-generation sequencing (NGS) (GENEWIZ; https://www.genewiz.com). The sequence of G542X and G542R are shown in Fig. 1b.

RT-qPCR

RNA was extracted from ALI cultures 28 days post plating using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. cDNA was generated using the Luna One-Step RT-qPCR kit (New England Biolabs) according to the manufacturer’s instructions alongside appropriate primers (Supplemental Table S2). Amplification was performed on mRNA extracted from fully differentiated ALI cultures four weeks post-editing using PCR protocol as described above and data shown as ΔΔCT = ΔCT(CFTR)-ΔCT(GAPDH).

Western blot

ALI cultures were lysed in NP-40 Lysis Buffer, incubated on ice for 20 min then centrifuged at 15,000×g for 20 min. Protein concentration was determined by Pierce™ BCA Protein Assay Kit and 20 µg protein was denatured with LDS sample buffer and reducing agent (NuPAGE) at 65 °C for 15 min [24]. Samples were resolved on NuPAGE 4–12% Bis-Tris Protein Gels with mass standards 10–250 kDa (LI-COR). Proteins were transferred to Immobilon®-FL PVDF membrane (Millipore). Membranes were blocked in Odyssey® Blocking Buffer (LI-COR), immunostained with anti-CFTR or anti-α Tubulin followed by IRDye® 800CW Goat anti-Mouse IgG (LI-COR), visualised and quantified on an Odyssey IR imager (LI-COR) (Supplemental Tables S3 and S4).

Ion transport

Transepithelial ion transport was measured in Ussing chambers using symmetrical bicarbonate buffered salt solution pH 7.4 (Supplemental Table S1), at 37 °C, bubbled with 21% O2, 5% CO2 and the following drugs were used: amiloride (apical, 100 μM) to inhibit the epithelial sodium channel (ENaC), forskolin (bilaterally, 10 μM) to activate CFTR and CFTRinh172 (apical, 10 μM) to inhibit CFTR as previously described [25] Data were analysed using LabChart 7 software (ADI Instruments). All drugs/chemicals were obtained from Sigma-Aldrich.

Airway surface liquid height

Airway surface liquid (ASL) was labelled with 2 mg/ml Texas red-dextran (10 kDa) in PBS (20 μl) and left for 24 h to equilibrate. Perfluorocarbon (PFC) was added to the mucosal surface to prevent evaporation of the ASL, and the culture was placed on the stage of the confocal microscope over a serosal reservoir containing 80 ml of bicarbonate buffered salt solution (Supplemental Table S1). To determine the average height of the ASL, eight predetermined points on the culture were scanned using a Leica SP8 confocal microscope with a × 63/1.3 numerical aperture (NA) glycerol immersion lens in XZ-scanning mode as previously described [26].

Measurement of ASL pH

ASL pH was measured using pH-sensitive pHrodo red dextran (10 mM), pH insensitive Alexa Fluor 647 dextran (10 mM) in bicarbonate buffered salt solution (20 μl) added to the apical surface of each transwell and incubated overnight in humidified air + 5% CO2 at 37 °C. Excitation/emission at 562/592nm and 650/668nm was measured the following day over a period of 8 h using a Tecan Spark. At 2 h 100 nM VIP was added basolaterally to activate CFTR. Apical pH was calculated as the fluorescence ratio pHrodo red dextran:Alexa Fluor 647 dextran less background fluorescence from non-labelled ALI cultures and results aligned to a standard curve generated from in situ controls of known pH 6.0–7.5) as previously described [27]

Immunohistochemistry

ALI cultures were fixed in 4% paraformaldehyde (PFA) for 25 min then permeabilised with 0.2% Triton-X100 at room temperature. Samples were incubated with blocking buffer prior to incubation with primary antisera, prepared in PBS with 5% normal goat serum and 0.1% Triton-X100, both overnight at 4 °C. Samples were washed with PBS 3 × 30 min before counterstaining with appropriate secondary antisera (Supplementary Table S3), prepared in PBS with 0.1% Triton-X100, for 1 h at room temperature. Samples were washed with PBS 3 × 30 min prior to mounting with ProLong Gold Mountant with DAPI (Invitrogen). Images were taken using a Nikon A1R confocal microscope with a × 100/1.25 NA oil immersion lens.

Scratch assay

Confluent basal airway epithelial cell cultures grown in PneumaCult™ Ex-Basal media were scratched mechanically using the AutoScratch tool. Images were taken every 25 min over 24 h using a LiveCyte 2 to assess collective migratory rate and cell directionality. Images were taken at three fixed points along the scratch edge for each well. Rate of wound closure was calculated using ImageJ software and cell directionality was tracked using LiveCyte Analyse and MATLAB software.

Inflammatory markers

Media samples were collected from non-transfected or transfected cultures 24 h prior to transfection and post-transfection (0–72 h) RTN plus ABE DNA or RTN alone. Treatment with IL-1β was used as a positive control. Cytokines were measured using human IL-6 and IL-8 Quantikine ELISA kit (Bio-techne).

Statistics and data analysis

Normally distributed data were analysed using ANOVA followed by Tukey’s test or unpaired t-test with Welch’s correction. Non-parametric equivalents (Mann–Whitney test, Kruskal–Wallis test with Dunn’s multiple comparisons test) were used when data were not normally distributed. Data are shown as individual points and/or mean ± standard deviation (SD). Significant differences are indicated with *p < 0.05; **p < 0.01 ***, p < 0.001, ****p < 0.0001. Data analyses were performed using GraphPad Prism v10.2.2 (GraphPad Software).