Lung Organoids from hiPSCs Can Be Efficiently Transduced by Recombinant Adeno-Associated Viral and Adenoviral Vectors

Abstract

Background: Organoids are a valuable model for studying hereditary diseases such as cystic fibrosis (CF). Recombinant adenoviral (rAdV) and adeno-associated viral (rAAV) vectors are promising tools for CF gene therapy and genome editing. Objective: This study aims to determine the most efficient viral vector (rAdV5, rAAV serotypes 5, 6 and 9) and transduction protocol for delivering transgenes to lung organoids (LOs), providing a foundation for future CF gene therapy development. Methods: Three transduction protocols were used taking into account the specificities of LOs' cultivation in specific matrices, both with and without organoid extraction from the matrix. This work was carried out on organoids from a healthy donor (LOs-WT) and on a patient with cystic fibrosis (LOs-CF). Results: High transduction efficiency was observed with rAdV5 (30% cells), rAAV6 (>80% cells), and rAAV9 (>40% cells). rAdV5 and rAAV9 transduced basal and secretory cells with >90% efficiency. For rAAV9, Protocol 1 (without extraction of organoids from the matrix) showed lower transduction efficiency (33% for LOs-WT, 9% for LOs-CF), significantly lower than that of Protocols 2 (60% for LOs-WT, 59% for LOs-CF) and 3 (46% for LOs-WT, 35% for LOs-CF) with organoid extraction from the matrix (p < 0.005). Conclusions: rAdV5 and rAAV9 are the most promising vectors for the delivery of transgenes to basal and secretory cells in a lung organoid model, providing a solid foundation for CF gene therapy development.

Keywords: adeno-associated viral vectors; adenoviral vector; airway basal cells; cystic fibrosis; induced pluripotent stem cells; lung organoids.

Figure 1

Scheme of LOs transduction using rAAV and rAdV vectors.

Figure 2

Efficiency of LOs-WT transduction using rAAV vectors. (A) Representative image of GFP fluorescence in LOs-WT on day six after transduction using rAAV5, 6 and 9 at MOI 1 × 10¹⁰ vg/organoid (Complex Lionheart FX, Magnification ×10). (B) rAAV dose-dependent transduction efficiency of LOs-WT using three different protocols of transduction. Data are presented as mean value ± SD; the differences were considered statistically significant when p value < 0.005 (**), p value < 0.0001 (****) using Dunnett’s T3 multiple comparison test.

Figure 3

Transduction efficiency of LOs-CF using three different infection protocols. (A) Representative image of LOs-CF on day six after transduction using rAAV vectors at MOI 1 × 10¹⁰ vg/organoid (Complex Lionheart FX, Magnification ×10). (B) GFP expression in LOs-CF after rAAV transduction using three infection protocols. Data are presented as mean value ± SD; the differences were considered statistically significant when p value < 0.05 (*), p value < 0.005 (**), p value < 0.0001 (****) using Dunnett’s T3 multiple comparison test.

Figure 4

Comparison of LOs-WT and LOs-CF transduction efficiency by rAAV vectors using three infection protocols. (A) The graphs provide a comparison of the efficiency of transduction protocols for LOs for each rAAV vector of serotypes 5, 6, and 9. (B) The present study compares the efficiency of transduction protocols for LOs-WT and LOs-CF using rAAV viral vectors. The data presented were obtained at an MOI of 1E10. Data are presented as mean value ± SD; the differences were considered statistically significant when p value < 0.005 (**), p value < 0.0005 (***) using Dunnett’s T3 multiple comparison test.

Figure 5

rAdV dose-dependent transduction efficiency of LOs-WT and LOs-CF using two transduction protocols. (A) Representative image of LOs-WT and LOs-CF on day six after transduction using rAdV5-mCherry at PFU 4 × 10⁶ (Complex Lionheart FX, Magnification ×10). (B) mCherry expression in LOs-WT and LOs-CF after rAdV5 transduction using two infection protocols. Data are presented as mean value ± SD; the differences were considered statistically significant when p value < 0.005 (**), p value < 0.0001 (****) using Dunnett’s T3 multiple comparison test.

Figure 6

Representative images from confocal microscopy of LOs stained against basal cell marker (TP63), secretory cell marker (SCGB3A2), GFP and mCherry proteins after transduction by rAAV6, rAAV9 and rAdV5 using Protocol 2. Nuclei were stained with DAPI (blue). Magnification ×63.