Shuttle peptide design and protein delivery to airway epithelia. A) Amino acid sequences of shuttle peptides. Sequences aligned to highlight structural similarities. Cationic residues are highlighted in blue; hydrophobic residues are in gray. Remaining residues are in green. B) Indel% in primary NK cells following Cas12a RNP delivery targeting NKG2A gene with indicated shuttle peptide ([Cas12a]: 1.33 uM; [crRNA]: 2.0 µM). Results quantified 48 h after delivery (mean ± SE; n = 3 independent experiments). C) Shuttle peptide-mediated delivery of nuclear-targeted GFP protein to human airway epithelia (HAE) and tracheal explants. Left panel: Schematic of primary HAE grown at air–liquid interface. ASL: airway surface liquid. Right panel: photomicrograph of well-differentiated primary HAE culture demonstrating pseudostratified columnar epithelium. Hematoxylin and eosin stain, ×40 magnification. D) Efficiency of GFP delivery by different shuttle peptides. Peptides applied at four concentrations (5, 10, 15, or 20 uM); GFP concentrations 10 or 20 uM. Left graph: Cells were transduced with indicated peptide and GFP combinations and GFP quantified by fluorescence reader. Results mean ± SE; n = 4 donors. *P < 0.05, **P < 0.005, ***P < 0.001, by one-way ANOVA with Tukey’s multiple comparison test. Right panels: Representative en face views of epithelial sheets following GFP delivery at the highest peptide:GFP concentration (20 uM:20 uM), ×20 magnification. Formulations were applied for 15 min. E) GFP localization in multiple epithelial cell types following shuttle peptide-mediated delivery. HAEs were transduced for 15 min with GFP protein and S10 peptide. Left panel: confocal image (XZ) shows co-localization of GFP and specific marker of ciliated cells (α-tubulin, red, indicated by white arrows), nonciliated cells (white arrowheads), F-actin (phalloidin stain, gray), nuclei (DAPI, blue). Right panel: co-localization of GFP and goblet cell marker (Muc5AC, red, indicated by white arrowhead), nuclei (DAPI, blue), and F-actin (phalloidin stain, gray). n ≥ 6 different donor epithelia; ×64 magnification. F) Shuttle peptide delivery of GFP to human tracheal explants. S10 (20 uM) and GFP protein (20 uM) applied to tracheal explant for 1 h. Left panel: both ciliated (αtubulin, red, white arrowhead) and non-ciliated (white arrow) cells were transduced. Right panel: non-ciliated goblet cells were transduced (Muc5AC, red, white arrows); ×40 magnification. https://doi.org/10.1038/s41467-019-12922-y
