Abstract
The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.
Highlights
The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells
While the peptides share features, including an improved hydrophobic cluster derived from the CM18 domain and an optimized hydrophilic/cationic tail derived from the cell-penetrating PTD4 peptide, they diverge in others
In addition to their variable length (25–34 amino acids), S10 contains five highly hydrophobic residues in the CM18-derived domain, while S18 and S85 have eight, the S85 linker was reduced to a single glycine, and the PTD4-derived domain of S18 contains six cationic residues compared to four in S10 and S85
Summary
The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells. Modifying airway epithelia with therapeutic proteins, gene expression cassettes, or genome-editing reagents offers great promise, but efficacious delivery is a common challenge. This is because the respiratory epithelium presents barriers through its specialized cell types, secreted host defense factors, and mucociliary transport[1]. Cas RNPs contain negatively charged gRNA that enables their delivery using cationic lipids or electroporation in responsive cell types[19] Other proteins, such as green fluorescent protein (GFP), antibodies, or luciferase, are poorly delivered using such procedures[20]. We demonstrate here that nextgeneration shuttles enable protein transfer to another refractory cell type by delivering reporter proteins and Cas RNPs in vitro and in vivo to differentiated epithelial cells lining the airways despite the physicochemical barriers imposed by these cells
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