Document Type

Article

Publication Date

10-28-2024

Keywords

JMG, Cystic Fibrosis Transmembrane Conductance Regulator, Humans, Epithelial Cells, Ribonucleoproteins, Cystic Fibrosis, Gene Editing, Animals, Peptides, Swine, Respiratory Mucosa, Mutation, Cell-Penetrating Peptides, Cell Line

JAX Source

Nucleic Acids Res. 2024;52(19):11911-25.

ISSN

1362-4962

PMID

39315713

DOI

https://doi.org/10.1093/nar/gkae819

Grant

[U42OD026635 to S.A.M.]

Abstract

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

Share

COinS