Journal article
Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells
Nucleic acids research, Vol.52(19), pp.11911-11925
10/28/2024
DOI: 10.1093/nar/gkae819
PMCID: PMC11514481
PMID: 39315713
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.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.
Details
- Title: Subtitle
- Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells
- Creators
- Katarina KulhankovaAnna X ChengSoumba TraoreMaud AugerMia PelletierMaxime HervaultKevin D WellsJonathan A GreenAddison ByrneBenjamin NelsonMariana SponchiadoChandra BoosaniCaleb S HeffnerKathy J SnowStephen A MurrayRaul A VillacresesMichael V RectorNicholas D GansemerDavid A StoltzChantal AllamargotFrédéric CoutureColin HemezStéphanie HalléeXavier BarbeauMario HarveyCoraline LauvauxBruno GailletGregory A NewbyDavid R LiuPaul B McCrayDavid Guay
- Resource Type
- Journal article
- Publication Details
- Nucleic acids research, Vol.52(19), pp.11911-11925
- DOI
- 10.1093/nar/gkae819
- PMID
- 39315713
- PMCID
- PMC11514481
- NLM abbreviation
- Nucleic Acids Res
- ISSN
- 1362-4962
- eISSN
- 1362-4962
- Publisher
- OXFORD UNIV PRESS
- Grant note
From the University of Iowa, we thank Amber Vu, Patrick Sinn, and Ashley Cooney for critically reviewing the manuscript and Linda S. Powers for technical advice. We acknowledge the support of the University of Iowa In Vitro Models and Cell Culture Core. From The Jackson Laboratory, we thank Ethan Saville, Yaned Gaitan, and Janet Grlica for their assistance with sample collection, shipment, and mouse colony maintenance. Some data presented herein were obtained at the Genomics Division of the Iowa Institute of Human Genetics which is supported, in part, by the University of Iowa Carver College of Medicine. This article is subject to HHMI's Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication. Graphic abstract contains images created with BioRender.com.
- Language
- English
- Electronic publication date
- 09/24/2024
- Date published
- 10/28/2024
- Academic Unit
- Molecular Physiology and Biophysics; Pulmonary, Critical Care, and Occupational Medicine; Microbiology and Immunology; Pulmonary Medicine; Stead Family Department of Pediatrics; Core Research Facilities; Internal Medicine
- Record Identifier
- 9984719238902771
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