Functional restoration of a CFTR splicing mutation through RNA delivery of CRISPR adenine base editor.
CRISPR-Cas9
RNA delivery
base editing
cystic fibrosis
gene editing
gene therapy
organoids
primary bronchial epithelial cells
Journal
Molecular therapy : the journal of the American Society of Gene Therapy
ISSN: 1525-0024
Titre abrégé: Mol Ther
Pays: United States
ID NLM: 100890581
Informations de publication
Date de publication:
07 06 2023
07 06 2023
Historique:
received:
04
11
2022
revised:
07
01
2023
accepted:
03
03
2023
pmc-release:
07
06
2024
medline:
12
6
2023
pubmed:
11
3
2023
entrez:
10
3
2023
Statut:
ppublish
Résumé
Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is a quite frequent defect causing an aberrant splicing and a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to correct the mutation in the absence of DNA double-strand breaks (DSB). To select the strategy, we developed a minigene cellular model reproducing the 2789+5G>A splicing defect. We obtained up to 70% editing in the minigene model by adapting the ABE to the PAM sequence optimal for targeting 2789+5G>A with a SpCas9-NG (NG-ABE). Nonetheless, the on-target base correction was accompanied by secondary (bystander) A-to-G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To decrease the bystander edits, we used a specific ABE (NG-ABEmax), which was delivered as mRNA. The NG-ABEmax RNA approach was validated in patient-derived rectal organoids and bronchial epithelial cells showing sufficient gene correction to recover the CFTR function. Finally, in-depth sequencing revealed high editing precision genome-wide and allele-specific correction. Here we report the development of a base editing strategy to precisely repair the 2789+5G>A mutation resulting in restoration of the CFTR function, while reducing bystander and off-target activities.
Identifiants
pubmed: 36895161
pii: S1525-0016(23)00125-9
doi: 10.1016/j.ymthe.2023.03.004
pmc: PMC10277887
pii:
doi:
Substances chimiques
Cystic Fibrosis Transmembrane Conductance Regulator
126880-72-6
RNA
63231-63-0
Adenine
JAC85A2161
CFTR protein, human
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1647-1660Informations de copyright
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of interests A.C. is a co-founder and holds stocks of Alia Therapeutics, a genome editing company.
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