Targeted insertion of partial
9 affiliations
- 1Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Center for Stem Cells and Regenerative Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, USA
- 2Department of Bioengineering, Rice University, Houston, TX 77030, USA
- 3Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA
- 4Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA
- 5Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA
- 6Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, USA
- 7Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, MA 02421, USA
- 8Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
- 9Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA 30322, USA
Abstract
Our objective is to develop a universal gene editing therapeutic for individuals with cystic fibrosis (CF). Due to the large number of CFTR variants, we sought to develop a correction methodology utilizing targeted integration of CFTR 2-27 sequence into CFTR intron 1 (TI-1). Following electroporation of CF primary airway basal stem cells with intron 1-targeted Cas9/gRNA, we delivered CFTR 2-27 donor, flanked by a splice acceptor and polyadenylation signal, via an integrase defective lentiviral vector. Molecular characterization revealed highly efficient integration of the CFTR 2-27 sequence into the native CFTR intron 1 with most insertion events reflecting the desired homology-directed repair. Fully differentiated airway epithelium, derived from TI-1 treated basal cells without enrichment for corrected cells, exhibited functional restoration of CFTR channel activity with significant improvement of mucus hydration and transport. ATAC-seq revealed that the native CFTR chromatin architecture was unaffected by the CFTR 2-27 sequence insertion, and importantly, scRNA-seq demonstrated that the cell-type-specific expression of the introduced transgene replicated that of the endogenous CFTR . In this report, we demonstrate an almost completely mutation-agnostic therapeutic approach that is capable of functional CFTR restoration. These results may also be relevant to CFTR 2-27 integration via other editing and delivery methodologies.
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BibTeX
@article{barilla2026targeted,
title = {{Targeted insertion of partial}},
author = {Cristina Barillà and Shingo Suzuki and So Hyun Park and Divyasri Nalluri and Hui Wen and Mohamed A. Hanafy and Daniel Betancourth and Ayushi Umrigar and Shih-Hsing Leir and Katreya Lovrenert and Andrey Sivachenko and Barbara Tabak and Zhong Liu and Rui Zhao and Hillary C. Valley and Andras Rab and Eric J. Sorscher and George M. Solomon and Martin Mense and John P. Carulli and Ann Harris and Gang Bao and Brian R. Davis},
journal = {Molecular Therapy: Nucleic Acids},
year = {2026},
month = oct,
doi = {10.1016/j.omtn.2026.103123},
url = {https://doi.org/10.1016/j.omtn.2026.103123},
note = {CC BY},
}
RIS
TY - JOUR
TI - Targeted insertion of partial
AU - Cristina Barillà
AU - Shingo Suzuki
AU - So Hyun Park
AU - Divyasri Nalluri
AU - Hui Wen
AU - Mohamed A. Hanafy
AU - Daniel Betancourth
AU - Ayushi Umrigar
AU - Shih-Hsing Leir
AU - Katreya Lovrenert
AU - Andrey Sivachenko
AU - Barbara Tabak
AU - Zhong Liu
AU - Rui Zhao
AU - Hillary C. Valley
AU - Andras Rab
AU - Eric J. Sorscher
AU - George M. Solomon
AU - Martin Mense
AU - John P. Carulli
AU - Ann Harris
AU - Gang Bao
AU - Brian R. Davis
PY - 2026
DA - 2026/10/01
JO - Molecular Therapy: Nucleic Acids
DO - 10.1016/j.omtn.2026.103123
UR - https://doi.org/10.1016/j.omtn.2026.103123
AB - Our objective is to develop a universal gene editing therapeutic for individuals with cystic fibrosis (CF). Due to the large number of CFTR variants, we sought to develop a correction methodology utilizing targeted integration of CFTR 2-27 sequence into CFTR intron 1 (TI-1). Following electroporation of CF primary airway basal stem cells with intron 1-targeted Cas9/gRNA, we delivered CFTR 2-27 donor, flanked by a splice acceptor and polyadenylation signal, via an integrase defective lentiviral vector. Molecular characterization revealed highly efficient integration of the CFTR 2-27 sequence into the native CFTR intron 1 with most insertion events reflecting the desired homology-directed repair. Fully differentiated airway epithelium, derived from TI-1 treated basal cells without enrichment for corrected cells, exhibited functional restoration of CFTR channel activity with significant improvement of mucus hydration and transport. ATAC-seq revealed that the native CFTR chromatin architecture was unaffected by the CFTR 2-27 sequence insertion, and importantly, scRNA-seq demonstrated that the cell-type-specific expression of the introduced transgene replicated that of the endogenous CFTR . In this report, we demonstrate an almost completely mutation-agnostic therapeutic approach that is capable of functional CFTR restoration. These results may also be relevant to CFTR 2-27 integration via other editing and delivery methodologies.
SN - 2162-2531
ER -