High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia

Sriram Vaidyanathan; Ameen A Salahudeen; Zachary M Sellers; Dawn T Bravo; Shannon S Choi; Arpit Batish; Wei Le; Ron Baik; Sean de la O; Milan P Kaushik; Noah Galper; Ciaran M Lee; Christopher A Teran; Jessica H Yoo; Gang Bao; Eugene H Chang; Zara M Patel; Peter H Hwang; Jeffrey J Wine; Carlos E Milla; Tushar J Desai; Jayakar V Nayak; Calvin J Kuo; Matthew H Porteus

doi:10.1016/j.stem.2019.11.002

High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia

Cell Stem Cell. 2020 Feb 6;26(2):161-171.e4. doi: 10.1016/j.stem.2019.11.002. Epub 2019 Dec 12.

Authors

Sriram Vaidyanathan¹, Ameen A Salahudeen², Zachary M Sellers¹, Dawn T Bravo³, Shannon S Choi², Arpit Batish², Wei Le³, Ron Baik¹, Sean de la O², Milan P Kaushik¹, Noah Galper¹, Ciaran M Lee⁴, Christopher A Teran³, Jessica H Yoo², Gang Bao⁴, Eugene H Chang⁵, Zara M Patel³, Peter H Hwang³, Jeffrey J Wine⁶, Carlos E Milla¹, Tushar J Desai⁷, Jayakar V Nayak⁸, Calvin J Kuo⁹, Matthew H Porteus¹⁰

Affiliations

¹ Department of Pediatrics, Stanford University, Stanford, CA 94304, USA.
² Department of Internal Medicine, Stanford University, Stanford, CA 94305, USA.
³ Department of Otolaryngology-Head and Neck Surgery, Stanford, CA 94305, USA.
⁴ Department of Bioengineering, Rice University, Houston, TX 77030, USA.
⁵ Department of Otolaryngology, University of Arizona, Tucson, Tucson, AZ 85724, USA.
⁶ Department of Psychology, Stanford University, Stanford, CA 94305, USA.
⁷ Department of Internal Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address: tdesai@stanford.edu.
⁸ Department of Otolaryngology-Head and Neck Surgery, Stanford, CA 94305, USA. Electronic address: jnayak@stanford.edu.
⁹ Department of Internal Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address: cjkuo@stanford.edu.
¹⁰ Department of Pediatrics, Stanford University, Stanford, CA 94304, USA. Electronic address: mporteus@stanford.edu.

Abstract

Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

Keywords: CF; CFTR; CRISPR; Cas9; F508del; airway stem cells; basal cells; cell therapy; cystic fibrosis; genome editing.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation
Cystic Fibrosis Transmembrane Conductance Regulator / genetics
Cystic Fibrosis* / genetics
Cystic Fibrosis* / therapy
Epithelial Cells
Epithelium
Humans
Stem Cells
Swine

Substances

CFTR protein, human
Cystic Fibrosis Transmembrane Conductance Regulator

Grants and funding

K08 DE027730/DE/NIDCR NIH HHS/United States