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Mol Ther Nucleic Acids. 2018 Dec 7;13:699-709. doi: 10.1016/j.omtn.2018.10.015. Epub 2018 Oct 30.

Overcoming the Undesirable CRISPR-Cas9 Expression in Gene Correction.

Author information

1
Translational Medicine, Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
2
Translational Medicine, Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada.
3
Translational Medicine, Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Paediatrics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
4
Translational Medicine, Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Paediatrics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address: jim.hu@utoronto.ca.

Abstract

The CRISPR-Cas9 system is attractive for gene therapy, as it allows for permanent genetic correction. However, as a new technology, Cas9 gene editing in clinical applications faces major challenges, such as safe delivery and gene targeting efficiency. Cas9 is a foreign protein to recipient cells; thus, its expression may prompt the immune system to eliminate gene-edited cells. To overcome these challenges, we have engineered a novel delivery system based on the helper-dependent adenoviral (HD-Ad) vector, which is capable of delivering genes to airway basal stem cells in vivo. Using this system, we demonstrate the successful co-delivery of both CRISPR-Cas9/single-guide RNA and the LacZ reporter or CFTR gene as donor DNA to cultured cells. HD-Ad vector genome integrity is compromised following donor DNA integration, and because the CRISPR-Cas9/single-guide RNA and donor DNA are carried on the same vector, CRISPR-Cas9 expression is concurrently eliminated. Thus, we show the feasibility of site-specific gene targeting with limited Cas9 expression. In addition, we achieved stable CFTR expression and functional correction in cultured cells following successful gene integration.

KEYWORDS:

CRISPR; cystic fibrosis; gene targeting; gene therapy; lung disease

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