CRISPR-Cas9 HDR system enhances AQP1 gene expression
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Zhimin Wang1, Yaohe Wang1,2, Songling Wang3, Li-Rong Zhang4, Na Zhang1, Zhenguo Cheng1, Qingshi Liu1, Kelly J. Shields5, Baoli Hu6,7 and Michael J. Passineau8
1Sino-British Research Centre for Molecular Oncology, National Centre for International Research in Cell and Gene Therapy, School of Basic Medical Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450052, China
2Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK
3Salivary Gland Disease Center and Molecular Laboratory for Gene Therapy and Tooth Regeneration, School of Stomatology, Capital Medical University, Beijing 100069, China
4Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450052, China
5Department of Medicine, Lupus Center of Excellence-Autoimmunity Institute, Allegheny Health Network, Pittsburgh, PA 15212, USA
6Division of Neurosurgery, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA 15224 USA
7Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
8Gene Therapy Program, Cardiovascular Institute, Allegheny Health Network, Pittsburgh, PA 15212, USA
Zhimin Wang, email: firstname.lastname@example.org
Keywords: replacement of promoter; gene editing; aquaporin 1; salivary gland dysfunction
Received: May 17, 2017 Accepted: November 16, 2017 Published: December 04, 2017
Ionizing radiation (IR) isthe primarytherapeutic tool to treat patients with cancerous lesions located in the head and neck. In many patients, IR results in irreversible and severe salivary gland dysfunction or xerostomia. Currently there are no effective treatment options to reduce the effects of xerostomia. More recently, salivary gland gene therapy utilizing the water-specific protein aquaporin 1 (AQP1) has been of great interest to potentially correct salivary dysfunction. In this study, we used CRISPR-Cas9 gene editing along with the endogenous promoter of AQP1 within theHEK293 and MDCK cell lines. The successful integration of the cytomegalovirus (CMV) promoterresultedin a significant increase of AQP1 gene transcription and translation. Additionalfunctional experiments involvingthe MDCK cell line confirmedthat over-expressed AQP1increasedtransmembrane fluid flux indicative of increased intracellular fluid flux. The off-target effect of designed guided RNA sequence was analyzed and demonstrateda high specificity for the Cas9 cleavage. Considering the development of new methods for robust DNA knock-in, our results suggest that endogenous promoter replacement may be a potential treatment forsalivary gland dysfunction.
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