1. CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism.
- Author
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Dorr CR, Remmel RP, Muthusamy A, Fisher J, Moriarity BS, Yasuda K, Wu B, Guan W, Schuetz EG, Oetting WS, Jacobson PA, and Israni AK
- Subjects
- Cell Line, Humans, Liver metabolism, Male, Middle Aged, Point Mutation genetics, RNA, Messenger genetics, Sequence Deletion genetics, CRISPR-Cas Systems genetics, Clustered Regularly Interspaced Short Palindromic Repeats genetics, Cytochrome P-450 CYP3A genetics, Hepatocytes metabolism, Midazolam metabolism, Tacrolimus metabolism
- Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 ( CYP3A5 *3/*3 ) has led to three CYP3A5 *1 cell lines by deletion of the exon 3B splice junction or point mutation. Cell lines CYP3A5 *1/*3 sd (single deletion), CYP3A5 *1/*1 dd (double deletion), or CYP3A5 *1/*3 pm (point mutation) expressed the CYP3A5 *1 mRNA and had elevated CYP3A5 mRNA ( P < 0.0005 for all engineered cell lines) and protein expression compared with HuH-7. In metabolism assays, HuH-7 had less tacrolimus (all P < 0.05) or midazolam (MDZ) (all P < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all P < 0.0005) or 4-OH (all P < 0.005) production in metabolism assays than all bioengineered cell lines. We confirmed CYP3A5 metabolic activity with the CYP3A4 selective inhibitor CYP3CIDE. This is the first report of genomic CYP3A5 bioengineering in human cell lines with drug metabolism analysis., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2017
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