RT Journal Article
SR Electronic
T1 CRISPR/Cas9 genetic modification of CYP3A5 *3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and tacrolimus metabolism
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP dmd.117.076307
DO 10.1124/dmd.117.076307
A1 Casey R Dorr
A1 Rory P Remmel
A1 Amutha Muthusamy
A1 James Fisher
A1 Branden Moriarity
A1 Kazuto Yasuda
A1 Baolin Wu
A1 Weihua Guan
A1 Erin G. Schuetz
A1 William S Oetting
A1 Pamala A Jacobson
A1 Ajay K Israni
YR 2017
UL http://dmd.aspetjournals.org/content/early/2017/05/22/dmd.117.076307.abstract
AB CRISPR/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 (CYP3A5 *3/*3) 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, 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 (Tac) (all p-values < 0.05) or midazolam (MDZ) (all p-values < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all p-values < 0.0005) or 4-OH (all p-values < 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.