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Research ArticleArticle

CRISPR/Cas9 genetic modification of CYP3A5 *3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and tacrolimus metabolism

Casey R Dorr, Rory P Remmel, Amutha Muthusamy, James Fisher, Branden Moriarity, Kazuto Yasuda, Baolin Wu, Weihua Guan, Erin G. Schuetz, William S Oetting, Pamala A Jacobson and Ajay K Israni
Drug Metabolism and Disposition May 22, 2017, dmd.117.076307; DOI: https://doi.org/10.1124/dmd.117.076307
Casey R Dorr
1 Minneapolis Medical Research foundation, Minneapolis,MN;
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Rory P Remmel
2 University of Minnesota, Minneapolis, MN;
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Amutha Muthusamy
3 Minneapolis Medical Research Foundation,Minneapolis,MN;
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James Fisher
2 University of Minnesota, Minneapolis, MN;
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Branden Moriarity
2 University of Minnesota, Minneapolis, MN;
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Kazuto Yasuda
4 St. Jude Children's Research Hospital, Memphis, TN;
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Baolin Wu
2 University of Minnesota, Minneapolis, MN;
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Weihua Guan
2 University of Minnesota, Minneapolis, MN;
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Erin G. Schuetz
5 St. Judes Children's Hospital;
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William S Oetting
6 University of Minnesota,Minneapolis, MN;
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Pamala A Jacobson
2 University of Minnesota, Minneapolis, MN;
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Ajay K Israni
7 Minneapolis Medical research Foundation, Minneapolis, MN
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  • For correspondence: isran001@umn.edu
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Abstract

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.

  • cell models
  • clinical pharmacology
  • cytochrome P450
  • enzyme engineering
  • hepatocytes
  • immunosuppression
  • pharmacogenetics/pharmacogenomics
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 51 (2)
Drug Metabolism and Disposition
Vol. 51, Issue 2
1 Feb 2023
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Research ArticleArticle

CRISPR/Cas9 genetic modification of CYP3A5 *3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and tacrolimus metabolism

Casey R Dorr, Rory P Remmel, Amutha Muthusamy, James Fisher, Branden Moriarity, Kazuto Yasuda, Baolin Wu, Weihua Guan, Erin G. Schuetz, William S Oetting, Pamala A Jacobson and Ajay K Israni
Drug Metabolism and Disposition May 22, 2017, dmd.117.076307; DOI: https://doi.org/10.1124/dmd.117.076307

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Research ArticleArticle

CRISPR/Cas9 genetic modification of CYP3A5 *3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and tacrolimus metabolism

Casey R Dorr, Rory P Remmel, Amutha Muthusamy, James Fisher, Branden Moriarity, Kazuto Yasuda, Baolin Wu, Weihua Guan, Erin G. Schuetz, William S Oetting, Pamala A Jacobson and Ajay K Israni
Drug Metabolism and Disposition May 22, 2017, dmd.117.076307; DOI: https://doi.org/10.1124/dmd.117.076307
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