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 S. Moriarity; Kazuto Yasuda; Baolin Wu; Weihua Guan; Erin G. Schuetz; William S. Oetting; Pamala A. Jacobson; Ajay K. Israni

doi:10.1124/dmd.117.076307

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 S. Moriarity, Kazuto Yasuda, Baolin Wu, Weihua Guan, Erin G. Schuetz, William S. Oetting, Pamala A. Jacobson, Ajay K. Israni

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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.

Original language	English (US)
Pages (from-to)	957-965
Number of pages	9
Journal	Drug Metabolism and Disposition
Volume	45
Issue number	8
DOIs	https://doi.org/10.1124/dmd.117.076307
State	Published - Aug 1 2017

Bibliographical note

Funding Information:
This study was funded by a Minneapolis Medical Research Foundation Postdoctoral Career Development Award (to C.D.) and grant support from United States National institutes of Health National Institute for Allergy and Infectious Diseases [Grant AI U19 AI070119] (to A.I.). The authors thank the University of Minnesota Genomics Center for numerous molecular biology services. Ajay Israni and Casey Dorr, along with Minneapolis Medical Research Foundation, have filed a provisional patent with the US Patent and Trademark Office titled: “Genetically Modified Cells for Metabolic Studies.” The patent application number is 62/459,749. The cell line CYP3A5 *1/*3 sd is deposited at American Type Culture Collection (ATCC) patent depository under the name “Human Liver Cell Line, 97 CYP3A5 *1/*3” with ATCC patent deposit designation PTA-123710.

Publisher Copyright:
Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

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Dorr, C. R., Remmel, R. P., Muthusamy, A., Fisher, J., Moriarity, B. S., Yasuda, K., Wu, B., Guan, W., Schuetz, E. G., Oetting, W. S., Jacobson, P. A., & Israni, A. K. (2017). CRISPR/Cas9 genetic modification of CYP3A5 *3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and tacrolimus metabolism. Drug Metabolism and Disposition, 45(8), 957-965. https://doi.org/10.1124/dmd.117.076307

Dorr, CR, Remmel, RP, Muthusamy, A, Fisher, J, Moriarity, BS, Yasuda, K, Wu, B, Guan, W, Schuetz, EG, Oetting, WS , Jacobson, PA & Israni, AK 2017, 'CRISPR/Cas9 genetic modification of CYP3A5 *3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and tacrolimus metabolism', Drug Metabolism and Disposition, vol. 45, no. 8, pp. 957-965. https://doi.org/10.1124/dmd.117.076307

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title = "CRISPR/Cas9 genetic modification of CYP3A5 *3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and 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.",

author = "Dorr, {Casey R.} and Remmel, {Rory P.} and Amutha Muthusamy and James Fisher and Moriarity, {Branden S.} and Kazuto Yasuda and Baolin Wu and Weihua Guan and Schuetz, {Erin G.} and Oetting, {William S.} and Jacobson, {Pamala A.} and Israni, {Ajay K.}",

note = "Funding Information: This study was funded by a Minneapolis Medical Research Foundation Postdoctoral Career Development Award (to C.D.) and grant support from United States National institutes of Health National Institute for Allergy and Infectious Diseases [Grant AI U19 AI070119] (to A.I.). The authors thank the University of Minnesota Genomics Center for numerous molecular biology services. Ajay Israni and Casey Dorr, along with Minneapolis Medical Research Foundation, have filed a provisional patent with the US Patent and Trademark Office titled: “Genetically Modified Cells for Metabolic Studies.” The patent application number is 62/459,749. The cell line CYP3A5 *1/*3 sd is deposited at American Type Culture Collection (ATCC) patent depository under the name “Human Liver Cell Line, 97 CYP3A5 *1/*3” with ATCC patent deposit designation PTA-123710. Publisher Copyright: Copyright {\textcopyright} 2017 by The American Society for Pharmacology and Experimental Therapeutics.",

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

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