CELLS HAVING DISRUPTED EXPRESSION OF PROTEINS INVOLVED IN ADME AND TOXICOLOGY PROCESSES
The present invention provides cells comprising disrupted expression of at least one membrane transporter, drug metabolizing enzyme, xenobiotic sensor, or cellular stress response pathway protein. Also provided are methods for assessing the effect of an agent in the cells disclosed herein relative to comparable control cells.
1 . A human cell line comprising disrupted expression of at least one protein involved in a drug absorption, distribution, metabolism and excretion and/or toxicology process, wherein the protein is chosen from a membrane transporter, a drug metabolizing enzyme, a xenobiotic sensor, a cellular stress response pathway protein, and combinations thereof.
2 . The human cell line of claim 1 , wherein the membrane transporter is chosen from ABCA2, ABCB1, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCC7, ABCC8, ABCC9, ABCC10, ABCC11, ABCC12, ABCC13, ABCC2, SLC10A1, SLC10A2, SLC15A1, SLC15A2, SLC16A1, SLC16A3, SLC16A4, SLC16A5, SLC16A7, SLC16A8, SLCO1A2, SLCO1B1, SLCO1B3, SLCO1C1, SLCO2B1, SLCO3A1, SLCO4A1, SLCO4C1, SLC22A1, SLC22A2, SLC22A3, SLC22A4, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLC22A9, SLC22A11, SLC47A1, SLC47A2, OSTA, and OSTB.
3 . The human cell line of claim 1 , wherein the drug metabolizing enzyme is chosen from CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, POR, FMO1, FMO2, FMO3, FMO4, FMO5, ADH, AOX, ALDH3A1, ALDH3A2, AKR, CES1, CES2, EPHX1, EPHX2, MAOA, MAOB, MPO, PTGS2, LPO, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B15, SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1E1, SULT2A1, NAT1, NAT2, TPMT, COMT, GSTA1, GSTA2, GSTA3, GSTA4, GSTK1, GSTM1, GSTM2, GSTM3, GSTM4, GSTM5, GSTP1, GSTT1, and GSTT2.
4 . The human cell line of claim 1 , wherein the xenobiotic sensor is chosen from AhR, PXR, CAR, LXR, FXR, and PPAR.
5 . The human cell line of claim 1 , wherein the cellular stress pathway protein is chosen from HSF1, Hsp90, Hsp27, Hsp70, HIF1, VHL, VEGF, PDK1, GLUT1, XBP1, ATF4, ATF6, BiP/Grp78, PERK, IRE1, CHOP, p58, p53, ATM, ATR, MDM2, p21, GADD45, BAX, RAD51, Nrf2, Keap1, HMOX1, PRDX1, GSTA2, p66Shc, NFκB, IκB, COX2, iNOS, ICAM, IL-6, and IL-8.
6 . The human cell line of claim 1 , further comprising expression of at least one reporter protein.
7 . The human cell line of claim 1 , wherein expression is disrupted such that the cell produces substantially no functional protein, a reduced level of the protein, an increased level of the protein, or an altered version of the protein.
8 . The human cell line of claim 1 , wherein a chromosomal sequence encoding the protein is modified by a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or a combination thereof.
9 . The human cell line of claim 8 , wherein the cell is heterozygous, homozygous, or hemizygous for the modified chromosomal sequence.
10 . The human cell line of claim 1 , wherein expression is disrupted by a technique chosen from targeting endonuclease mediated genome editing, RNA interference, and homologous recombination.
11 . The human cell line of claim 10 , wherein the targeting endonuclease is a zinc finger nuclease.
12 . The human cell line of claim 1 , wherein the cell comprises an inactivated ABCG2 chromosomal sequence such that it produces substantially no BCRP protein and/or the cell comprises an inactivated ABCC1 chromosomal sequence such that it produces substantially no MDR1 protein.
13 . The human cell line of claim 12 , further comprising expression of at least one reporter protein.
14 . The human cell line of either claim 12 or claim 13 , further comprising disrupted expression of at least one additional protein chosen from a membrane transporter, a drug metabolizing enzyme, a xenobiotic sensor, a cellular stress response pathway protein, and combinations thereof.
15 . A method for assessing the effect of an agent, the method comprising:
a) contacting a cell comprising disrupted expression of at least one protein involved in a drug absorption, distribution, metabolism and excretion and/or toxicology process, wherein the protein is chosen from a membrane transporter, a drug metabolizing enzyme, a xenobiotic sensor, a cellular stress response pathway protein, and combinations thereof with the agent; and
b) comparing results of a selected parameter to results obtained from contacting a comparable wild-type cell with the same agent.
16 . The method of claim 15 , wherein the agent is chosen from a pharmaceutically active ingredient, a therapeutic agent, a toxin, and a chemical.
17 . The method of claim 15 , wherein the membrane transporter is chosen from ABCA2, ABCB1, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCC7, ABCC8, ABCC9, ABCC10, ABCC11, ABCC12, ABCC13, ABCC2, SLC10A1, SLC10A2, SLC15A1, SLC15A2, SLC16A1, SLC16A3, SLC16A4, SLC16A5, SLC16A7, SLC16A8, SLCO1A2, SLCO1B1, SLCO1B3, SLCO1C1, SLCO2B1, SLCO3A1, SLCO4A1, SLCO4C1, SLC22A1, SLC22A2, SLC22A3, SLC22A4, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLC22A9, SLC22A11, SLC47A1, SLC47A2, OSTA, and OSTB.
18 . The method of claim 15 , wherein the drug metabolizing enzyme is chosen from CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, POR, FMO1, FMO2, FMO3, FMO4, FMO5, ADH, AOX, ALDH3A1, ALDH3A2, AKR, CES1, CES2, EPHX1, EPHX2, MAOA, MAOB, MPO, PTGS2, LPO, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B15, SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1E1, SULT2A1, NAT1, NAT2, TPMT, COMT, GSTA1, GSTA2, GSTA3, GSTA4, GSTK1, GSTM1, GSTM2, GSTM3, GSTM4, GSTM5, GSTP1, GSTT1, and GSTT2.
19 . The method of claim 15 , wherein the xenobiotic sensor is chosen from AhR, PXR, CAR, LXR, FXR, and PPAR.
20 . The method of claim 15 , wherein the cellular stress pathway protein is chosen from HSF1, Hsp90, Hsp27, Hsp70, HIF1, VHL, VEGF, PDK1, GLUT1, XBP1, ATF4, ATF6, BiP/Grp78, PERK, IRE1, CHOP, p58, p53, ATM, ATR, MDM2, p21, GADD45, BAX, RAD51, Nrf2, Keap1, HMOX1, PRDX1, GSTA2, p66Shc, NFκB, IκB, COX2, iNOS, ICAM, IL-6, and IL-8.
21 . The method of claim 15 , wherein the cell further comprises expression of at least one reporter protein.
22 . The method of claim 15 , wherein expression is disrupted such that the cell produces substantially no functional protein, a reduced level of the protein, an increased level of the protein, or an altered version of the protein.
23 . The method of claim 15 , wherein a chromosomal sequence encoding the protein is modified by a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or a combination thereof.
24 . The method of claim 23 , wherein the cell is heterozygous, homozygous, or hemizygous for the modified chromosomal sequence.
25 . The method of claim 15 , wherein the cell comprises an inactivated ABCG2 chromosomal sequence such that it produces substantially no BCRP protein and/or the cell comprises an inactivated ABCC1 chromosomal sequence such that it produces substantially no MDR1 protein is.
26 . The method of claim 25 , wherein the cell further comprises expression of at least one reporter protein.
27 . The method of either claim 25 or claim 26 , wherein the cell further comprises disrupted expression of at least one additional protein chosen from a membrane transporter, a drug metabolizing enzyme, a xenobiotic sensor, a cellular stress response pathway protein, and combinations thereof.