Reversal of different drug-resistant phenotypes by an autocatalytic multitarget multiribozyme directed against the transcripts of the ABC transporters MDR1/P-gp, MRP2, and BCRP  Petra Kowalski, Pawel Surowiak, Hermann Lage  Molecular Therapy  Volume 11, Issue 4, Pages 508-522 (April 2005) DOI: 10.1016/j.ymthe.2004.11.016 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Schematic structure of the anti-ABC-transporter MTMR containing the following components: anti-MRP2 ribozyme, MDR1/P-gp spacer 1, anti-BCRP ribozyme, MDR1/P-gp spacer 2, anti-MDR1/P-gp ribozyme 1, MDR1/P-gp spacer 3, anti-MDR1/P-gp ribozyme 2, and anti-MDR1/P-gp ribozyme 3. The arrows inside indicate self-cleavage in cis. The arrows outside indicate cleavage of target mRNAs in trans. For clarity, the different components of the MTMR are colored: red, anti-ABC-transporter monoribozymes; green, MDR1/P-gp spacer sequences and, thereby, cleavage in cis; blue, ABC-transporter mRNAs and, thereby, cleavage in trans. Molecular Therapy 2005 11, 508-522DOI: (10.1016/j.ymthe.2004.11.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 (A) Polyacrylamide gel electrophoresis of the in vitro transcription products of the RNA substrate synthesis, i.e., BCRP substrate, MRP2 substrate, and MDR1/P-gp substrate, as well as of the MTMR synthesis. The MTMR already undergoes autocatalytic cleavage into distinct fragments (MTMR fragment a (MTMRa) to MTMR fragment e (MTMRe)) during in vitro transcription. (B) Autocatalytic self-cleavage activity of the full-length MTMR incubated in the presence of Mg2+ ions at 37°C for 2 h. The reaction was size-fractionated by polyacrylamide gel electrophoresis and identical distinct MTMR fragments, MTMRa to MTMRe, appeared as during the in vitro transcription reaction. Molecular Therapy 2005 11, 508-522DOI: (10.1016/j.ymthe.2004.11.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 (A) Representative autoradiography of the cleavage analysis of MTMRa to MTMRe with the 343-nt BCRP-specific substrate BCRPsub. The RNA marker was applied on lane 1. Specific MTMR fragment-mediated digestion products after 2-h cleavage reactions at 37°C were loaded in the following tracks: lane 2, free; lane 3, 1 pmol BCRPsub; lane 4, 1 pmol MTMRa; lane 5, 1 pmol BCRPsub + 1 pmol MTMRa; lane 6, 1 pmol MTMRb; lane 7, 1 pmol BCRPsub + 1 pmol MTMRb; lane 8, 1 pmol MTMRc; lane 9, 1 pmol BCRPsub + 1 pmol MTMRc; lane 10, 1 pmol MTMRd; lane 11, 1 pmol BCRPsub + 1 pmol MTMRd; lane 12, 1 pmol MTMRe; lane 13, 1 pmol BCRPsub + 1pmol MTMRe. The arrows indicate the occurrence of cleavage of the BCRP-specific RNA into the 259-nt 5′ product and the 84-nt 3′ product. (B) Representative autoradiography of the cleavage analysis of MTMRa to MTMRe with the 233-nt MRP2-specific substrate MRP2sub. The RNA marker was applied on lane 1. Specific MTMR fragment-mediated digestion products after 2-h cleavage reactions at 37°C were loaded in the following tracks: lane 2, free; lane 3, 1 pmol MRP2sub; lane 4, 1 pmol MTMRa; lane 5, 1 pmol MRP2sub + 1 pmol MTMRa; lane 6, 1 pmol MTMRb; lane 7, 1 pmol MRP2sub + 1 pmol MTMRb; lane 8, 1 pmol MTMRc; lane 9, 1 pmol MRP2sub + 1 pmol MTMRc; lane 10, 1 pmol MTMRd; lane 11, 1 pmol MRP2sub + 1 pmol MTMRd; lane 12, 1 pmol MTMRe; lane 13, 1 pmol MRP2sub + 1pmol MTMRe. The arrows indicate the occurrence of cleavage of the MRP2-specific RNA into the 134-nt 5′ product and the 99-nt 3′ product. (C) Representative autoradiography of the cleavage analysis of MTMRa to MTMRe with the 241-nt MDR1/P-gp-specific substrate MDR1sub. The RNA marker was applied on lane 1. Specific MTMR fragment-mediated digestion products after 2-h cleavage reactions at 37°C were loaded in the following tracks: lane 2, free; lane 3, 1 pmol MDR1sub; lane 4, 1 pmol MTMRa; lane 5, 1 pmol MDR1sub + 1 pmol MTMRa; lane 6, 1 pmol MTMRb; lane 7, 1 pmol MDR1sub + 1 pmol MTMRb; lane 8, 1 pmol MTMRc; lane 9, 1 pmol MDR1sub + 1 pmol MTMRc; lane 10, 1 pmol MTMRd; lane 11, 1 pmol MDR1sub + 1 pmol MTMRd; lane 12, 1 pmol MTMRe; lane 13, 1 pmol MDR1sub + 1pmol MTMRe. The arrows indicate the occurrence of cleavage of the MDR1/P-gp-specific RNA into the 152-nt 5′ product and the 89-nt 3′ product. Molecular Therapy 2005 11, 508-522DOI: (10.1016/j.ymthe.2004.11.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 (A) RT-PCR confirmation of MTMR expression in the MDR1/P-gp-overexpressing gastric carcinoma cell line 257RDB/MTMR, the BCRP-overexpressing variant 257RNOV/MTMR, and the MRP2-overexpressing ovarian carcinoma cell line A2780RCIS/MTMR. In the empty expression vector-transfected control clones (257RDB/control, 257RNOV/control, A2780RCIS/control), a specific amplification product of a plasmid-encoded transcript is visible, whereas no RT-PCR signal could be obtained in parental and drug-resistant nontransfected cell lines (257P, 257RDB, 257RNOV, A2780, A2780RCIS). As control, an RT-PCR directed against the mRNA encoding the housekeeping enzyme aldolase was performed. (B) Analysis of MTMR-mediated silencing of ABC-transporter mRNA expression determined by quantitative real-time RT-PCR using a LightCycler instrument. Relative ABC-transporter mRNA expression levels were normalized against aldolase mRNA expression. In each case, the ABC-transporter/aldolase expression ratio in drug-sensitive, parental cells (257P, A2780) was set at 1. Relative MDR1/P-gp mRNA expression in MTMR-treated cells: 257P, parental drug-sensitive cells; 257RDB, drug-resistant cells overexpressing MDR1/P-gp; 257RDB/MTMR, MTMR-treated cells; 257RDB/control, control transfectants. (C) Relative BCRP mRNA expression in MTMR-treated cells: 257P, parental drug-sensitive cells; 257RNOV, drug-resistant cells overexpressing BCRP; 257RNOV/MTMR, MTMR-treated cells; 257RNOV/control, control transfectants. (D) Relative MRP2 mRNA expression in MTMR-treated cells: A2780, parental drug-sensitive cells; A2780RCIS, drug-resistant cells overexpressing MRP2; A2780RCIS/MTMR, MTMR-treated cells; A2780RCIS/control, control transfectants. (E) Characterization of MTMR-mediated decrease in cellular ABC-transporter content determined by Western blot analysis using the mAb C219 directed against MDR1/P-gp: 257P, parental drug-sensitive cells; 257RDB, drug-resistant cells overexpressing MDR1/P-gp; 257RDB/MTMR, MTMR-treated cells; 257RDB/control, control transfectants. As control for equivalent protein loading, membranes were simultaneously incubated with a GAPDH-specific mAb. (F) Western blot analysis using BXP-21 directed against BCRP: 257P, parental drug-sensitive cells; 257RNOV, drug-resistant cells overexpressing BCRP; 257RNOV/MTMR, MTMR-treated cells; 257RNOV/control, control transfectants. For control, the blot was simultaneously incubated with a mAb directed against actin. Molecular Therapy 2005 11, 508-522DOI: (10.1016/j.ymthe.2004.11.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Effect of the MTMR on ABC-transporter protein expression measured by immunocytochemistry using the mAbs C219 directed against MDR1/P-gp, BXP-21 directed against BCRP, and MRP2clone4-2 directed against MRP2. (A) MDR1/P-gp protein expression in MTMR-treated cells: 257P, parental drug-sensitive cells; 257RDB, drug-resistant cells overexpressing MDR1/P-gp; 257RDB/MTMR, MTMR-treated cells; 257RDB/control, control transfectants. (B) BCRP protein expression in MTMR-treated cells: 257P, parental drug-sensitive cells; 257RNOV, drug-resistant cells overexpressing BCRP; 257RNOV/MTMR, MTMR-treated cells; 257RNOV/control, control transfectants. (C) MRP2 protein expression in MTMR-treated cells: A2780, parental drug-sensitive cells; A2780RCIS, drug-resistant cells overexpressing MRP2; A2780RCIS/MTMR, MTMR-treated cells; A2780RCIS/control, control transfectants. Molecular Therapy 2005 11, 508-522DOI: (10.1016/j.ymthe.2004.11.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Reversal of drug-resistant phenotypes of human carcinoma cells by the MTMR as measured by a monolayer proliferation assay to assess drug-specific IC50 values (A, C, E) and increase of relative drug accumulation by the MTMR as measured by flow cytometry. In each case, the accumulation value in drug-sensitive, parental cells was set at 1 (B, D). (A) Daunorubicin-specific IC50 values of MTMR-treated cells: 257P, parental drug-sensitive cells; 257RDB, drug-resistant cells overexpressing MDR1/P-gp; 257RDB/MTMR, MTMR-treated cells; 257RDB/control, control transfectants. (B) Daunorubicin accumulation of MTMR-treated cells: 257P, parental drug-sensitive cells; 257RDB, drug-resistant cells overexpressing MDR1/P-gp; 257RDB/MTMR, MTMR-treated cells; 257RDB/control, control transfectants. (C) Mitoxantrone-specific IC50 values of MTMR-treated cells: 257P, parental drug-sensitive cells; 257RNOV, drug-resistant cells overexpressing BCRP; 257RNOV/MTMR, MTMR-treated cells; 257RNOV/control, control transfectants. (D) Mitoxantrone accumulation of MTMR-treated cells: 257P, parental drug-sensitive cells; 257RNOV, drug-resistant cells overexpressing BCRP; 257RNOV/MTMR, MTMR-treated cells; 257RNOV/control, control transfectants. (E) Cisplatin-specific IC50 values of MTMR-treated cells: A2780, parental drug-sensitive cells; A2780RCIS, drug-resistant cells overexpressing MRP2; A2780RCIS/MTMR, MTMR-treated cells; A2780RCIS/control, control transfectants. Molecular Therapy 2005 11, 508-522DOI: (10.1016/j.ymthe.2004.11.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions