Volume 16, Issue 1, Pages 178-186 (January 2008) Complete In Vivo Reversal of the Multidrug Resistance Phenotype by Jet-injection of Anti-MDR1 Short Hairpin RNA-encoding Plasmid DNA  Ulrike Stein, Wolfgang Walther, Alexandra Stege, Alexander Kaszubiak, Iduna Fichtner, Hermann Lage  Molecular Therapy  Volume 16, Issue 1, Pages 178-186 (January 2008) DOI: 10.1038/sj.mt.6300304 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Characterization of RNA interference-mediated decrease of multidrug resistance 1 (MDR1) messenger RNA (mRNA) expression and cellular MDR1/P-gp content in drug-sensitive MaTu and multidrug-resistant MaTu/ADR cells. (a) Northern blot analysis of MDR1 mRNA expression in MaTu/ADR cells after treatment with control RNAs. (b) Western blot analysis of cell membranous MDR1/P-gp content in MaTu/ADR cells after treatment with control RNAs. (c) Northern blot analysis depicting the time course of MDR1 mRNA expression in the MaTu/ADR cells during silencing using the MDR-A small interfering RNA (siRNA construct). (d) Western blot analysis of MDR1/P-gp content in cell membranes after MDR-A siRNA exposure. (e) Northern blot analysis depicting the time course of MDR1 mRNA expression in MaTu/ADR cells during silencing using the MDR-B siRNA construct. (f) Western blot analysis of cell membranous MDR1/P-gp content after MDR-A siRNA exposure. (g) Northern blot analysis of MDR1 mRNA expression in MaTu/ADR cells after treatment with short hairpin RNA (shRNA) encoding expression vectors. (h) Western blot analysis of cell membranous MDR1/P-gp content after treatment with shRNA encoding expression vectors. In each case, Northern blot membranes were stripped and reprobed for aldolase-encoding mRNA as loading control. As control for equivalent protein loading, Western blot membranes were simultaneously incubated with an actin-specific monoclonal antibody. MDR-A siRNA, double-stranded biologically active siRNA; MDR-A sense, single-stranded MDR-A RNA in sense orientation; MDR-A antisense, single-stranded MDR-A RNA in antisense orientation; MDR-B siRNA, double-stranded biologically active siRNA; MDR-B sense, single-stranded MDR-B RNA in sense orientation; MDR-B antisense, single-stranded MDR-B RNA in antisense orientation; Luciferase siRNA, double-stranded biologically active siRNA; MDR-C, MDR-C shRNA expression vector; vector, psiRNA-hH1zeo containing an active LacZ cassette producing the EM7-lacZ α-peptide; n.d., non detected. Molecular Therapy 2008 16, 178-186DOI: (10.1038/sj.mt.6300304) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Relative resistance to doxorubicin in multidrug-resistant (MDR) MaTu/ADR cells after treatment with (a) small interfering RNAs (siRNAs), or (b) short hairpin RNA (shRNA) expression vectors. Resistance value in drug-sensitive, parental MaTu cells was set at 1. MDR-A siRNA, double-stranded biologically active siRNA; MDR-A sense, single-stranded MDR-A RNA in sense orientation; MDR-A antisense, single-stranded MDR-A RNA in antisense orientation; MDR-B siRNA, double-stranded biologically active siRNA; MDR-B sense, single-stranded MDR-B RNA in sense orientation; MDR-B antisense, single stranded MDR-B RNA in antisense orientation; Luciferase siRNA, double-stranded biologically active siRNA; MDR-C, MDR-C shRNA expression vector; vector, psiRNA-hH1zeo containing an active LacZ cassette producing the EM7-lacZ α-peptide; n.s., non-significant difference to the multidrug-resistant cell line; *P< 0.05; **P < 0.01; ***P < 0.001. Molecular Therapy 2008 16, 178-186DOI: (10.1038/sj.mt.6300304) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Jet-injection device and schematic representation of intratumoral jet-injection gene transfer. (a) The jet-injector consisting of a control unit (1) for pressure adjustment and the handpiece with nozzle (2) for application of naked DNA. (b) The jet-injection applies the DNA directly into the subcutaneous tumor. The air pressure forces the DNA-containing liquid at high speed (>300 m/s) through the nozzle into the tissue. The application sites in the animal are indicated by white arrows. Panel (c) shows the appearance of transgene expression in tumor tissue jet-injected with a LacZ-expressing reporter plasmid. The picture indicates the inhomogeneous, spotted distribution of the transgene expression. Molecular Therapy 2008 16, 178-186DOI: (10.1038/sj.mt.6300304) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Relative expression of anti-multidrug resistance 1 (anti-MDR1) short hairpin RNAs (shRNAs) in jet-injected tumors measured by real-time reverse transcription polymerase chain reaction. The relative change of shRNA expression was calculated in relation to an endogenous control miR-16 RNA set at 1, using the the 2−ΔΔCT method at days 1, 2, and 3 after injection. Each expression value was determined in triplicate in tumor xenografts growing in two different mice. As control, shRNA expression was measured in two NaCl injected tumors of two different mice I day after injection. Error bars show SD. Molecular Therapy 2008 16, 178-186DOI: (10.1038/sj.mt.6300304) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Intratumoral jet-injection of anti-multidrug resistance 1 (anti-MDR1) short hairpin RNA (shRNA) leads to down-regulation of MDR1/P-gp expression in MaTu/ADR-derived tumors in mice. (a) MaTu/ADR-derived tumors were intratumorally jet-injected with anti-MDR1 shRNA, control vector, or phosphate-buffered saline (PBS). At days 1, 2, and 3 after jet-injection, the animals were killed and the tumors were removed. The relative MDR1 messenger RNA (mRNA) expression was determined by quantitative real time reverse transcriptase polymerase chain reaction; values represent the ratios of MDR1 mRNA to housekeeping mRNA. Two experiments, each performed in duplicate, were averaged. Broken line, average of relative MDR1 mRNA expressions after jet-injection of PBS. The strong reduction in MDR1 mRNA expression after jet-injection of the anti-MDR1 shRNA is shown. (b) The relative MDR1 mRNA expressions in fractions of MaTu/ADR-derived tumors after jet-injection of anti-MDR1 shRNA. Consecutive series of cryosections were made of at least four fractions of each tumor in order to identify tumor areas containing MDR1 mRNA down-regulation caused by anti-MDR1 shRNA, as exemplified for one animal per group at days 2 and 3 after jet-injection. The intratumoral differences in down-regulation of MDR1 mRNA expression within the tumor fractions are shown. Molecular Therapy 2008 16, 178-186DOI: (10.1038/sj.mt.6300304) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 MDR1/P-gp protein expression in MaTu/ADR-derived tumors after jet-injection of anti-MDR1 short hairpin RNA (shRNA) or control vector, as exemplified for one representative animal per group at days 1, 2, 3, 5, 8, and 40 after jet-injection (bars, 50 μm). MDR1/P-gp was detected by immunohistochemistry using the C219 antibody. The strong reduction of MDR1/P-gp expression after jet-injection of anti-MDR1 shRNA at days 1, 2, and 3 is shown. MDR1, multidrug resistance 1. Molecular Therapy 2008 16, 178-186DOI: (10.1038/sj.mt.6300304) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Intratumoral jet-injection of anti-MDR1 short hairpin RNA (shRNA) combined with doxorubicin leads to growth inhibition of MaTu/ADR-derived tumors in mice. (a) Treatment schedule: intratumoral jet-injection of anti-MDR1 shRNA vectors, control vectors, or phosphate-buffered saline (PBS) was performed at days 19 and 27. Intravenous (IV) application of doxorubicin or PBS was carried out 3 days after each jet-injection, at days 22 and 30. The mice were killed at day 40. (b) Growth of MaTu- and MaTu/ADR-derived tumors. Mice harboring MaTu/ADR-derived tumors were jet-injected intratumorally with anti-MDR1 shRNA, control vector, or PBS, and subsequently treated with either doxorubicin or PBS (IV). As controls, mice with MaTu-derived tumors were jet-injected with anti-MDR1 shRNA or PBS, and subsequently treated with either doxorubicin or PBS. Significant reductions in the volume of MaTu/ADR-derived tumors are observed on comparing the values for anti-MDR1 shRNA and doxorubicin with those for control vector and doxorubicin (*P = 0.0156), as well as on comparing the values for anti-MDR1 shRNA and doxorubicin with those for PBS and doxorubicin (**P = 0.0156). The reduction in growth in MaTu/ADR-derived tumors after treatment with anti-MDR1 shRNA and doxorubicin was comparable to the levels achieved for the chemosensitive MaTu-derived tumors treated with PBS and doxorubicin. MDR1, multidrug resistance 1. Molecular Therapy 2008 16, 178-186DOI: (10.1038/sj.mt.6300304) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 8 Jet-injection of anti-MDR1 short haripin RNA (shRNA) restores drug sensitivity in MaTu/ADR-derived tumors. Doxorubicin toxicity was calculated at day 40, in terms of the ratios of the volumes of MaTu/ADR-derived tumors treated with phosphate-buffered saline (PBS) and PBS to those treated with PBS and doxorubicin (white bar), control vector and PBS versus control vector and doxorubicin (gray bar); and the ratio of the volumes of tumors treated with anti-MDR1 shRNA and PBS to those treated with anti-MDR1 shRNA and doxorubicin (black bar). Broken line, doxorubicin toxicity in MaTu-derived tumors (PBS and PBS versus PBS and doxorubicin). MDR1, multidrug resistance 1. Molecular Therapy 2008 16, 178-186DOI: (10.1038/sj.mt.6300304) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions