RNA TECHNOLOGIES FOR REVERSAL OF ABC TRANSPORTER-MEDIATED MULTIDRUG-RESISTANCE IN CANCER

ABC transporters can mediate the multidrug-resistance (MDR) phenotype of human cancer cells. Thus, disruption of ABC transporter-mediated drug extrusion results in a re-sensitization of tumor cells to drug treatment. Low molecular weight compounds may circumvent MDR by inhibiting the efflux pump activities of the transporters. However, the innate side-effects of these compounds must be carefully considered. Thus, experimental gene therapeutic approaches using RNA technologies have been applied for inhibition of different ABC transporters. These techniques include antisense oligonucleotides, ribozymes and chemically synthesized small interfering RNAs (siRNAs) or expression cassette encoded short hairpin RNAs (shRNAs) mediating the RNA interference (RNAi) phenomenon. In order to reverse different types of MDR, different ribozymes and siRNAs were designed to inhibit the expression of the ABC transporters MDR1/P-gp, MRP2, and BCRP. These RNA constructs were used to treat different ABC transporter expressing cancer cell lines derived from various tissues. All RNA constructs decreased the level of specific ABC transporter mRNA and protein expression dramatically, and reduced the cellular resistance to drug treatment by enhancing the cellular drug accumulation in vitro. Since all of the anti-ABC transporter RNA constructs showed gene-silencing activity, plasmid expression vectors were designed for stable expression of shRNA molecules. Furthermore, expression vectors were designed that encode a multitarget multiribozyme (MTMR) or a multitarget-multi-siRNA/ribozyme (MTMsiR) simultaneously directed against three different ABC transporters, MDR1/P-gp, MRP2, and BCRP. Transfection of these RNA constructs into different MDR cell lines fully inhibited ABC transporter expression at the mRNA and protein level and completely reversed the drug-resistant phenotypes. For in vivo application, shRNA encoding DNA was designed to reverse ABC transporter-mediated MDR phenotype. An adenovirus- and an Escherichia coli-basing strategy, as well as a nonviral jet-injection technology were developed for in vivo delivery of shRNA-vector constructs.