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Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug

Abstract

The dose-limiting toxicity of chemotherapeutics, heterogeneity and drug resistance of cancer cells, and difficulties of targeted delivery to tumors all pose daunting challenges to effective cancer therapy. We report that small interfering RNA (siRNA) duplexes readily penetrate intact bacterially derived minicells previously shown to cause tumor stabilization and regression when packaged with chemotherapeutics. When targeted via antibodies to tumor-cell-surface receptors, minicells can specifically and sequentially deliver to tumor xenografts first siRNAs or short hairpin RNA (shRNA)–encoding plasmids to compromise drug resistance by knocking down a multidrug resistance protein. Subsequent administration of targeted minicells containing cytotoxic drugs eliminate formerly drug-resistant tumors. The two waves of treatment, involving minicells loaded with both types of payload, enable complete survival without toxicity in mice with tumor xenografts, while involving several thousandfold less drug, siRNA and antibody than needed for conventional systemic administration of cancer therapies.

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Figure 1: siRNA packaging in minicells, loading efficiency and BsAb targeting of the siRNA-packaged minicells to tumor cells.
Figure 2: Intracellular fate of siRNA-containing minicells targeted to human cancer cells and cytoplasmic release of siRNA.
Figure 3: PLK1 knockdown following EGFRminicellssiPLK1-delivery to tumor cells, inhibition of tumor cell proliferation after minicell-mediated siRNA delivery and reversal of drug resistance in vitro.
Figure 4: Inhibition of xenograft tumor–growth following minicell-mediated delivery of si/sh RNAs against cell cycle–associated proteins and target mRNA/protein knockdown in vivo.
Figure 5: Inhibition or regression of xenograft tumor–growth by minicell delivery of shRNA or chemotherapeutic drug and reversal of multidrug resistance in vivo by sequential treatment with targeted, shMDR1-containing minicells followed by targeted, chemotherapeutic drug-containing minicells.
Figure 6: Reversal of multidrug resistance in MDR1-overexpressing aggressive uterine cancer xenografts with complete survival of mice administered dual sequential treatments.

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Acknowledgements

This work was supported in part by a Commercial Ready Grant from AusIndustry, Australia. We appreciate the assistance of R.P. Paulin, G. Al Bakri, M. Harrison and R. Emira in minicell purification. We thank S. Pattison and N. Grimes for technical assistance. We are grateful to S. Friend, L. Sepp-Lorenzino and W. Tao (Merck & Co., West Point, PA, USA) for the permission to publish the work associated with the kinesin spindle protein (KSP) siRNA sequence. The authors dedicate this paper to R.M.G.'s mother, who died of metastatic renal cell carcinoma on August 5, 2008.

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Contributions

H.B. and J.A.M. conceived the project and co-invented the minicell delivery technology. They designed the experiments and analyzed the data and wrote the manuscript. J.A.M. also carried out critical breakthrough experiments. N.B.A.-M. constructed the shRNA plasmids, packaged the siRNA into minicells and carried out the quantification studies. J.M-W. and S.W. packaged drugs into minicells and performed in vitro transfection studies and in vivo xenograft studies. I.S., K.K. and C.P. carried out intracellular kinetic studies and established target knockdown in vivo. S.T.P. performed the immune response studies and V.N.B. constructed the bispecific antibodies and targeted the minicells. L.P. estimated minicell packaged drug quantification. R.M.G. and B.S. provided helpful discussion and R.M.G. also assisted in writing the manuscript.

Corresponding author

Correspondence to Himanshu Brahmbhatt.

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Competing interests

H.B. and J.A.M. are the scientific founders of EnGenIC, which is developing bacterial minicells as delivery vehicles for drugs and nucleic acids. All authors apart from R.M.G. and B.S. are employed by EnGenIC. H.B., J.A.M., R.M.G. and B.S. hold shares in EnGenIC.

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MacDiarmid, J., Amaro-Mugridge, N., Madrid-Weiss, J. et al. Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug. Nat Biotechnol 27, 643–651 (2009). https://doi.org/10.1038/nbt.1547

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