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TAG-RNAi overcomes off-target effects in cancer models

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Abstract

RNA interference offers therapeutic opportunities for the clinical targeting of otherwise undruggable oncogenes. However RNAi can have off-target effects that considerably increase treatment risks. To manage these side effects and allow an easy subtraction of their activity in healthy tissues, we present here the TAG-RNAi approach where cells that are not designated targets do not have the mRNA tag. Using TAG-RNAi we first established the off-target signatures of three different siRNAs specific to the Cyclin D1 oncogene by RNA-sequencing of cultured cancer cells expressing a FLAG-HA-tagged-Cyclin D1. Then, by symmetrical allografts of tagged-cancer cells and untagged controls on the left and right flanks of model mice, we demonstrate that TAG-RNAi is a reliable approach to study the functional impact of any oncogene without off-target bias. Finally we show, as examples, that mutation-specific TAG-RNAi can be applied to downregulate two oncogenic mutants, KRAS-G12V or BRAF-V600E, while sparing the expression of the wild-type proteins. TAG-RNAi will thus avoid the traditional off-target limitations of RNAi in future experimental approaches.

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Acknowledgements

For kind gifts we thank: P. Sicinski for the tagged-CycD1 animals, Ccnd1−/− cells, and pBABE-PURO vector, S. Chouaib for the MCF7 cells, Montpellier SIRIC for the kind gift of HT-29 and SW-620 cells, L. Le Cam for the pL56-Ras vector, O. Ayrault for the kind gift of MSCV vector, and J. Pannequin for the kind gift of BRAF antibodies. We thank Dr J.C. Maurel, Dr P. Maurel, Dr Elsa Compte, Dr Caroline Bauer, and Mrs Lorraine Benigno for their help with in vivo siRNA delivery. The Aonys® vector, used to administer the siRNAs in mice, was kindly provided, for free, by the Medesispharma company. Medesispharma did not financially contribute to this work and has no conflict of interest in this study.

Funding

This work was supported by the Atip-Avenir program (RSE11003FSA), the Merieux Research Grant (060805), the FP7 Marie-Curie European IRG (277118), the EpiGenMed LabEx (“investissements d’avenir” référence ANR-10-LABX-12–01), the Fondation pour la Recherche Médicale, the Ligue Nationale and Régionale contre le Cancer, the University of Montpellier, the SATT AxLR, the Montpellier SIRIC, and the Cancéropôle GSO, all to FB. JC is the recipient of a Ligue Nationale contre le Cancer Ph.D. fellowship.

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Authors and Affiliations

  1. CNRS, UMR-5203, Institut de Génomique Fonctionnelle, F-34094, Montpellier, France

    Julien Champagne, Benjamin Maurel, Alexandre Zampieri, Maeva Moreno, Ivanna Fuentes, Adrien Decorsière & Frédéric Bienvenu

  2. INSERM, U1191, F-34094, Montpellier, France

    Julien Champagne, Benjamin Maurel, Alexandre Zampieri, Maeva Moreno, Ivanna Fuentes, Adrien Decorsière & Frédéric Bienvenu

  3. Université de Montpellier, UMR-5203, F-34094, Montpellier, France

    Julien Champagne, Benjamin Maurel, Alexandre Zampieri, Maeva Moreno, Ivanna Fuentes, Adrien Decorsière & Frédéric Bienvenu

  4. IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U896, Université de Montpellier, Institut régional du Cancer de Montpellier, F-34298, Montpellier, France

    Laetitia K. Linares

  5. MGX-Montpellier GenomiX, c/o Institut de Génomique Fonctionnelle, 141 rue de la cardonille, 34094, Montpellier Cedex 5, France

    Emeric Dubois & Dany Severac

  6. Laboratory of Excellence From Genome and Epigenome to Molecular Medicine (EpiGenMed), F-34094, Montpellier, France

    Frédéric Bienvenu

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  1. Julien Champagne
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  2. Laetitia K. Linares
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  3. Benjamin Maurel
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  10. Frédéric Bienvenu
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Contributions

JC performed experiments with the help of BM, LKL, MM, AZ, IF, AD, and FB. IF performed the screening for siRNAs specific to FLAG and HA tags helped by AZ. AZ did the tandem-HTRF measures with the help of MM and JC. AD did the clonogenic assays and D-Cyclins immunoblots with the help of JC. LKL performed the in vivo experiments in immune-compromised mice helped by BM, JC, and MM. JC did the RNAi treatments for RNA-Seq experiments and prepared total RNAs. DS prepared and processed the RNA-Seq libraries and ED processed the raw RNA-Seq data. FB analyzed the DESeq2 data with the help of AD. MM did the screening for G12V and V600E-specific siRNAs with the help of AZ and JC. FB designed the TAG-RNAi strategy, directed the study, analyzed the data, and wrote the manuscript. FB is the inventor of patents WO2018134305A1, WO2017009588A1, WO2017009587A1, and WO 2016046508A1.

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Correspondence to Frédéric Bienvenu.

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Champagne, J., Linares, L.K., Maurel, B. et al. TAG-RNAi overcomes off-target effects in cancer models. Oncogene 39, 935–945 (2020). https://doi.org/10.1038/s41388-019-1020-2

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