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Published March 8, 2022 | Supplemental Material + Published
Journal Article Open

Programmable siRNA pro-drugs that activate RNAi activity in response to specific cellular RNA biomarkers

Abstract

Since Paul Ehrlich's introduction of the "magic bullet" concept in 1908, drug developers have been seeking new ways to target drug activity to diseased cells while limiting effects on normal tissues. In recent years, it has been proposed that coupling riboswitches capable of detecting RNA biomarkers to small interfering RNAs (siRNAs) to create siRNA pro-drugs could selectively activate RNA interference (RNAi) activity in specific cells. However, this concept has not been achieved previously. We report here that we have accomplished this goal, validating a simple and programmable new design that functions reliably in mammalian cells. We show that these conditionally activated siRNAs (Cond-siRNAs) can switch RNAi activity against different targets between clearly distinguished OFF and ON states in response to different cellular RNA biomarkers. Notably, in a rat cardiomyocyte cell line (H9C2), one version of our construct demonstrated biologically meaningful inhibition of a heart-disease-related target gene protein phosphatase 3 catalytic subunit alpha (PPP3CA) in response to increased expression of the pathological marker atrial natriuretic peptide (NPPA) messenger RNA (mRNA). Our results demonstrate the ability of synthetic riboswitches to regulate gene expression in mammalian cells, opening a new path for development of programmable siRNA pro-drugs.

Additional Information

© 2022 The Author(s). Pblished by Elsevier Under a Creative Commons license - Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Received 26 August 2021, Accepted 31 December 2021, Available online 3 January 2022. This research was supported by grants from the NSF (NSF EFRI-ODISSEI 1332411 and CMMI-SNM 1120890), the NIH (NIH R01 AI042552, NIH/NCI P30 CA033572 and NIH R35HL150807), and the Margaret E. Early Medical Research Trust and by gifts to the MSC and to the Gehr Family Center for Leukemia Research. We thank Paul Rothemund for useful comments on the manuscript and feedback and advice during the course of the research. We thank Erik Winfree for helpful comments on the manuscript. Author contributions: S.-p.H., L.S., M.G., A.M.S., M.B.H.S., R.M., S.S., R.H., J.D., Y.-H.K., and G.M. conducted experiments. S.-p.H., L.S., S.D., J.J.R., and W.A.G. designed the experiments. S.-p.H. and S.D. wrote the manuscript. S.D., J.J.R., and W.A.G. provided critical editing and revision of the manuscript. S.D., J.J.R., and W.A.G. were responsible for funding of the project. Declaration of interests: S.-p.H., L.S., W.A.G., S.D., and J.J.R. hold financial interests in Switch Therapeutics, a company formed to develop therapeutic applications of conditional siRNAs. S.-p.H. and L.S. are now employees of Switch Therapeutics. Switch Therapeutics played no role in the funding, design, or analysis of any of the studies described here. The authors are inventors on granted and pending patents covering novel aspects of conditional RNAi technologies and applications: US 9,029,524 (granted; Caltech; S.-p.H., Robert D. Barish, and W.A.G.; fundamental concepts and designs for Cond-siRNAs), US 9,115,355 (granted; Caltech, City of Hope; S.-p.H., W.A.G., L.S., and J.J.R.; Exonuclease blocking domain for released siRNA), US 9,725,715 (granted; Caltech, City of Hope; S.-p.H., W.A.G., L.S., and J.J.R.; design of Cond-siRNA), PCT/US2019/046075 (pending; Caltech, City of Hope; M.B.H.S., S.-p.H., W.A.G., L.S., and J.J.R.; chemical modifications for Cond-siRNAs), and PCT/US2018/046379 (pending; Caltech, City of Hope, Massachusetts General Hospital; S.D., A.M.S., S.-p.H., L.S., J.D., R.H., S.S., W.A.G., and J.J.R.; Cond-siRNAs for treatment of cardiac hypertrophy).

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Supplemental Material - 1-s2.0-S216225312200004X-mmc1.pdf

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Additional details

Created:
August 22, 2023
Modified:
October 23, 2023