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

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.

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© 2022 The Authors.

Déclaration de conflit d'intérêts

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).