Abstract
RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.
Highlights
RNA interference (RNAi) is a highly conserved endogenous cellular process whereby small interfering RNAs regulate post transcriptional gene expression via sequence-specific mRNA degradation and translational blocking
Three days after treatment, normalized luciferase protein activity was significantly reduced in the ventricles of mice treated with luciferase small interfering RNAs (siRNA)-loaded MBs compared to ventricles of mice treated with control siRNA-loaded MBs (p = 0.03, Fig 3B)
Normalized ventricular luciferase expression at the mRNA level was significantly less in mice treated with luciferase siRNA-loaded MBs than in mice treated with control siRNAloaded MBs (p
Summary
RNA interference (RNAi) is a highly conserved endogenous cellular process whereby small interfering RNAs (siRNA) regulate post transcriptional gene expression via sequence-specific mRNA degradation and translational blocking. Due to its potent specific gene knockdown effects and efficiency, RNAi has emerged as a promising approach to treat molecular targets in a spectrum of human disease, including cardiac disease [1], but clinical translation has been stymied by inefficient delivery vectors and techniques. Viral vectors have been successful in mediating RNAi in cardiac tissues [2], but are limited by adverse off-target effects [3]. Custom designed microbubbles (MBs) are emerging as gene therapy vectors that may overcome several limitations of current RNAi delivery platforms and function as true theranostic agents [4,5,6]. The unique properties that make MBs ideal ultrasound contrast agents for echocardiography–intravascular kinetics comparable to that of erythrocytes and MB expansion and contraction in response to ultrasound– confer capabilities for targeted molecular therapeutics [9, 10]. Ultrasound targeted MB destruction (UTMD) has been shown by us and others to deliver nucleic acids to cells and tissues in vitro and in vivo [5, 14,15,16,17]
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