A rapid, targeted, neuron-selective, in vivo knockdown following a single intracerebroventricular injection of a novel chemically modified siRNA in the adult rat brain

Abstract

There has been a dramatic expansion of the literature on RNA interference and with it, increasing interest in the potential clinical utility of targeted inhibition of gene expression and associated protein knockdown. However, a critical factor limiting the experimental and therapeutic application of RNA interference is the ability to deliver small interfering RNAs (siRNAs), particularly in the central nervous system, without complications such as toxicity and inflammation. Here we show that a single intracerebroventricular injection of Accell siRNA, a new type of naked siRNA that has been modified chemically to allow for delivery in the absence of transfection reagents, even into differentiated cells such mature neurons, leads to neuron-specific protein knockdown in the adult rat brain. Following in vivo delivery, targeted Accell siRNAs were incorporated successfully into various types of mature neurons, but not glia, for 1 week in diverse brain regions (cortex, striatum, hippocampus, midbrain, and cerebellum) with an efficacy of delivery of approximately 97%. Immunohistochemical and Western blotting analyses revealed widespread, targeted inhibition of the expression of two well-known reference proteins, cyclophilin-B (38–68% knockdown) and glyceraldehyde 3-phosphate dehydrogenase (23–34% knockdown). These findings suggest that this novel procedure is likely to be useful in experimental investigations of neuropathophysiological mechanisms.

Introduction

Gene silencing by RNA interference has emerged as a promising new method of inhibiting the expression of targeted genes and inducing knockdown of associated proteins both in vitro and in vivo (Akhtar and Benter, 2007, de Fougerolles et al., 2007, Elbashir et al., 2001, Shim and Kwon, 2010). RNA interference has been applied in experimental investigations of physiological and pathophysiological mechanisms in animal models, and also has been considered as a potential clinical tool in the treatment of intractable illnesses including cancer, infectious diseases, and neurodegenerative and neuropsychiatric disorders (Akhtar and Benter, 2007, Chen and Zhaori, 2011). Thus, findings from studies involving in vivo application of small interfering RNAs (siRNAs) in animal models may be useful in inspiring clinical studies and predicting unintended off-target effects and adverse reactions such as recruitment of immune responses (Akhtar and Benter, 2007, Chen and Zhaori, 2011).

Unfortunately, naked siRNAs generally are not stable enough to exert their expected effects in vivo because they are degraded rapidly by endo- and exonucleases (Shim and Kwon, 2010). Various delivery systems have been developed to circumvent this problem, including liposomes (Zimmermann et al., 2006), cationic polymers (Pulford et al., 2010), viruses (Dreyer, 2011), chemical modifications (Walton et al., 2010), short peptide-conjugations (Kumar et al., 2007), electroporation (Zhao et al., 2005), and exosomes (Alvarez-Erviti et al., 2011). In the central nervous system (CNS), topical injection of naked siRNAs has been used successfully to induce gene silencing, although the efficacy of siRNA delivery is adequate only immediately surrounding the injection site and not at more remote locations (Jean et al., 2007, Lingor et al., 2005, Makimura et al., 2002, Manrique et al., 2009). Intracerebroventricular (i.c.v.) injection of siRNAs conjugated to a lipid-based reagent (e.g., Lipofectamine 2000) has also shown promise, but this approach may possibly raise concerns about toxicity and recruitment of immune responses induced by the lipid-based reagent (Chen et al., 2009, Hu et al., 2009, Thakker et al., 2004, Thakker et al., 2005).

Thermo Scientific Dharmacon Accell siRNA is a new type of naked siRNA that has been modified chemically to allow for delivery without requiring transfection reagents, resulting in robust silencing of selected genes and knockdown of associated proteins. Accell siRNA is designed to minimize off-target effects, toxicity, and recruitment of immune responses (Baskin et al., 2008). To date, there have been several studies in which Accell siRNA has been used successfully to induce robust gene silencing and knockdown of targeted genes and proteins in neurons, but these have been exclusively in vitro cell culture studies (Dolga et al., 2008, Dreses-Werringloer et al., 2008, Sebeo et al., 2009, Suzuki et al., 2010). A few reports have appeared in which Accell siRNA was delivered in vivo via intravenous and nasal routes (Bonifazi et al., 2010, Difeo et al., 2009), but none of these studies involved delivery into the adult brain.

In the present study we demonstrate that rapid, targeted, neuron-selective, in vivo knockdown can be achieved in the adult rat brain following a single i.c.v. injection of Accell siRNA. This novel methodology has considerable potential utility as an experimental tool.