A rapid, targeted, neuron-selective, in vivo knockdown following a single intracerebroventricular injection of a novel chemically modified siRNA in the adult rat brain
Highlights
► Accell siRNA is a new type of naked siRNA without requiring transfection reagents. ► We indicated a gene silencing of adult rat brain by Accell siRNA for the first time. ► The Accell siRNA was specifically incorporated into neuron, not into glia. ► Two proteins resulted in knockdown in wide regions of brain by the procedure. ► The presented method enables an in vivo gene silencing of brain easily and quickly.
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.
Section snippets
Materials
Chemicals: Unless otherwise noted, reagents were of analytical grade.
Accell siRNA: The sequences of the siRNAs were as follows:
Accell carboxyfluroescence (FAM)-labeled control siRNA and Accell control siRNA, 5′-UGGUUUACAUGUCGACUAA-3′; Accell rat cyclophilin-B siRNA, 5′-CCUUUGGACUCUUUGGAAA-3′; Accell rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA, 5′-UCUACAUGUUCCAGUAUGA-3′.
A single i.c.v. injection of Accell siRNA
The experiments were performed in accordance with the guidelines of the Animal Ethical Committee of Osaka
Delivery of Accell siRNA to brain by i.c.v. injection
To address whether Accell siRNA is effective in the adult rat brain, we injected Accell FAM-labeled control siRNA (5 μg/rat) i.c.v. as shown in Fig. 1A. The optimal concentration of siRNA was determined through preliminary experiments and is in accordance with published studies (Thakker et al., 2004, Thakker et al., 2005). To investigate the time course of siRNA incorporation, we analyzed a cortical region (layer V; indicated by the green inset in Fig. 1A) in rats sacrificed 2, 4, or 7 days
Discussion
We have shown that a single i.c.v. injection of Accell siRNA into the adult rat brain leads to siRNA incorporation (Fig. 1, Fig. 2, Fig. 3) and knockdown of cyclophilin-B (Fig. 5, Fig. 6; Supplementary Information) and GAPDH (Fig. 7) in neurons, not glia (Fig. 4). To our knowledge, ours is the first study to demonstrate successful in vivo, intra-CNS delivery of Accell siRNA.
Although we observed widespread incorporation of siRNA in diverse brain regions, we also found that certain cell types
Conclusion
In the present study, we have developed a new procedure involving a single i.c.v. injection of Accell siRNA into the adult rat brain to induce neuron-specific protein knockdown in the CNS. The method is simple, produces rapid effects, lends itself well to experimental investigations of neural pathophysiology, and provides a hint to potential clinical utility as a tool in the treatment of neurodegenerative and neuropsychiatric disorders.
Acknowledgements
We thank Dr. Akira Sawa (Johns Hopkins University School of Medicine) and Miyuu Tanaka (Osaka Prefecture University) for helpful discussion. This work was supported in part by a Grant-in-Aid (22580339) from the Japan Society for the Promotion of Science (to H.N.).
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These authors equally contributed to this study.