Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain

Authors: R.L. Rungta, H.B. Choi, P.J. Lin, R.W. Ko, D. Ashby, J. Nair, M. Manoharan, P.R. Cullis and B.A. MacVicar

Journal: Molecular Therapy—Nucleic Acids

DOI: 10.1038/mtna.2013.65

Publication - Summary

December 03, 2013


The ability to manipulate gene expression and regulation in neurons is an invaluable asset to advancement of neuroscience and developing therapeutics for neurological disorders. Gene knockdown via small interference RNA (siRNA) is attracting a great deal of interest due to simplicity of siRNA sequence design and the safety profile compared to other tools of gene therapy. Viral vectors for example, can be used to manipulate gene expression in the nervous system, but they are labour intensive and time consuming to construct and they could potentially threaten host genome integrity via genome insertion. Additionally, viral vectors cause immunogenic responses and adverse reaction, making the treatment more complicated. siRNA does not cause those complications, however a delivery vehicle is needed for circulation longevity and cellular transfection. Lipid Nanoparticles (LNPs) are drug delivery vehicles that can deliver a wide range of molecules, including nucleic acids. LNPs have been previously extensively optimized for siRNA delivery and several siRNA-LNP formulations are currently undergoing clinical trials. siRNA-LNPs have minimal toxicity and immunogenicity and are highly efficacious in intracellular delivery. The MacVicar lab from the University of British Columbia, in conjunction with Alnylam Pharmaceuticals from Cambridge, MA, have developed an siRNA-LNP formulation that can deliver siRNA to neurons in vitro and in vivo. Neuron transfection using siRNA-LNP has not been greatly studied previously due to the inability of LNPs to cross the blood-brain barrier. In this article featured in Molecular Therapy- Nucleic Acids, 2013, the authors exploit the potency of siRNA-LNP to knockdown genes in the brain.

The authors first incubated primary rat hippocampal neurons with a fluorescently labeled siRNA-LNP and found that 100% of cells took up the nanoparticles and the fluorescent label was detected in endosomes and lysosomes. Additionally, no toxicity was observed in the neurons treated with siRNA-LNP and 48 hours of incubation led to 80% protein knockdown.. Further investigation indicated that much like the thoroughly studied LNP uptake mechanism in the liver, neuronal uptake is also facilitated by Apolipoprotein E (ApoE), a component heavily involved in lipoprotein metabolism and transport. To test in vivo knockdown, LNPs containing siRNA targeting phosphatase and tensin homolog 1 (PTEN) was then injected directly into the cerebral cortex. 5 days following treatment, brain slices were harvested and observed by fluorescence microscopy. The fluorescently labeled siRNA-LNP was detected in the injection site neurons and a single injection resulted in knockdown of PTEN protein. Further studies indicated no significant toxicity in the brain and the proinflammatory cytokine TNF-α levels were not elevated. The authors then tested the siRNA-LNP knockdown of other targets in vitro and in vivo. Specifically, they targeted the GluN1 subunit of the NMDAR ion channel and measured the synaptic response in brain slices using patch-clamp electrophysiology. The functional knockdown of NMDAR currents indicated that siRNA-LNP can be used to diminish protein level and function in the brain.

This article is the first example of use of siRNA-LNP in the brain. The authors successfully delivered siRNA intracellularly and knockdown genes both in vitro and in vivo. They reported siRNA-LNP transfection efficiency toxicity profile was significantly superior to other in vitro transfection methods such as lipofectamine. In vivo, siRNA-LNP caused no significant toxicity or immunogenicity, which is a concern with other gene delivery systems such as viral vectors. Additionally, using the NanoAssemblr Benchtop instrument, siRNA-LNPs can be prepared significantly faster than viral vectors, and the same LNPs can be used in vitro and in vivo.  siRNA-LNP can therefore be a highly potent gene delivery agent both for functional genome studies or development of new therapeutics for brain and neurological disorders.


Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E–dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs. Effective targeted knockdown was demonstrated by showing that intracortical delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor (NMDAR)) selectively reduced synaptic NMDAR currents in vivo as compared with synaptic AMPA receptor currents. Therefore, LNP delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling the development of gene therapies for neurological disorders.

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