Nonviral Vectors for the Delivery of Small Interfering RNAs to the CNS

Inmaculada Posadas; Francisco Javier Guerra; Valentín Ceña


Nanomedicine. 2010;5(8):1219-1236. 

In This Article

Abstract and Introduction


While efficient methods for cell line transfection are well described, for primary neurons a high-yield method different from those relying on viral vectors is lacking. Viral vector-based primary neuronal infection has several drawbacks, including complexity of vector preparation, safety concerns and the generation of immune and inflammatory responses, when used in vivo. This article will cover the different approaches that are being used to efficiently deliver genetic material (both DNA and small interfering RNA) to neuronal tissue using nonviral vectors, including the use of cationic lipids, polyethylenimine derivatives, dendrimers, carbon nanotubes and the combination of carbon-made nanoparticles with dendrimers. The effectiveness, both in vivo and in vitro, of the different methods to deliver genetic material to neural tissue is discussed.


The ability to introduce genetic material into cells (i.e., transfection) to either overexpress a protein or inhibit its expression has boosted developments in both therapeutic and cell biological exploitation of this technology.[1–4] Therapeutic applications have focused on attempting to introduce the defective gene(s) into malfunctioning target cells from patients suffering from diseases of genetic origin or interfering with signaling pathways involved in the genesis of different diseases by selectively knocking down different proteins using RNAi technology or generating knockout (KO) mice.

This approach has been shown to be especially useful in tumoral cell lines[4] and blood cells.[5] However, nonmitotic cells,[6,7] especially neurons, are very difficult to transfect. Efficient transfection methods of established neural-derived cell lines or postmitotic neurons would markedly improve the ability to regulate the different signaling pathways activated in neuronal death during different neurological diseases, including neurodegenerative diseases.

The study of the role of different proteins in neuronal physiology or pathology requires an approach that should include the selective KO of such proteins to study the lack-of-function effect. One traditional approach is to generate KO mice for the selective protein,[8] but this is a time-consuming method and the function of the removed protein can occasionally be replaced by another protein during development, thus leading to a lack of phenotype. Another approach is to generate conditional knockdown or knock-in mice that only remove or express the protein following a specific treatment.[9] This procedure prevents compensation of function during development by another protein, but it is very time consuming and technically difficult to achieve. RNAi technology has emerged as a good alternative to KO mice to study the role that certain proteins play in physiological and pathological mechanisms because it is specific, it does not allow for the genesis of compensatory pathways during mouse development, and it is cheaper and less time consuming than preparing KO mice.

RNAi is a powerful gene silencing mechanism that operates in most eukaryotic cells.[10] The effector molecules comprise short duplex RNA sequences of 21–23 bp that cause direct inhibition of homologous genes. Naturally, the pathway is important for the processing of regulatory micro RNAs (miRs).[11,12] These noncoding cellular sequences regulate several pathways, such as cell differentiation, metabolism, proliferation and malignant transformation. The miR processing pathway is initiated by transcription of miR-encoding cellular genes to produce hairpin-containing primary miRs. Within the nucleus, these primary miRs are processed to form precursor miR hairpins of 60–80 nucleotide in length, which are then processed by Dicer to form a staggered RNA duplex of 21–24 bp. This duplex is handed on to the RNA-induced silencing complex that is responsible for direct target-specific silencing. Exogenous activators of RNAi may be used for therapeutic applications by silencing specific sequences that cause pathology. One of these is small interfering RNAs (siRNAs); double stranded synthetic mimics (~21 nucleotides) of the staggered RNA duplex formed after Dicer processing of pre-miRs. This topic has been covered by recent reviews.[13–15]

However, the general use of siRNA technology in postmitotic neuronal cells has been scarce owing to the low efficiency of the different vectors in delivering significant amounts of siRNA to neurons.

In neural cells, due to the lack of transfection efficiency of nonviral vectors,[16,17] the initial strategy to deliver genetic material to neurons has relied on the use of viral vectors. These have displayed the highest transfer efficiency in introducing siRNA into neurons, achieving approximately 80% inhibition of specific protein levels, which is certainly considered enough to perform lack-of-function studies.[18]

However, the use of viral vectors has specific drawbacks, such as:

  • The high cost of production;

  • Host immune response against the viral agents: basically a humoral response that produces neutralizing antibodies, decreasing the effectiveness of the vector.[19] The presence of neutralizing antibodies against adeno-associated virus in vivo, owing to prior infection, has been shown;[20]

  • Safety concerns, especially with the use of oncoretroviruses, such as the occurrence of insertional mutagenesis during human gene therapy trials.[21]

These drawbacks have generated a huge barrier to the use of these vectors in nonlethal non-Mendelian diseases. Moreover, these problems have been aggravated by the death of patients participating in clinical trials using these vectors,[22–24] thus posing a serious limitation to the use of this strategy in humans.

The use of nonviral vectors was proposed to overcome the aforementioned drawbacks of viral vectors. Nonviral vectors should share features common to other drugs, such as easy scale-up production, long-term stability and minimal safety risks. In spite of the decrease in the toxicity of nonviral vectors, these compounds display a lower gene transfer rate than viral vectors, with this low efficiency being the most important weakness for the use of these compounds in gene therapy in the nervous system.[25,26] In the past decade, the amount of research performed in the area of nonviral vectors for gene delivery has increased and very useful reviews have been published.[27–33]

This article will focus on the use of different types of nonviral vectors commonly used for evaluation of gene transfer effectiveness, focusing on their efficiency as delivery agents and their toxicity in the nervous system. The use of cationic lipids, polyethylenimine (PEI) derivatives, dendrimers and carbon-based nanoparticles (NPs) will be discussed.


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