Vehicles for Small Interfering RNA Transfection: Exosomes Versus Synthetic Nanocarriers

RNA NANOTECHNOLOGY Review Article • DOI: 10.2478/rnan-2013-0002 • RNAN • 2013 • 16-26 Vehicles for Small Interfering RNA transfection: Exosomes versus Synthetic Nanocarriers Abstract Markus Duechler* Therapies based on RNA interference (RNAi) hold a great potential for targeted interference of the expression of specific genes. Small-interfering RNAs (siRNA) and micro-RNAs interrupt protein synthesis by inducing the Department of Bioorganic Chemistry, degradation of messenger RNAs or by blocking their translation. RNAi- Centre of Molecular and Macromolecular based therapies can modulate the expression of otherwise undruggable Studies, Polish Academy of Sciences, 112 Sienkiewicza Street, 90-363 Lodz, Poland target proteins. Full exploitation of RNAi for medical purposes depends on efficient and safe methods for delivery of small RNAs to the target cells. Tremendous effort has gone into the development of synthetic carriers to meet all requirements for efficient delivery of nucleic acids into particular tissues. Recently, exosomes unveiled their function as a natural communication system which can be utilized for the transport of small RNAs into target cells. In this review, the capabilities of exosomes as delivery vehicles for small RNAs are compared to synthetic carrier systems. The step by step requirements for efficient transfection are considered: production of the vehicle, RNA loading, protection against degradation, lack of immunogenicity, targeting possibilities, cellular uptake, cytotoxicity, RNA release into the cytoplasm and gene silencing efficiency. An exosome- based siRNA delivery system shows many advantages over conventional transfection agents, however, some crucial issues need further optimization before broad clinical application can be realized. Keywords siRNA • exosomes • liposomes • polycations • transfection Received 01 March 2013 Accepted 19 April 2013 © Versita Sp. z o.o. 1. RNA interference miRNAs add another layer to the complex process of gene regulation in eukaryotes. Primary miRNAs are transcribed Gene expression is regulated at many levels in eukaryotes. RNA from introns of coding genes or from noncoding sequences interference (RNAi) constitutes a post-transcriptional regulation as longer precursors and are further processed through a mechanism constraining the translation of messenger RNA series of endonucleolytic steps [1]. A RNase called Drosha (mRNA). Central to the RNAi gene silencing mechanism is a cleaves the primary transcript inside the nucleus to yield the large protein complex called the RNA-induced silencing complex approximately 70 nucleotide long precursor miRNA (pre- (RISC) which is loaded with small RNA oligonucleotides of 19 miRNA). After the export from the nucleus, Dicer cleaves the – 25 nucleotides in length [1]. Through complementary base- pre-miRNA resulting in the 22 nucleotide double-stranded pairing the RISC binds to the cognate mRNA and induces mRNA miRNA duplex. One strand, the guide strand, becomes degradation or translation inhibition. The RNA oligonucleotides incorporated into the RISC to target the 3’ untranslated region are either derived from double stranded RNAs (siRNA pathway) of specific mRNAs for degradation or translation repression. or from larger precursors of the microRNA (miRNA) pathway. Perfectly matching miRNAs induce mRNA cleavage, while Small-interfering RNAs are processed by the cytoplasmic RNase imperfectly matching miRNAs stall the translation process. Dicer into pieces of suitable size. They present a perfect match Due to imperfect match interactions a particular miRNA is for the target mRNAs and induce their cleavage. siRNA double able to repress the expression of many target genes. Over- strands contain a phosphate group at both the 5′ ends along expression or loss of miRNAs can be the cause for a variety of with hydroxyl groups and two nucleotide overhangs at both the diseases including cancer. miRNAs can act both as ‘oncomirs’ 3′ ends, which are important for proper function [2]. One strand and tumor suppressors, so carcinogenesis may occur through becomes associated with the RISC to guide the cleavage of the loss of tumor suppressing miRNAs or over-expression of complementary mRNA. tumor-promoting miRNAs [3,4]. * E-mail: [email protected] 16 Exosomes for siRNA transfection The potential of RNAi for efficient downregulation of gene Exosomes might be taken up by their target cells through expression is being increasingly utilized for the development endocytosis, or by direct fusion with the plasma membrane of experimental tools and promising therapies against a wide [10]. Uptake seems to depend on specific lipid or ligand- array of diseases [5]. siRNAs can be easily synthesized and the receptor interactions. In cases of direct fusion, the exosomal delivery of these biomolecules into the desired cells and tissues membrane proteins become part of the host membrane while can specifically inhibit the expression of any target protein the exosomal content is delivered to the cytosol of the target [6]. The major challenge in the use of siRNA as a therapy lies cell. miRNAs transported by exosomes might epigenetically in the safe and efficient in vivo transfer into specified target reprogram recipient cells [6]. Interestingly, some viruses cells. A variety of synthetic drug carriers which have been hijack the exosomal pathway of intercellular communication designed for DNA and RNA transfection experiments in cell to facilitate their spreading and to impede anti-viral immune culture have been adapted for siRNA delivery and further reactions. miRNAs from Epstein-Barr-Virus (EBV) infected cells developed for therapeutic applications [7]. The most commonly are transferred via exosomes to uninfected recipient cells to shut used siRNA carriers include liposomes, poly-cationic polymer down immunoregulatory genes [18]. Also, cancer cell derived nanoparticles, and attenuated viruses. Successful introduction exosomes were shown to manipulate their environment for the of siRNAs to the liver was accomplished with all three optimization of growth conditions. They can promote therapy systems. However, the liver seems to be a preferred place for resistance, angiogenesis, tumor cell invasion and immune nanoparticle accumulation, while efficient targeting of siRNA suppression [10]. Cancer cell released exosomes can contribute to other target tissues is still a pending problem [8]. Viruses to the inhibition of natural killer cell activity [19] or the preparation show high potency for gene transfection but are not well of niches for metastatic spread [20]. To what extent miRNAs are accepted for therapeutic use due to associated risks such involved in the observed effects is not yet clear. as immunogenicity, oncogenesis, the potential reversion to virulence and high cost of production. 3. Synthetic carriers for nucleic acid transfection Recently, exosomes were exploited for the transport of small RNAs into target cells [9]. These naturally produced nano- The major classes of synthetic carriers are shown in Table 1, vesicles are able to incorporate functional small RNAs and to together with the most pronounced advantages and drawbacks deliver them to target cells. In this review, the characteristics of in their usage for siRNA transfection [21]. The number of exosomes and synthetic scaffolds as two classes of delivery synthetic molecules for nucleic acid transfection is large and vehicles for small RNAs are compared in terms of vehicle continuously increasing as their performance is enhanced production, siRNA loading and protection, clearance and toxicity by chemical modifications. The potential to improve crucial of the transfection complexes, cell targeting, cellular penetration, functions for successful siRNA transfer such as stability during endosomal release, and gene silencing efficiency. circulation, effective cell penetration, cytoplasmic delivery, and tissue distribution by creating derivatives is actually one of the 2. Exosomes biggest advantages of synthetic drug carrier systems. Current efforts aim at the integration of these functions into one single Exosomes are nanoparticles, about 30–100 nm in size and are system. Besides chemical derivatisation, also mixtures of generated constitutively by most cell types. They are formed by molecules derived from different molecular classes (polycations, the inward budding of the membrane of multivesicular bodies lipids, endosomolytic substances, etc.) are very successfully (MVBs), a specialized compartment of late endosomes. Through employed for nucleic acid transfections. fusion of MVBs with the plasma membrane, exosomes are released into the surrounding environment, for instance the 4. Comparison of exosomes and synthetic extracellular matrix or circulatory system [10]. The production and drug carriers for siRNA transfection secretion of exosomes constitutes a quite recently recognized mechanism for intercellular communication [6]. 4.1 Production Exosomes contain proteins, lipids, mRNA and miRNAs. Exosomes are produced by most cell types and can be Several proteins are specifically enriched in exosomes as harvested from cell culture supernatants. For their usage as compared to their parent cells, including tetraspanins (CD9, drug delivery vesicles, a high-yielding source and a reproducible CD63, CD81, CD82) and heat shock proteins (Hsp 90, Hsp 70) scalable purification procedure are desired [22]. Currently, all [11]. The same is true for miRNAs, exosomes are enriched in methods for the preparation of a highly

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