Investigators develop degradation-resistant microspheres composed of RNA as both carrier and cargo.
Scientists have developed a completely new way of delivering extremely high concentrations of gene-silencing siRNAs to target cells, based on nanoscale microspheres composed entirely of tightly packed, double-stranded RNA. The RNAi-microsponges developed by a multidisciplinary team at the Massachusetts Institute of Technology (MIT) are constructs of long RNA polymers comprising repeating sequences of the designated siRNA. These RNA strands self assemble into incredibly dense crystalline microspheres, which resist degradation, are efficiently taken up into cells, and then are simply chopped up by the cell’s own enzymes into vast numbers of active siRNAs.
The investigators, headed by Professor Paula T. Hammond, at MIT’s Koch Institute for Integrative Cancer Research, and Department of Chemical Engineering, say the technology enables delivery of more than half a million copies of an siRNA to a cell in a single RNAi-microsponge. They report their achievement in Nature Materials, in a paper titled “Self-assembled RNA interference microsponges for efficient siRNA delivery.”
Short interfering RNAs (siRNAs) represent a promising therapeutic option for gene silencing, but one of the major hurdles to their clinical success is the difficulty in designing an efficient delivery method. siRNA carrier or encapsulation vehicles such as lipids or inorganic nanoparticles have shown some promise, but drawbacks with these techniques include the inability to load large amounts of siRNA into the carriers, because the short siRNA strands don’t pack tightly, professor Hammond and team report. Moreover, producing the carriers is often costly, encapsulation isn’t necessarily very efficient, and the large amounts of carrier required can lead to toxicity or immune response issues in vivo.
The ideal siRNA delivery approach would instead use the host cell’s own oligonucleotide processing machinery to generate polymeric RNAi that could then be folded into dense particles representing both the carrier and the cargo, and which would be cleaved into its siRNA subunits within the target cells, the researchers suggest.
With this concept as their goal, the MIT researchers applied an enzymatic RNA polymerization technique, known as rolling circle transcription (RCT), to generate elongated polymers of pure RNA that could self assemble into organized nano- to microstructures. For their proof of principle work the team designed the RNA polymer to encode repeating units of an anti-luciferase siRNA, which they could then test in experimental models.
The construction technique first involved preparing long linear single-stranded DNA encoding complementary sequences of both the antisense and sense sequences of anti-luciferase siRNA. This long strand was then hybridized with short DNA strands containing the T7 promoter sequence, to form circular DNA. The circle was closed using T4 DNA ligase, and the circular DNA then used to produce RNA transcripts by RCT, which encode both antisense and sense sequences of the anti-luciferase siRNA. This effectively generates nanoscale pleated sheets of hairpin RNA that self-assemble into sponge-like microspheres. Critically, once internalized in cells, the RNA is split up into the 21 nucleotide-long RNA component units by activity of the enzyme Dicer, and then converted to active siRNAs by the RNA-induced silencing complex.
When the MIT team analyzed their RNA-microsponges, they estimated that each spherical particle contained about half a million tandem copies of Dicer-cleavable RNA strands. Scanning electron micrograph (SEM) showed that as the RCT polymerization process progressed, the products first formed fiber-like structure, then a sheet-like structure, which subsequently thickened and started to exhibit a densely packed internal organization. By 12 hours the sheet structures began to develop wrinkled and semi-spherical constructs, and after 16 hours the RNA polymer continued to morph into interconnected globular superstructures. These spherical structures then start to separate into individual particles, and at about 20 hours the finished spherical 2 μm sponge-like structures were seen. These were confirmed by polarizing optical microscopy to have a crystalline structure.
Because the RNAi-microsponges are essentially highly concentrated balls of nearly 100% interfering RNA (RNAi), they should be an effective means of delivering siRNA to target cells. Analyses of Dicer enzyme-treated RNAi-microspheres confirmed that they yielded 21-bp products, and about 21% of the cleavable double-stranded RNA was actually chopped into siRNA. “With these results, we estimate that each individual RNAi-microsponge can yield about 102,000 siRNA copies,” the authors write.
To boost cellular uptake of the RNA particles, the basic RNAi-microsponges were wrapped in polyethylenimine (PEI), which generated a net positively charged outer layer. The polymer coat also resulted in the RNA compacting down even further, so the particle size actually shrank from 2 μm down to just 200 nm, resulting in even higher RNA density. “To the best of our knowledge, this represents the highest number of siRNA molecular copies encapsulated in a nanoparticle,” Professor Hammond et al state.
To test whether the particles were efficiently taken up by cells and could silence their target genes, the team incubated fluorescence-labelled RNAi-microsponge/PEI constructs with T22 cancer cells. They found that compared with the uncoated RNAi-microsponges, the compacted PEI-coated constructs were much more efficiently taken up by the cells, and reduced firefly luciferase expression down to 42.4%, at a concentration of 980 fM. In contrast, at the same concentration the uncoated RNAi-microsponges were only capable of slightly reducing firefly luciferase expression, while naked siRNA demonstrated very little gene silencing even at concentrations of up to 100 nM siRNA.
The investigators then tested the level of gene knockdown in vivo by imaging firefly luciferase expression in tumors injected directly with RNAi-microsponge/PEI. Encouragingly, they found that even extremely low numbers (2.1 fmol) of RNAi-microsponge/PEI particles were capable of significantly silencing firefly luciferase gene expression. “Roughly three orders of magnitude less carrier was required to achieve the same degree of gene silencing as a conventional particle-based vehicle,” they claim.
The authors believe their RNAi-microsponge could represent a more cost-effective and efficient means of delivering high concentrations of siRNAs to target cells for therapeutic applications. The crystalline form of the polymeric RNA protects the RNA itself from degradation during delivery, while the polymerization approach can easily be modified, which means multiple RNA species could be included in the constructs for combination therapy. “The RNAi-microsponge presents a novel materials system in general owing to its unique morphology and nanoscale structure within the polymer particle,” the team concludes, “and provides a promising self-assembling material that spontaneously generates a dense siRNA carrier for broad clinical applications of RNAi delivery using the intrinsic biology of the cell.”