(Contributed by Precision NanoSystems Inc.)
The use of siRNA for gene silencing is now common practice in many labs, and can be quite effective in cultured cells. However, academic and corporate researchers continue to lament the difficulties of delivering siRNAs in vivo, or to difficult-to-transfect cells, such as neurons.
Precision NanoSystems Inc (PNI) technology represents a new family of reagents that effectively delivers RNA into a cell using a natural pathway, with over 95% transfection efficiency. Scientists can now focus on studying the effects of gene knockdown without concern for carrier effects and limitations such as poor performance in postmitotic cells orcellular toxicity.
Precision NanoSystems nanoparticles
PNI has developed unique, proprietary equipment (NanoAssemblr™) and reagent kits (SUB9KITS™) that enable the simple assembly of novel nanoparticles that greatly improve the delivery of RNA payloads. The foremost of the SUB9KITS is the Neuro9™ Kit, which has been validated for delivery of RNA to neurons, in vitro and in vivo, with high efficiency. Nanoparticle preparation by precipitation has long been considered more art than science until PNI’s recent application of microfluidics to the encapsulation process. Precise control of microfluidic injection parameters enables RNA to be encapsulated with little to no waste material, and with consistently tuned particle size.
These factors allow PNI’s nanoparticles to outperform traditional transfection approaches such as use of cationic lipids, and without the cell wall disruption often incurred with electroporation. Furthermore, the semi-automated microfluidic process is more consistent across experiments and facilitates a quicker experimental cycle. Thus, researchers can focus on more important factors, such as RNA selection and gene knockdown.
IDT DsiRNA silencing
An effective delivery tool is the ideal complement to a well-designed and selected payload. Therefore, the SUB9KITS use dicer-substrate RNAs (DsiRNA) from IDT as active agent for RNA interference. DsiRNAs are chemically synthesized 27mer duplex RNAs that have increased potency in RNA interference compared to traditional 21mer siRNAs. Dicer-substrate siRNAs are designed to be optimally processed by Dicer and show increased potency by engaging this natural processing pathway .
Using this approach, sustained knockdown has been regularly achieved using sub-nanomolar DsiRNA concentrations. The SUB9KITS can be ordered with either 3 or 5 DsiRNA duplexes, allowing for experimental identification of the DsiRNA sequence that most effectively silences the gene of interest.
Delivery of DsiRNAs in primary neurons using nanoparticles
The biotech industry is filled with lofty claims, and it can be difficult to convince researchers that a new method is truly an improvement over existing tools. However, researchers who adopt SUB9KIT nanoparticles have been pleasantly surprised by their easy implementation. This has allowed PNI to continually attract new users in the RNA delivery community, as labs using traditional methods learn of SUB9KIT advantages, and the ability to run more successful experiments in a shorter amount of time. Examples of 2 labs that have switched to the Neuro9 Kit are provided below.
Consider Dr Craig’s experience. As professor in the department of psychiatry at the University of British Columbia, Prof Craig focuses her research on neural communication within the brain, and mechanisms that disrupt this communication, leading to psychiatric disorders. A Postdoctoral Fellow in Prof Craig’s laboratory approached PNI to discuss the potential for the Neuro9 Kit to simultaneously knock down 3 genes that are expressed in primary neurons. His attempts to use plasmid vector delivery of shRNA was of limited success using either traditional cationic transfection reagents, or electroporation with primary neurons. He was also interested in a lower cost alternative to viral vectors.
Even after these considerations, Dr Craig recounts, “We were planning to transfect HEK 293 cells with cDNA expressing the neuronal genes of interest and then using a traditional cationic transfection reagent to identify siRNA sequences that would knockdown expression of the cDNA-expressed genes. The Neuro9 Kit nanoparticles allowed us to use the more relevant primary hippocampal neuron cells to identify potent DsiRNA sequences, designed by IDT, against the target genes.” Prof Craig’s group was able to use primary neurons to successfully screen a total of 15 DsiRNA sequences designed against the 3 target genes (5 DsiRNA sequences per gene). These results underscore the benefits of the Neuro9 delivery mechanism and IDT RNA selection algorithms, both instrumental in moving the work forward.
A second example of the benefits of PNI and IDT technologies is illustrated by the work of Dr Bamji, an associate professor in the department of cellular and physiological sciences at UBC. Dr Bamji has also used a similar experimental model in the study of changing synaptic connections. The Bamji lab was introduced to the SUB9KITS technology through use of the Neuro9 in vitro Demo Kit. The in vitro Demo Kit comprises a DsiRNA targeted against a housekeeping gene (HPRT, PTEN, or GAPDH) encapsulated in fluorescent Neuro9™ nanoparticles. The kit enables users to validate their experimental protocols and ensure the particles reach their targets, which they did with >90% transfection efficiency in Prof. Bamji’s studies. Further work progressed through IDT’s established gene knockdown workflow, whereby in vitro duplexes were screened, identified for potent knockdown and then modified and tested in vivo. From the beginning, the use of Demo Kits enabled PNI and IDT to introduce Prof Bamji to the value of each product.
The IDT-PNI partnership, with IDT offering their highly potent DsiRNAs and PNI providing a successful delivery mechanism into hard-to-transfect cells enables many new and exciting research projects that were previously limited by the low transfection efficiency and/or high toxicity of traditional reagents. For more information contact firstname.lastname@example.org, or visit the PNI website at www.precisionnanosystems.com..