The advancement of more than 20 therapeutic siRNAs into the clinic illustrates that RNAi-based medicine holds a pivotal place in the future treatment of human diseases. However, there remain several obstacles in the clinical development of RNAi-based therapeutics. One of the greatest challenges in RNAi therapy continues to be the delivery method of the therapeutic siRNA to the target cells.
The advancements of synthetic carriers of siRNA have broadened the usage of siRNA therapeutics for a variety of tissue-specific diseases and tumors. By providing a delivery vehicle for the siRNA payload to the target cell, nanoparticles can prevent non-specific delivery of the siRNA drug and may also protect the siRNA during transport. Moreover, the efficacy and safety of siRNA drugs heavily depends on its delivery to the intended target [87
]. Lipid-based nanoparticles like SNALP from Tekmira can deliver siRNA to various tissues and tumors by IV injection. Other promising advancements in nanoparticle delivery include the cyclodextrin carrier from Calando and the LODER delivery system from Silence. Such biodegradable polymers can be engineered to release the siRNA over a localized area of tissue for an extended and controllable duration. Novel synthetic carriers, such as RNA-aptamers conjugated with siRNAs and next-generation lipid-based carriers will further drive siRNA drugs into clinical applications [1
Naked siRNAs have been extensively tested, particularly for therapies that allow for administration through topical or IVT methods. Due to the fragility of RNA molecules and their susceptibility to degradation, prolonged expression of RNAi-based therapeutics has been notoriously difficult. Chemical modifications to siRNA molecules, including modifications to the base, the ribose sugar, and phosphodiester backbone may alleviate undesired inflammatory responses while also extending the half-life of the therapeutic drug for days or weeks [87
In addition to the delivery methods based on synthetic materials, viruses, and bacteria offer an alternative for shRNA-based therapeutics encoded in DNA vectors. Bacterial or lentiviral shRNA-expressing vectors offer a long-term delivery method, in which the expression of the shRNA is designed to persist indefinitely. However, both delivery methods are currently limited to packaging of nucleic acids that encode shRNA precursors of siRNA, and in vivo delivery of RNAi viral vectors has not yet been clinically tested.
Other major challenges for RNAi-based therapeutics include controlling the specificity of the siRNA and minimizing potential off-target effects related to the sequence of both dsRNA strands. siRNAs are known to initiate off-target gene silencing by functioning like microRNAs (miRNAs) [89
]. This effect may occur when the antisense strand of the siRNA is perfectly matched at positions 2–7 or 2–8 to the 3’ UTR sequence of a nonspecific mRNA [90
]. Asymmetric 25-nt/27-nt dicer substrates can be designed to preferentially load the guide ssRNA strand of the dsRNA duplex, thus mitigating the potential off-target effects from the passenger ssRNA strand [91
]. Dual-targeting siRNAs are designed so that both strands target different sites within a single mRNA target or two separate target mRNAs [93
]. Thus, dual-targeting siRNAs may reduce the potential for off-target gene silencing, increase the opportunity to knockdown the desired target gene(s), and potentially provide additive or even synergistic effects by both strands.
As observed with the two VEGF siRNA drugs that were terminated during clinical testing, siRNAs may also have the tendency to activate TLRs to lead to inflammation and other off-target effects. Despite the promising advancement of the drugs to Phase II and III trials, these setbacks have drawn public concern and attention. However, it should be noted that despite the drawbacks, more than a thousand patients were treated with siRNAs and no major negative effects or adverse events were detected. Moreover, the pro-inflammatory effects of TLR activation may be beneficial for treatment of certain diseases, so these properties of siRNA drugs may eventually be therapeutically exploited.
Like most novel therapies, the progress of siRNA drugs in clinical trials has encountered challenges along the way. Encouraging signs have developed in a number of trials, most notably for the treatment of various cancers, acute kidney injury, RSV, and HIV. While the setbacks from the AMD and DME trials deserve thoughtful scrutiny, lessons have quickly been learned, including validating siRNA-directed mRNA cleavage using an advanced technology known as 5’-RACE PCR. Thus, despite mixed results for the 21 siRNA/shRNA drugs in clinical trials, the potential of RNAi therapeutics in the field remains strong. With the emergence of newer technologies, such as chemical modifications to siRNA and more advanced delivery systems, the discontinuation of a few drugs has not impeded the clinical progress or the pre-clinical developments of other RNAi-based therapeutics.