Therapeutic siRNA: Recent Advances and Prospects

The concept of RNA interference (RNAi) was discovered in 1998 and is a naturally occurring defense mechanism against exogenous nucleic acid invasion and control of gene expression. Soon thereafter, small interfering RNAs (siRNAs) were identified as mediators of RNAi in mammalian cells. Then, siRNA delivery was found to be necessary and sufficient to induce RNAi-mediated knockdown because the cell can provide the other components of RNAi. Therefore, RNAi based on the direct application of siRNA opens up new avenues for innovative therapies and attempts to explore the use of siRNA as a therapeutic agent.

RNAi mechanism

The mechanism of RNAi has been extensively studied. The endoribonuclease Dicer processes longer double-stranded RNA or short hairpin RNAs into mature siRNA. This siRNA can also be delivered directly to cells as a chemically synthesized molecule and introduced in the RNA-induced silencing complex (RISC), which consists of several different proteins including Argonaute-2(Ago-2) and Dicer. After activating the siRNA by removing its sense or passenger strand, the remaining antisense or guide strand directs RISC to bind to target messenger RNA (mRNA), and Ago-2 in RISC mediates cleavage. In contrast to antisense techniques, RNAi relies on a catalytic mechanism since, upon cleavage of the target mRNA, the siRNA-loaded RISC can dissociate and bind to another mRNA molecule.

The mechanism should also be distinguished from microRNA (miRNA), which only bind to partial complementarity whereas siRNAs rely on 100% complementarity for action. Thus, siRNA acts on proteins post-transcriptionally rather than post-translationally at the mRNA level.

Synthesis of small interfering RNA (siRNA) -mediated knockdown mechanism Fig. 1 Synthesis of small interfering RNA (siRNA) -mediated knockdown mechanism (Friedrich M, 2022)

The challenges of siRNA

Optimal siRNA should have the following characteristics:

  • Does not activate the innate immune system
  • Cleaves its target site efficiently and specifically
  • No off-target (i.e. effects on non-target genes) or other toxic effects
  • Long half-life/slow degradation in systemic circulation and inside target cells

Selecting the right target for silencing to treat a particular disease is of vital importance.

Currently used siRNAs are 19 to 29 nucleotides in length. Short siRNAs may tend to bind more non-specifically, but siRNAs with 19 to 25 nucleotides show similar efficiency in gene silencing. Short siRNAs are preferred because longer siRNAs can provoke an inflammatory antiviral immune response. However, the introduction of chemical nucleotide modifications is now effective in preventing this unwanted side effect.

Many studies have disclosed the direct correlation between local target secondary structure and target site accessibility on RNAi efficiency, although long-standing controversy regarding the effect of target mRNA structure on siRNA efficiency. In this case, the 5' UTR and 3' UTR of mRNA and sequences close to the start codon are not recommended as siRNA targets because binding of regulatory proteins in this region may impede RISC binding and thus silencing effects. Instead, it is recommended to select regions in the open reading frame approximately 50 to 100 nucleotides downstream of the start codon. In addition, siRNAs closer to the start codon appear to be more efficient than siRNAs further downstream.

siRNA delivery system

RNA molecules must be considered to be unstable and prone to rapid enzymatic and nonenzymatic degradation. The half-life of unmodified bare siRNAs in blood flow is only about 5 min, and only a few cell types such as neurons or retinal ganglion cells can take up naked siRNAs. Therefore, the development of delivery strategies has been a major bottleneck for the use of siRNAs in vivo.

Commonly used siRNA drug delivery systems Fig. 2 Commonly used siRNA drug delivery systems

The future of siRNA

  • Metabolic disease

Nedosiran (DCR-PHXC; Dicerna Therapeutics) is another drug developed for the treatment of PH. It specifically inhibits the expression of major LDH isoforms in the liver. Cemdisiran is another liver-targeting GalNAc-siRNA drug that results in the knockdown of complement 5 (C5) protein.

  • Hematology

Hemophilia A and B are indications for fitusiran (ALN-AT3), a GalNAc-siRNA conjugate targeting SERPINC1 mRNA.

  • Infectious diseases

The siRNA drug RG6346 mediates the selective knockdown of the hepatitis B surface antigen required for the hepatitis B virus life cycle in hepatocytes.

  • Oncology

Mutant KRAS is the most prominent oncogenic driver in many cancers. siRNA targeting the KRAS G12D mutant has been formulated in a biodegradable polymer matrix for sustained local release (LODER; The topical drug EluteR).

  • Eye disease

Several siRNA drug developments have been investigated for eye diseases. These include glaucoma, age-related macular degeneration (AMD), dry eye (dry eye syndrome), diabetic macular edema (DME), and various inherited retinal diseases.


More than two decades after the discovery of RNAi, siRNA drugs have opened new avenues for innovative therapies for many diseases. Although there are several significant challenges and limitations, including siRNA delivery and side effects, that have slowed the clinical translation of this novel class of pharmacological compounds, several siRNA drugs have been approved for clinical use and several more are in late-stage clinical studies. siRNA targeting any target gene also highlights the need to identify the best target.


  1. Friedrich M; et al. Therapeutic siRNA: State-of-the-Art and Future Perspectives. BioDrugs. 2022 Sep; 36(5): 549-571.
* Only for research. Not suitable for any diagnostic or therapeutic use.
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