Overcoming Barriers for siRNA Therapeutics: From Bench to Bedside

Abstract

The RNA interference (RNAi) pathway possesses immense potential in silencing any gene in human cells. Small interfering RNA (siRNA) can efficiently trigger RNAi silencing of specific genes. FDA Approval of siRNA therapeutics in recent years garnered a new hope in siRNA therapeutics. However, their therapeutic use is limited by several challenges. siRNAs, being negatively charged, are membrane-impermeable and highly unstable in the systemic circulation. In this review, we have comprehensively discussed the extracellular barriers, including enzymatic degradation of siRNAs by serum endonucleases and RNAases, rapid renal clearance, membrane impermeability, and activation of the immune system. Besides, we have thoroughly described the intracellular barriers such as endosomal trap and off-target effects of siRNAs. Moreover, we have reported most of the strategies and techniques in overcoming these barriers, followed by critical comments in translating these molecules from bench to bedside.

Keywords: barriers to siRNA delivery; chemical modifications; endosomal escape; immune system activation; membrane impermeability; off-target effects; renal clearance; reticuloendothelial system entrapment; siRNA delivery systems.

Figure 1

Timeline highlighting the major historical events leading up to siRNA therapeutics.

Figure 2

General mechanism of action for siRNA induced gene silencing. (1) Synthetic siRNA successfully enters the cell via endocytosis. (2) siRNAs will then travel in the cytosol until it comes into contact with cytosolic RNAi enzymes–dicer and Tar RNA Binding Protein (TRBP) to form RISC Loading Complex (RLC). (3) Strand selection is done. (4) Production of mature RISC following successful guiding strand selection. (5) siRNA guiding strand with full complementarity to a single target mRNA induces potent and targeted gene silencing. (6) Mature RISC can regulate gene expression via inhibition of mRNA translation, inducing mRNA sequestration in cytoplasmic P bodies and or GW bodies to promote mRNA degradation. (7) Degraded mRNA is no longer useful and cannot go through translation to produce protein.

Figure 3

Chemical modifications of siRNA.

Figure 4

Surface modification of siRNA carriers to decrease opsonization, renal clearance, and degradation.

Figure 5

Carriers to overcome the impermeability of the siRNA.

Figure 6

Mechanism of action of PEG-based cleavable polymeric system that responds to decreased pH in the endosomal environment.

Figure 7

Ways to overcome endosomal entrapment.

Figure 8

General mechanism of ligand-receptor mediated targeting and siRNA delivery. Nanoparticle carriers holding siRNA will be surface modified with complementary ligands of receptors overexpressed on the damaged or diseased cell. Once internalized, it will incorporate with the RNAi pathway and efficiently silence the gene of interest.