Small interfering RNA, or siRNA, represents a class of small, double-stranded RNA molecules. These molecules play a role in a natural cellular process known as RNA interference (RNAi). By leveraging this process, siRNA can specifically “turn off” or silence particular genes. This gene-silencing capability positions siRNA as a powerful tool in medicine, offering a targeted approach to treating diseases that involve faulty or overactive genes.
Understanding siRNA and Its Potential
RNA interference (RNAi) is a biological mechanism in cells that regulates gene expression, controlling which genes are active and to what extent. siRNA molecules harness this natural pathway, measuring between 20 to 24 base pairs in length. When introduced into a cell, siRNA is incorporated into a protein complex called the RNA-induced silencing complex (RISC). Within this complex, one strand of the siRNA, known as the guide strand, directs the RISC to a complementary messenger RNA (mRNA) molecule.
Once the RISC-siRNA complex binds to its target mRNA, an enzyme within the RISC, Argonaute-2, cleaves and degrades the mRNA. This degradation prevents the mRNA from being translated into a protein, effectively silencing the gene. This gene-silencing ability holds promise for treating a range of diseases. For example, siRNA could address genetic disorders, combat viral infections by targeting replication genes, or suppress uncontrolled cell growth in certain cancers.
The Hurdles of Delivering siRNA
Despite its therapeutic potential, delivering siRNA to target cells presents several biological challenges. One primary hurdle is the rapid degradation of siRNA by enzymes called nucleases, which are abundant in the bloodstream and inside cells. These enzymes can quickly break down the RNA molecules, limiting their stability and effectiveness before they reach their intended targets.
Beyond degradation, getting siRNA into target cells is difficult because siRNA molecules are relatively large and carry a negative electrical charge. This charge prevents them from easily crossing the cell membrane. Even if siRNA enters a cell, it often becomes trapped within cellular compartments called endosomes. If siRNA remains trapped in endosomes, it is degraded before it can escape into the cytoplasm, where the RNAi machinery resides.
Off-target effects are a concern, where siRNA might bind to unintended mRNA sequences that share partial similarity with the target, leading to side effects. This can disrupt the expression of other genes, potentially altering cellular function. The immune system can also recognize foreign siRNA molecules, potentially reducing therapeutic effect or causing adverse reactions.
Key Strategies for siRNA Delivery
To overcome the inherent challenges of siRNA delivery, various strategies have been developed to enhance its stability, facilitate cellular uptake, and ensure its precise delivery to target cells.
Lipid Nanoparticles (LNPs)
One approach involves Lipid Nanoparticles (LNPs), which are fat-based vesicles that encapsulate siRNA. LNPs protect siRNA from enzymatic degradation in the bloodstream and facilitate its entry into cells through endocytosis. Once inside the cell, ionizable lipids within the LNP help siRNA escape from endosomes into the cytoplasm, allowing it to engage with the gene-silencing machinery. LNPs have been successfully employed in approved siRNA therapies, demonstrating effectiveness in delivering these molecules, particularly to the liver due to their affinity for apolipoprotein E (ApoE) receptors on liver cells.
Polymeric Nanoparticles
Polymeric Nanoparticles utilize biodegradable polymers like poly(lactic-co-glycolic acid) (PLGA) to encapsulate and protect siRNA. These nanoparticles can be engineered to condense and stabilize siRNA, improving its cellular uptake and controlled release within the cell. Incorporating cationic polymers, such as polyethyleneimine (PEI), into PLGA nanoparticles can further enhance siRNA encapsulation and delivery to the cytoplasm. These systems offer versatility in design and can be modified with targeting ligands to improve specificity for certain cell types.
GalNAc Conjugation
siRNA can be directly linked, or conjugated, to molecules that facilitate targeted delivery. A notable example is N-acetylgalactosamine (GalNAc) conjugation, highly effective for delivering siRNA to liver cells. GalNAc is a sugar molecule that specifically binds to the asialoglycoprotein receptor (ASGPR), a protein abundantly expressed on the surface of hepatocytes (liver cells). This binding triggers the uptake of the GalNAc-siRNA conjugate into the cell via endocytosis. Trivalent GalNAc conjugates, which feature three GalNAc molecules clustered together, show high affinity for ASGPR, promoting efficient liver delivery and prolonged gene silencing.
Chemical Modifications
Chemical modifications to the siRNA molecule can improve its therapeutic properties. Modifying the nucleobases or the phosphate backbone of siRNA can increase its stability against nuclease degradation and reduce off-target effects. For instance, certain chemical alterations, particularly within the “seed region” of the siRNA guide strand, can suppress its binding to unintended mRNA sequences, minimizing side effects. These modifications also help reduce the likelihood of the immune system recognizing the siRNA as foreign, decreasing potential immune responses.
Exosomes
An emerging approach for siRNA delivery involves Exosomes, naturally occurring nano-sized vesicles released by various cell types. Exosomes possess a hollow, aqueous core enveloped by a phospholipid bilayer, providing stability in biological fluids and an inherent ability to transport genetic material between cells. Their natural origin contributes to their low immunogenicity and biocompatibility, making them promising carriers for siRNA. Researchers are exploring methods to load siRNA into exosomes, such as electroporation or incubation, and can modify exosome surfaces with targeting ligands to enhance specific delivery to desired cells.