Small interfering RNA (siRNA) represents an advancement in medicine, offering the ability to precisely control gene expression. This capability holds potential for treating a wide array of diseases by selectively turning off genes that contribute to illness. However, realizing this potential in living organisms faces a hurdle: effectively and safely delivering siRNA to its intended cellular targets. The body’s natural defenses and cellular barriers make this a complex undertaking, requiring sophisticated delivery methods.
What is siRNA and How Does it Work?
Small interfering RNA, or siRNA, consists of short, double-stranded RNA molecules, typically 20-25 nucleotides in length. These molecules are involved in RNA interference (RNAi), a process that regulates gene expression within cells. SiRNA silences specific genes by targeting their messenger RNA (mRNA) for degradation.
When siRNA enters a cell, it is incorporated into a complex called the RNA-induced silencing complex (RISC). The RISC unwinds the double-stranded siRNA, retaining one strand (the guide strand). The guide strand directs the RISC to a complementary mRNA sequence. Upon binding, the RISC cleaves the mRNA, which is then degraded by the cell. This prevents protein translation and effectively turns off the gene.
The Hurdles of Delivering siRNA in the Body
Delivering siRNA effectively within a living organism presents challenges due to the body’s protective mechanisms. One obstacle is the rapid degradation of naked siRNA by enzymes (nucleases) in the bloodstream. Unmodified siRNA has a short half-life, often less than ten minutes, in human serum. This rapid breakdown means little siRNA reaches target cells without protection.
The kidneys also swiftly clear siRNA, reducing its availability to target tissues. The immune system can recognize exogenous nucleic acids like siRNA, triggering inflammatory responses. Beyond these systemic issues, siRNA struggles to cross the cell membrane to reach the cytoplasm, where the RNA interference machinery resides. SiRNA is large, negatively charged, and hydrophilic, making passive diffusion through the cell’s lipid bilayer challenging. Overcoming these barriers requires specialized delivery systems.
Key Strategies for siRNA Delivery
To overcome the challenges of delivering siRNA in vivo, researchers have developed various delivery vehicles. These strategies broadly fall into two categories: viral and non-viral methods, each with advantages and considerations. Viral vectors use modified viruses to introduce siRNA into cells, leveraging their natural efficiency in gene delivery. However, concerns like immune responses and production scale limitations can arise.
Non-viral methods are widely explored for siRNA delivery, offering advantages like easier synthesis and scalability. Lipid-based nanoparticles (LNPs) are a prominent non-viral delivery technology. These fatty particles encapsulate siRNA through electrostatic interactions, shielding it from degradation and facilitating cellular entry. LNPs typically range from 10 to 150 nanometers for systemic intravenous delivery. Their internalization into cells often occurs through endocytosis, requiring endosomal escape mechanisms to release siRNA into the cytoplasm.
Polymer-based nanoparticles are another non-viral strategy. Synthetic polymers, often positively charged, complex with negatively charged siRNA to form stable nanoparticles. For example, poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles, often combined with cationic polymers like polyethylenimine (PEI), improve siRNA encapsulation and release. These polymeric systems protect siRNA and enhance cellular uptake, leading to effective gene silencing.
Conjugates involve directly attaching siRNA to molecules like antibodies, peptides, or small chemical ligands. For instance, N-acetylgalactosamine (GalNAc) is conjugated to siRNA to specifically target liver cells, leveraging GalNAc’s high affinity for the asialoglycoprotein receptor on liver cell surfaces. This direct attachment enhances siRNA stability, improves pharmacokinetic behavior, and guides it to specific cell types, increasing cellular uptake and target specificity.
Targeting and Safety in siRNA Delivery
Beyond simply delivering siRNA into the body, a focus is on ensuring it reaches the correct cells or tissues and minimizes unintended effects. This involves targeting and safety strategies. Active targeting functionalizes delivery vehicles with specific molecules (e.g., antibodies, peptides, aptamers) that bind to receptors overexpressed on diseased cells, such as cancer cells. This binding enhances the specificity and selectivity of siRNA delivery to intended cellular targets.
Passive targeting relies on the physiological characteristics of diseased tissues, such as tumors. Tumors often have leaky blood vessels with larger pore sizes (typically 100-800 nm), allowing nanoparticles to accumulate in the tumor microenvironment through the enhanced permeability and retention (EPR) effect. This mechanism allows nanoparticles, especially those smaller than 100 nm, to accumulate in tumors, hepatocytes, and inflamed tissues. Strategies to reduce off-target gene silencing and immune responses involve careful siRNA design, chemical modifications, and specialized delivery systems that prevent non-specific interactions.
Therapeutic Applications of siRNA
SiRNA technology holds potential for treating a variety of diseases by turning off specific disease-causing genes. Several siRNA-based therapies have already received approval, marking milestones in gene therapy. These approved agents address rare metabolic disorders such as hereditary transthyretin amyloidosis (hATTR), acute hepatic porphyria (AHP), and primary hyperoxaluria type 1 (PH1).
Beyond rare genetic conditions, siRNA therapies are also being explored for common ailments. For example, some siRNA drugs are used to lower low-density lipoprotein cholesterol (LDL-C), a factor in cardiovascular disease. Research is ongoing into their application in cancers, viral infections like hepatitis B, and ocular diseases. The ability of siRNA to specifically target and silence genes offers a versatile approach for developing new treatments across many medical conditions.