siRNA Drugs: Targeting Tissues for Better Treatment

Small interfering RNA (siRNA) drugs have emerged as a promising therapeutic approach for treating diseases at the genetic level. By silencing specific genes, these therapies target disease-causing proteins once considered undruggable. However, their success depends on efficient delivery to the right tissues while avoiding off-target effects and degradation. Researchers are refining strategies to enhance stability, improve cell entry, and direct siRNA molecules to affected organs.

Molecular Basis of RNA Interference

RNA interference (RNAi) regulates gene expression by degrading specific messenger RNA (mRNA) molecules before they can be translated into proteins. This process is mediated by siRNA and microRNA (miRNA), which guide the RNA-induced silencing complex (RISC) to complementary mRNA sequences, leading to cleavage or translational repression. The discovery of RNAi revolutionized molecular biology and therapeutic development, offering a precise method to silence disease-associated genes without altering the genome.

The RNAi pathway begins with the introduction of double-stranded siRNA into the cytoplasm, where it is processed by the endoribonuclease Dicer. Dicer cleaves the duplex into 21-23 nucleotide fragments with characteristic 2-nucleotide overhangs at the 3′ ends. These fragments are loaded onto the Argonaute (AGO) protein, a core component of RISC. One strand—the guide strand—is retained, while the passenger strand is discarded. The guide strand directs RISC to its target mRNA by complementary base pairing, leading to AGO-mediated cleavage and preventing protein synthesis.

RNAi efficiency depends on sequence specificity, guide strand stability within RISC, and target mRNA accessibility. Mismatches can reduce silencing efficiency or cause unintended gene suppression, known as off-target effects. Bioinformatics tools help design siRNA sequences that optimize target selection while minimizing homology with unintended transcripts. Chemical modifications, such as 2′-O-methylation and phosphorothioate linkages, enhance stability and reduce degradation by cellular nucleases.

Structural Properties of siRNA

The molecular architecture of siRNA is fundamental to its function. Each siRNA consists of a short, double-stranded RNA (dsRNA) sequence, typically 21-23 nucleotides long, with 2-nucleotide overhangs at the 3′ ends. These overhangs aid in recognition and processing by RISC. A 5′ phosphate group on the guide strand is essential for efficient loading onto AGO proteins. Without these structural features, siRNA may fail to engage the RNAi machinery effectively.

Unmodified siRNA is rapidly degraded by nucleases, limiting its therapeutic viability. To counteract this, researchers introduce chemical modifications such as 2′-O-methyl (2′-OMe) modifications, locked nucleic acids (LNAs), and phosphorothioate linkages. These alterations improve stability while preserving interaction with RISC. Excessive modifications, however, can compromise siRNA’s ability to guide RISC to its target mRNA.

Duplex thermodynamic stability is also crucial. Asymmetry between the two strands determines which is preferentially incorporated into RISC. A lower thermodynamic stability at the 5′ end of the guide strand favors its selection, ensuring proper strand bias. If the passenger strand is mistakenly retained, silencing efficiency diminishes. Computational algorithms and empirical studies help design siRNA sequences with optimal thermodynamic properties, balancing stability with efficient RISC loading.

Cell Entry Mechanisms

siRNA’s ability to enter cells is a major determinant of its therapeutic efficacy. Due to its negatively charged phosphate backbone and large molecular size (~13 kDa), siRNA cannot passively diffuse across cell membranes. Instead, it relies on endocytosis, including clathrin-mediated, caveolae-dependent, and macropinocytosis pathways, with the specific route influenced by the chemical properties of the delivery vector. The efficiency of these pathways varies between cell types, necessitating tailored approaches for different tissues.

Once inside the cell, siRNA must escape the endosome to reach the cytoplasm and engage RISC. Without effective endosomal escape, most internalized siRNA is degraded in lysosomes before reaching its target. Strategies to enhance cytoplasmic delivery include pH-responsive lipids that destabilize endosomal membranes, fusogenic peptides that mimic viral entry mechanisms, and nanoparticles engineered to induce osmotic swelling and rupture of endosomal vesicles. Some formulations achieve escape rates as high as 10–20%, while others remain below 1%, limiting therapeutic potential.

Tissue Targeting Principles

Achieving precise tissue targeting requires balancing stability, biodistribution, and cellular uptake. The physicochemical properties of siRNA, including charge, size, and hydrophilicity, influence how it navigates the body. Naked siRNA molecules are rapidly cleared due to renal filtration and nuclease degradation, making unmodified delivery inefficient. Chemical modifications and conjugation strategies improve circulation time and facilitate recognition by specific cell types.

Ligand-mediated targeting is a widely used approach. For example, siRNA conjugated to N-acetylgalactosamine (GalNAc) selectively binds to asialoglycoprotein receptors on hepatocytes, significantly improving liver delivery. Tissue vascular architecture also plays a key role in siRNA distribution. Highly fenestrated capillaries, such as those in the liver and spleen, allow easier siRNA passage, while tightly regulated endothelial barriers, such as the blood-brain barrier, require specialized transport mechanisms. Modulating nanoparticle size and surface charge can enhance penetration into dense tissues.

Organ Specific Dynamics

siRNA biodistribution is influenced by organ physiology, including vascular permeability, receptor expression, and enzymatic degradation. Some organs, like the liver, provide favorable conditions for siRNA uptake, while others, such as the brain, present significant delivery challenges. Understanding these dynamics is essential for designing siRNA therapies that maximize target engagement while minimizing off-target effects.

Liver

The liver is an ideal target for siRNA drugs due to its endothelial architecture and asialoglycoprotein receptors (ASGPR) on hepatocytes. These receptors exhibit high affinity for GalNAc-conjugated siRNA, enabling rapid uptake. This strategy has been successfully employed in FDA-approved therapies such as inclisiran, which lowers LDL cholesterol by silencing the PCSK9 gene. The liver’s fenestrated capillaries further facilitate siRNA accumulation. However, the high metabolic activity of hepatocytes requires siRNA molecules to evade degradation by hepatic nucleases. Chemical modifications like 2’-O-methylation and phosphorothioate linkages improve stability and prolong therapeutic effects.

Kidney

Renal targeting presents challenges due to the kidney’s filtration and excretion functions. Small, unmodified siRNA molecules are rapidly cleared by the glomerulus, limiting systemic availability. Enhancing kidney-specific delivery involves peptide and antibody conjugates that bind to proximal tubular epithelial cells, key players in kidney diseases. Cyclic peptide-conjugated siRNA has shown preferential uptake in these cells, offering potential treatments for conditions such as autosomal dominant polycystic kidney disease (ADPKD). Nanoparticles with optimized size and charge properties can prolong renal retention, increasing intracellular delivery. However, achieving sustained gene silencing remains a challenge due to endosomal escape and intracellular stability limitations.

Lung

Lung-targeted siRNA therapies must overcome mucosal barriers and rapid clearance. Inhalation-based delivery allows localized deposition while minimizing systemic exposure. Lipid nanoparticles (LNPs) and polymeric micelles enhance siRNA stability in the respiratory tract, protecting against enzymatic degradation. Surface modifications with polyethylene glycol (PEG) improve retention by reducing mucociliary clearance. Pulmonary diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD) are key targets, with preclinical studies showing promising results in reducing inflammation and mucus hypersecretion. However, achieving uniform lung distribution remains a challenge, requiring optimized aerosolized formulations for efficient uptake.

Brain

Delivering siRNA to the brain is difficult due to the blood-brain barrier (BBB), which restricts large and hydrophilic molecules. Receptor-mediated transcytosis, where siRNA is conjugated to ligands that bind to transport proteins like transferrin or insulin receptors, has shown promise in preclinical neurodegenerative disorder models. Exosome-based delivery, leveraging naturally occurring vesicles, is another emerging approach. Intrathecal and intracerebroventricular injections bypass systemic circulation to deliver siRNA directly to the central nervous system. While these methods enhance brain targeting, challenges related to dosing frequency and long-term safety remain.

Delivery System Approaches

Effective siRNA therapeutics rely on delivery systems that protect molecules from degradation, facilitate cellular uptake, and promote endosomal escape. Lipid nanoparticles (LNPs) are among the most effective carriers, encapsulating siRNA and shielding it from enzymatic degradation. Composed of ionizable lipids, LNPs undergo charge transitions at physiological pH, enhancing interaction with cell membranes and promoting endosomal release. This technology has been instrumental in the success of siRNA-based drugs such as patisiran, which targets transthyretin amyloidosis.

Polymer-based carriers, such as poly(lactic-co-glycolic acid) (PLGA), offer an alternative approach, enabling controlled siRNA release. These carriers can be engineered to respond to physiological stimuli, such as pH changes or enzymatic activity, allowing targeted release in specific tissues. Another promising strategy involves conjugating siRNA to aptamers—short, structured RNA or DNA sequences that bind to specific cell surface receptors—enhancing specificity while reducing off-target interactions, making it particularly valuable for precision medicine.