siRNA, composed of sense and antisense strands, is a synthetic double-stranded RNA with a typical length of 21 nucleotides. The antisense strand complements and fully pairs with mRNA, leading to mRNA degradation and exerting post-transcriptional regulatory functions. Synthesized siRNA enters cells through endocytosis, with a small fraction escaping endosomes to enter the cytoplasm. In the cytoplasm, siRNA forms an RISC-loading complex (RLC) with Dicer and transactivating response RNA-binding protein (TRBP). The RLC recruits AGO2, initiating the degradation of the sense strand. The antisense strand then binds to the complementary mRNA sequence, and AGO2 cleaves the mRNA, ultimately causing mRNA degradation.
Reviewing the developmental history of RNAi therapy—from the first observation of RNAi in 1998, the Nobel Prize in 2006, to the approval of the first siRNA drug in 2017—siRNA has undergone significant development, overcoming several main challenges:
Addressing these challenges involves optimizing siRNA sequences and utilizing chemical modifications and delivery systems for improvement.
Chemical modifications, such as glycol nucleic acid (S-GNA), locked nucleic acid (LNA), and 2′-methoxyethyl (2'-OMe), effectively enhance siRNA's immunogenicity, reduce off-target toxicity, and increase efficacy. Chemical modifications, categorized into phosphate, ribose, and base modifications, are typically present in siRNA simultaneously.
The standard template chemistry (STC) uses ribose and phosphate modifications, including 2'-deoxy-2'-fluoro (2'-F) and 2'-OMe. While STC modification is a universal approach enhancing siRNA stability and mRNA affinity, it raised safety concerns in subsequent clinical trials. The next-generation modification, enhanced stablilization chemistry (ESC), emerged with a reduction in the number of 2'-F modifications to enhance siRNA efficacy and stability. ESC significantly improved siRNA's effectiveness and half-life, leading to better pharmacokinetics (PK) and pharmacodynamics (PD). Alnylam's GIVLAARI, used to treat acute hepatic porphyria, employs the ESC modification. Advanced ESC further reduced 2'-F modifications and optimized the ratio and modification sites of 2'-F and 2'-OMe. Advanced ESC enhances siRNA stability without compromising efficacy. Alnylam later developed the ESC+, adding GNA to the antisense strand's seed sequence, addressing miRNA-like off-target effects and liver toxicity. Several siRNAs, including ALN-HBV02 and ALN-AGT, use the ESC+ modification.
Currently, various delivery systems are employed for siRNA drugs, including antibodies, GalNAc, liposomes, exosomes, polymers, etc., with LNP and GalNAc being clinically approved.
Antibody-oligonucleotide conjugates (AOC), linking siRNA to antibodies, leverage the high specificity, good drugability, and convenient production characteristics of antibody molecules. This facilitates liver-specific targeting and diversified tissue delivery of siRNA. A notable AOC product is Avidity Biosciences' AOC 1001, currently in phase 1/2 clinical studies for myotonic dystrophy type 1 (DM1).
GalNAc serves as the ligand for the asialoglycoprotein receptor (ASGPR), highly expressed in liver cells but nearly absent in other cells. ASGPR, mediating endocytosis, efficiently transports GalNAc-conjugated siRNA from the cell surface to the cytoplasm. Hence, ASGPR is an ideal liver-targeting receptor. Covalently connecting GalNAc to siRNA enables efficient liver delivery. Various connection modes exist, such as GalNAc linked to the 3' or 5' end of the sense strand. The entry of GalNAc-siRNA into cells is regulated by ASGPR through a natural mechanism, providing higher safety compared to lipid-based delivery. Many GalNAc-siRNA conjugates are currently in clinical research.
LNP primarily consists of cationic lipids, cholesterol, helper lipids, and PEG-lipids. Ionic polymers form the core component of LNP. LNP effectively protects siRNA from degradation by nucleases and renal clearance, facilitating efficient transfer into target organs and cells. Packaged LNP in circulation remains nearly neutral, with PEG-lipids gradually losing during circulation, replaced by serum proteins, especially apolipoprotein E (ApoE). ApoE's function is to transport lipids into the liver, leading to LNP absorption by liver cells. After absorption, LNP enters endosomes. The endosomal pH is acidic, causing lipid deionization and LNP breakdown. The disintegrated LNP interacts electrostatically and hydrophobically with the endosomal membrane, aiding siRNA escape into the cytoplasm.
Most cells release extracellular vesicles (EV), including apoptotic bodies, microvesicles (MV), and exosomes. Exosome formation depends on the cell's physiological and pathological environment. Exosomes, with a diameter similar to nanocarriers, carry various substances such as proteins, lipids, and different types of nucleic acids. The content of exosomes entering the circulatory system varies between healthy individuals and different patient types, making them potential diagnostic biomarkers for diseases. In addition to their role as disease biomarkers, exosomes, as endogenous extracellular vesicles, have the potential to serve as drug delivery carriers. Compared to external nanocarriers, exosomes have advantages such as low immunogenicity and no biological toxicity.