The length of siRNA is 7-8 nm, the diameter is 2-3 nm, the molecular weight is 13-14 kDa, and it is hydrophilic. Therefore, unmodified and naked siRNA cannot passively cross the cell membrane and is easily cleared by the kidney. If an effective gene silencer cannot be delivered to its intended cell type, tissue, or organ, it remains ineffective. Therefore, how to deliver siRNA to target cells and translate it into the clinic has been a major bottleneck in the development of siRNA.
Challenges of siRNA Drug Development
- Route of administration limitation
Due to the instability of siRNA in the intestinal environment, the subcutaneous route is limited by lipophilicity and carrier size. In addition, siRNA is a negatively charged molecule, making it nearly impossible for siRNA to cross biomembranes. The half-life of most naked or unmodified siRNAs is between 5-10 minutes. Therefore, the intravenous injection is usually chosen for therapeutic siRNA therapy, and a suitable delivery system needs to be selected to protect naked siRNA from endogenous enzyme degradation of unmodified siRNA.
- Vascular limitation
Due to the discontinuous and rapid angiogenesis of endothelial cells, the blood vessel wall within the tumor has multiple vascular pores ranging in diameter from 100 nm to 780 nm. Blood vessels in healthy tissue have tight junctions between endothelial cells, which greatly reduces the leakage of the vessel wall. Extravasation of tumor blood vessels can lead to drug concentration gradient-dependent diffusion, convection and transcellularization, drug extravasation, etc. Thus, tumor leakage can enhance siRNA/vector extravasation, but may also lead to increased interstitial fluid pressure.
- Renal clearance
The length of the siRNA is 7-8 nm and the diameter is 2-3 nm. Molecules that are typically less than 8 nm in size are easily filtered by the renal system. Therefore, unmodified or naked siRNA is easily cleared by the kidney. Studies have shown that increasing the molecular weight of siRNA by linking ligands, incorporating larger particles, or binding to plasma proteins can effectively avoid renal clearance of siRNA.
- Destruction by the reticuloendothelial cells
The reticulo-endothelial system (RES) consists of monocyte progeny cells, including phagocytes and macrophages. Nanoparticles larger than 100 nm have been reported to be trapped by RES in liver, bone marrow, spleen, and lung, and thus degraded by activated monocytes and macrophages. It can be seen that nanoparticles loaded with siRNA are easily taken up and destroyed by the reticuloendothelial system. Therefore, for target organs rich in RES, surface modification and charge modification of nanoparticles loaded with siRNA can interfere with the uptake of RES.
- Off-target effects
The introduction of siRNA can lead to off-target effects, i.e. inhibition of genes other than the intended gene target, leading to dangerous mutations in gene expression and unintended consequences. Currently, chemically modified siRNAs, such as 2′-O-methylation of the lead siRNA strand, can reduce miRNA-like off-target effects and immunostimulatory activity without losing the silencing effect on target genes. Therefore, off-target effects should be minimized when designing therapeutic siRNAs.
- Induced immune response
Studies have found that 23nt long siRNA can activate the release of IFN-α, IFN-β and other cytokines, which means that siRNA drugs can trigger a powerful innate immune response in the body.
In view of the above problems and challenges, it can be solved and improved in three aspects: 1. Sequence optimization and selection 2. Chemical modification 3. Delivery system.
Sequence optimization and selection
The sequence selection of siRNA is mainly to achieve two effects: one is to reduce the possibility of its off-target. The second is that it has a good knockdown effect on the target protein, and it has a small EC50. There are three ways for siRNA to be off-target. The first is the miRNA-like off-target effect of the antisense strand of siRNA, that is, siRNA has a form of action similar to miRNA, and binds specifically to mRNA sequence through seed sequence, thus leading to mRNA degradation or translation obstruction. The second is the antisense strand of siRNA which causes off-target effect due to mismatching. When siRNA binds to mRNA, certain mismatch is allowed to occur, which leads to the degradation of mismatched mRNA sequence. The third, the sense strand that would otherwise be degraded, enters the RISC complex, causing knockdown of non-target genes.
Alnylam summarized several general characteristics of siRNAs that may have good gene knockout ability: 1. The first position at 5′ is usually a GC base. 2. The 13th and 14th positions in 5’ are usually not G. 3. There are 3 or more U and A bases in the seed sequence. These characteristics can be used to predict the effectiveness of the designed siRNA, and some siRNA sequences that do not meet the characteristics can be deleted.
Chemical modification of siRNA
Chemical modifications, such as GNA, LNA and 2′-MOE, can effectively improve the immunogenicity of siRNA, reduce the off-target toxicity of siRNA, and increase the effectiveness of siRNA. Chemical modifications can be divided into three different modification types according to their sites: 1. Phosphate modification 2. Ribose modification 3. Base modification. These three modification schemes are usually present in siRNA at the same time.
Delivery system
At present, the delivery systems used for siRNA drugs include liposomes, GalNAc, exosome, peptides, conjugated polymers, etc., among which LNP and GalNAc have been approved for clinical use.
- LNP
LNP is mainly composed of 4 substances: cationic lipids, cholesterol, auxiliary lipids, and PEG-lipids. Among them, the LNP used by various companies is mainly different in cationic lipids and PEG-lipids. Cationic polymer is the core component of LNP, and different companies have their own unique cationic polymer.
LNP can effectively protect siRNA from nuclease degradation and kidney clearance, and can effectively transfer siRNA into target organs and target cells. LNP packaged with siRNA is almost uncharged during the cycle, and the PEG-lipids (PEG2000-C-DMG) in them are gradually lost during the cycle and replaced by serum proteins, especially apolipoprotein E (ApoE), whose function is to transport lipids into the liver. Therefore, LNP is absorbed by hepatocytes and enters into endosomes after absorption. The endosome pH is acidic, and in an acidic environment, lipids reionize, resulting in the decomposition of LNP. Electrostatic and hydrophobic interactions occur between the disintegrated LNP and the endosome membrane, thereby helping the siRNA escape into the cytoplasm.
GalNAc is a ligand for the asialoglycoprotein receptor (ASGPR), an endocytic receptor that is specifically highly expressed in hepatocytes but barely expressed in other cells. ASGPR and clathrin-mediated endocytosis can efficiently transport galactose-derived ligands from the cell surface to the cytoplasm. During this process, ASGPR has a cycle time of approximately 15 minutes, making ASGPR an ideal liver-targeting receptor. Efficient liver delivery of siRNA can be achieved by covalently linking siRNA to trivalent or tetravalent GalNAc.
In recent years, exosomes have received increasing attention as an important tool for intercellular communication. Most cells are capable of releasing many small vesicles, which are loaded with many substances such as proteins, liposomes, and various types of nucleic acids. According to the formation process of these small vesicles, extracellular vesicles can be broadly divided into apoptotic bodies, cell microvesicles, and exosomes. The formation of exosome depends on the physiological and pathological environment of the cell itself, so the RNA (miRNA, mRNA, lncRNA) and protein in the exosome carried in the circulatory system by healthy individuals and different kinds of patients are different. This makes it a potential biomarker for diagnosing diseases. Tumor-derived exosome contain a large number of miRNAs, and the types of miRNAs secreted by different tumors are different, so the types and content of miRNAs in the circulatory system are an important tumor marker. Exosome is also a biomarker for many non-neoplastic diseases, such as cardiovascular disease, kidney disease, and neurodegenerative diseases, etc. In addition to being biomarkers of disease, exosomes act as endogenous extracellular vesicles with diameter and size similar to nanoscale carriers. And the exosome can carry different signaling molecules (RNA and protein), so it has a potential ability to act as a drug delivery. Compared with exogenous nanocarriers, exosome has the advantages of no immune response and no biological toxicity.
Antibody drug conjugates (ADCs) bind biologically active small-molecule drugs to monoclonal antibodies through a linker, and the monoclonal antibodies act as carriers to deliver the small molecules to the target cells. Currently, antibody-oligonucleotide conjugate (AOC) drugs that attach siRNA to antibodies have also been extended through ADC drugs. AOC drugs take advantage of the high specificity of antibody molecules, good druggability, and convenient production to realize the ability of siRNA extrahepatic targeting and diverse tissue delivery.
- PNP
PNP is an artificially designed and synthesized lys-histidine peptide copolymer. The advantage of the PNP delivery system is that each PNP nanoparticle can carry multiple siRNA molecules for multi-target combined drug delivery. At the same time, the second advantage of PNP is that its components are natural amino acids, so its toxic and side effects are small.