siRNA, also known as small interfering RNA or silencing RNA, is non-coding double-stranded RNA that operates within the RNA interference (RNAi) pathway. This blog will provide a brief summary of siRNA from different aspects.
- Structure of siRNA
siRNA consists of double-stranded RNA with 20-25 base pairs. It comprises a guide strand and a passenger strand, as shown in Figure 1 below. Two nucleotides are overhung at the 3′ side of both strands.
- Molecular mechanism of siRNA
Figure 2 depicts the naturally occurring RNAi pathway. Double-stranded RNA or hairpin-shaped RNA existing in the body is cleaved by an endo-ribonuclease, Dicer, into small double-stranded siRNA that are 20-25 bps long. siRNA can also be chemically synthesized directly. The siRNA is then incorporated into the RNA-Induced Silencing Complex (RISC), where the two strands are separated, leaving only the guide strand within RISC. The RISC identifies target mRNA with complementary sequences, binds to it, and cleaves it with Argonaute, the enzyme within RISC. Once cleaved, the target mRNA is degraded and is no longer functional for further translation into protein, thus silencing the gene that encodes the protein.
- How to produce siRNA?
- Designing siRNA – Identify the target gene and its specific region crucial to its function. Create the siRNA guide strand sequence, 20-25 nucleotides long, complementary to the target gene sequence. The siRNA passenger strand should be complementary to the guide strand, with two nucleotides overhung on each strand. Design the siRNA carefully to avoid off-target effects.
- Chemical Synthesis – Chemical synthesis is used to produce the siRNA. This step involves using a solid support and adding one nucleotide each cycle until reaching the full length. The coupling efficiency of RNA is about 98%. For a 25-nucleotide-long siRNA strand, the yield would be approximately 60%. Two strands are synthesized separately in two reactions and then annealed together. Purification is necessary to remove product- and process-based impurities.
- Annealing siRNA – After synthesizing the two single-stranded RNA molecules, annealing is completed by mixing an annealing buffer, equimolar amounts of the two RNA strands, and incubating at high temperature for a certain amount of time to facilitate the formation of the double-stranded siRNA.
- Challenges with siRNA as a therapeutic tool
Although siRNA shows promise as a therapeutic tool for various applications, there are some challenges that need addressing. They can be categorized into four major challenges:
- Stability: Naked siRNA is subject to degradation by exo- and endonucleases and has a very short half-life within the bloodstream or cells. Therefore, one of the most significant challenges is stabilizing siRNA to target cells and tissues. Different delivery platforms are continuously being developed to protect siRNA from degradation and enhance siRNA half-life.
- Delivery and Tissue Specificity: Delivery efficiency and tissue specificity are crucial to ensure that siRNA molecules are delivered to target tissues and taken up by target cells. siRNA molecules are stabilized through different delivery platforms, such as LNPs and ligand conjugations, to ensure they can travel and function without being digested. For LNPs, there is generally no good tissue specificity. For various ligands, like GalNAc and fatty acids, multiple cells have receptors for the ligands, which may interfere with the specificity of siRNA.
- Off-target: Off-target effects of siRNA occur when siRNA interacts with genes other than the intended target, impacting the expression and function of off-target genes, causing unwanted side effects. Researchers have been exploring various ways to address this problem, such as chemical modifications and rational RNA design.
- Immune Response: Exogenous siRNA can trigger an immune response, causing inflammation and reducing the effectiveness of the therapeutic approach. It’s especially crucial to reduce the immune response when multiple dosing is necessary.
- Approved siRNA therapeutics
Alnylam is the first and only company to bring RNAi therapeutics to the market so far. It has already commercialized multiple products, including Onpattro (patisiran, 2018), Givlaari (givosiran, 2019), Oxlumo (lumasiran, 2020), Vutrisiran (vutrisiran, 2022), and Leqvio (inclisiran, 2022). The first four products target genetic medicines, and the last one targets cardio-metabolic diseases.
- siRNA Companies
Here is a non-exhaustive list of siRNA companies for comparison.
| Company | Therapeutic Areas | Delivery Platform | Pipeline |
| Alnylam | Genetic medicines, cardio-metabolic diseases, infectious diseases, and central nervous system (CNS) and ocular diseases | Lipid nanoparticles (LNPs); Conjugates with GalNAc ligand or short lipid chain | Five approved, multiple in early/late stages |
| DTx Pharma | Peripheral nervous system, muscular and CNS disorders | Conjugates with fatty acids | Four in R&D/preclinical |
| Arrowhead Pharmaceuticals | Intractable diseases, like liver disease, cardiovascular disease, CNS disease, etc. | Conjugates with ligand | Multiple in various stages from preclinical to phase 3 |
| Dicerna | Mainly liver diseases | Conjugates with GalXC ligand | Four in preclinical to phase 2 |
| Silence Therapeutics | Hematology, cardiovascular disease, and rare diseases | Conjugates with GalNAc ligand | Three in preclinical to phase 2 |
- Difference between siRNA and mRNA
| siRNA | mRNA | |
| Structure | Double-strand | Single-strand |
| Size | 20-25 bp | a few hundred to several thousand of nucleotides |
| Mechanism | Identify and bind onto mRNA that can translate to target protein through complementary sequence, and cleave the mRNA so protein couldn’t be produced, thus the gene expressing the protein is silenced. | 1) vaccine: trigger immune response 2) therapeutics: express a specific protein within body to replace problematic protein |
| Production Method | Chemical Synthesis | In Vitro Transcription (IVT) |
| Application Areas | Have potential therapeutic applications in neurological diseases, viral infections, liver diseases, ocular diseases, and cancer, etc. | Have potential vaccine and therapeutic applications in infectious diseases, cancer, rare diseases, cardiovascular disorders, metabolic diseases, and neurological diseases, etc. |
| Delivery Platform | Lipid nanoparticles (LNPs), conjugates with sugar, lipid chain, fatty acids, ligands, etc. | Mostly LNPs |
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