RNAi therapy "picture"

RNA interference (RNA interference, RNAi) is a natural defense mechanism against invasion of foreign genesRNAi forms, including small interferoRNA (siRNA) and microRNA (miRNA), are able to strike low-target gene expression in a sequence-specific manner, by leading target mRNA degradation or inhibiting mRNA translationBecause of the subtle differences between siRNA and miRNA (a miRNA can affect the expression of several different target genes at the same time), siRNA is often able to trigger more effective and specific gene silencing than miRNA triggering, so siRNA and miRNA have different effects in drug practice (Figure 1)Figure 1 miRNA (a) and siRNA (b) Working Mechanism Schematic (Source: Trans Signaling and Targeted Therapy) Since the establishment of the RNAi concept in 1998, siRNA therapy has experienced many ups and downsIn 2001, Sayda MElbashir et alsuccessfully silenced the expression of a particular gene by introducing chemically synthesized siRNAs into mammalian cellsThis breakthrough led to a wave of research and development, and by 2003 there were a number of companies that had developed RNAi therapyUnfortunately, the first clinical trial siRNA using unmodified siRNA produced immuno-related toxicity and the suspect (uncertain) RNAi effectThe second wave of clinical trials, which used systematically administered siRNA nanoparticle formulations, showed significant dose-limiting toxicity and ineffectiveness despite significant advances (e.g., the first evidence that siRNA nanoparticle system administration can produce RNAi effects in humans)The problems exposed in these developments led most pharmaceutical companies to pull out of the RNAi field in the early 2010sHowever, RNAi therapy is gradually returning to the "center" of drug development as scientists make new breakthroughs in chemical modification and deliveryIn 2018, the field reached a milestone: the U.SFDA approved the first RNAi therapy (ONPATTRO ®) for the treatment of nerve damage caused by hereditary transthyroxine protein amyloid degeneration (hATTR)In November 2019, the FDA approved a second RNAi therapy GIVLAARI ™ for the treatment of acute hepatic dystrophy (hepatic porphyria, AHP)SiRNA has an innate advantage over small molecules and monoclonal antibody drugs because siRNA achieves its function by pairing with mRNA with AWatson-Crick base, while small molecules and monoclonal antibody drugs require the identification of complex spatial structures of certain proteins There are many diseases that cannot be treated with small molecules and monoclonal antibodies because highly active, affinity- and specific molecules for disease-causing proteins cannot be identified Instead, in theory, siRNA can target any gene of interest, simply selecting the correct nucleotide sequence on the target mRNA This advantage makes siRNA shorter in development cycles and a wider range of treatments than small molecules or antibody drugs However, while siRNA has broad prospects for drug development, multiple barriers in and outside cells limit its wide range of clinical applications For example, unmodified siRNAs have disadvantages such as poor stability, poor pharmacokinetic characteristics, and possible induced off-target effects In addition, siRNA's phosphate dilated bonds are susceptible to damage to RNases and phosphatase Once the drug is administered through the system into the cycle, endoenzymes or exsotoses throughout the body will quickly degrade siRNA into fragments, preventing the accumulation of complete therapeutic siRNAs in the desired tissue On the other hand, in theory, siRNA can only function if its antonyms are paired with the target mRNA's complete base However, the RNA-induced silent complex (RISC) can tolerate a small number of mismatched genes, which can lead to unexpected silence in a small number of nucleotide mismatched genes In addition, the righteous chain of siRNA may also reduce the expression of other unrelated genes Finally, unmodified siRNA may also cause the activation of Toll-like receptor 3 (TLR3) and adversely affect the blood and lymphatic system The findings raise a number of concerns about the safety and medicinal nature of siRNA To maximize therapeutic effectiveness, reduce or avoid side effects of siRNA, researchers have done a lot to investigate various chemical modification patterns and develop different dosing systems The effects of a range of modification patterns on the activity, stability, specificity and biosafety of siRNA therapy have been evaluated both preclinical lying and clinically Delivery materials based on lipids, lipid-like materials (lipidoids), polymers, peptides, exosomes, inorganic nanoparticles, etc have also been investigated Source: Signal Transduction and Targeted Therapy June 19, several researchers from the National Nanoscience Center, Guangxi Medical University, and Beijing Polytechnic University published a new review entitled "Therapeutic siRNA: State of the Art" in the journal TransSignalandand And Targeted Therapy, which discusses the detailed development of siRNA modification and delivery technology At the same time, this article also provides a historical review of the development of siRNA therapy The following reference seduing charts, excerpted from some of the main points: First, siRNA modification As mentioned earlier, in the early stages of the development of siRNA therapy, many drugs are designed to be based on completely unmodified or slightly modified siRNA to reach the appropriate tissue and then silencing the target gene These molecules can mediate gene silencing in the body, especially in tissues receiving local drug therapy, such as the eyes However, using these completely unmodified or slightly modified siRNAs may not only have limited efficacy, but also potential off-target effects For example, Kleinman and colleagues observed that siRNA therapy bevasiranib (targeted veGFA) and AGN211745 (targetve VEGFR1) developed to treat age-dependent macular degeneration triggered significant activation of TLR3 and its adapter molecule TRIF, inducing the secretion of interleukin-12 and interferon-gamma Chemical modification siRNAs, such as replacing 2'-OH's siRNA with 2'-O-methyl (2'-OMe) or 2'-methoxyethyl (2'-MOE) groups, and locknucleic nucle acid, LNA) and non-locking nucleic acid (unlocked nucleic acid, UNA) or glycol nucleic acid (glycol nucleic acid, GNA) replace certain nucleotides siRNA, which effectively inhibits immune stimulation of immune stimulus siRNA-driven innate immune activation, improves activity and specificity, and reduces off-target-induced toxicity (Figure 2) Figure 2 For chemical modifications and similarstructures of siRNA and antisense oligonucleotide (ASO) modified (Source: Signal Transduction and Targeted Therapy) Scientists have developed and tested a number of chemical modification patterns to improve the effectiveness and reduce their potential toxicity According to the modification site in nucleotides, the structure of chemical modification and analogues used for siRNA can be divided into three main categories: 1) glycemic modification, 2) ribose modification, and 3) base modification, represented in red, purple and blue, respectively Typically, these modifications are introduced into siRNA at the same time For example, ONPATTRO ® used both 2'OMe and 2'-deoxy-2'fluoro (2'-F) retouching; Moiety" and an L-DNA cytonucleotide were modified, and inclisiran (ALN-PCssc) was retouched simultaneously with phosphorothioate (PS), 2'-OMe, 2'-F, and 2'-deoxy( Figure 3) a representative design of the chemical modification of Figure 3 siRNA, including the sequence and modification details of ONPATTRO ®, QPI-1007, GIVLAARI ™, and inclisiran (Source: Signal Transduction and Targeted Therapy) II, siRNA delivery system siRNA needs to bypass a number of barriers to gene silophin, such as nuclease degradation, transient circulation, immune identification in blood circulation, accumulation in target tissue, effective transmembrane transport, and escape from endosomes and lysozyes to cytoplasm The challenges of nuclease degradation and immunoidentification have been well addressed by adding chemical modifications, but some other obstacles still need to be overcome In blood circulation, nonspecific binding and glomerular filtration block the accumulation of siRNA in the target tissue The neutral surface charge of the nano-formulation loaded with siRNA helps to avoid adverse binding in the cycle The ligand-siRNA coupling siRNA couple siRNA transports siRNA to the desired tissues and cells through specific identification and interaction between the ligand (e.g carbohydrate, peptides, antibodies, ligands, small molecules, etc.) and the surface receptor This proactive targeting strategy not only reduces the accumulation of siRNA in unexpected tissues, thereby reducing or avoiding adverse side effects and toxicity, but also enables effective gene silencing at low doses Figure 4 Representative siRNA preparations and their pharmacoeutic properties in the clinical development phase , in which a-g is 7 delivery systems, followed by the lipid nanoparticles (LNPs), DPC ™/EX-1 ™, TRiM ™, GalNAc-siRNA Conjugates, LODER™, iExososme Gal, iExOsosme Gal, ™ (Source: Signal The molecule is too large to pass through the cell membrane, but small enough to be freely removed by a glomerular As a result, once siRNAs leave the blood, they accumulate in the bladder and quickly drain from the body within a few minutes to half an hour, preventing them from accumulating in the target tissue or cells Sealing siRNA in a vesicle or associating it with a specific ligand can effectively prevent kidney removal and, more importantly, deliver siRNA to the desired tissue or cells Lipid nanoparticles (LNPs), dynamic polyconjugates (DPC ™) and GalNAc-siRNA conjugates are all able to effectively deliver siRNAs to liver cells (Figure 4, Figure 5) And SiRNA delivery platforms that have been evaluated both preclinically and clinically A wide range of lipids, lipids, siRNA conjugates, peptides, polymers, exosomes, tree-like macromolecules, etc have been explored and applied to the development of siRNA therapy by biotechnology companies or research institutes (Source: Signal TransandIng And Targeted The) due to relatively high molecular weight (-13-16 kD) and net negative charges prevent artificial siRNAs from passing through cell membranes Therefore, the researchers tried to determine whether cells could internalize siRNA without a vector The study concluded that naked siRNA can only be absorbed by a small number of cells, such as retinal nerve cells (RGCs) and neurons In addition, researchers are working to identify a variety of vectors for efficient cross-membrane transmission Cation cell penetration peptides (CPPs) became the first choice for early research The tail of CPPs usually has a fine-rich sequence that can form a double-tooth bond by interacting with the negative phosphate, sulfate, and pholates on the cell surface through the argon salt group This interaction causes membrane holes to form, causing cells to absorb siRNA Another strategy for cross-membrane transport is to neutralize the negative charge of siRNA with a positively charged lipid or polymer, making it easier for siRNA to bind to the membrane and easier to internalize by adsorption of cell drinking In the final step, siRNAs must effectively escape from the endothelial and lysozyme to the cytoplasm Most non-viral vectors mainly enter the cells through the internal swallowing process, the carrier after internal swallowing will be assembled with the biofilm components into the endosome, and then develop into lysosomes through a variety of mechanisms, and the lysosome has a large number of degradation enzymes, will cause the gene drugs contained in it to degrade and fail, therefore, an effective non-viral vector should be able to promote the rapid flight of gene drugs from the endosome Many delivery systems use a pH sensitive unit to respond to pH changes in the endothelial and lysozymes, where they absorb H-plus and present a positive charge on the surface Then, the osmotic pressure of the endothelial or lysozyme increases, causing Cl-and-H2O to flow internally Finally, these changes can cause membrane rupture and siRNA to be released into the cytoplasm However, the potential mechanism for internal release has yet to be elaborated Because only 1% to 2% of internalized LNP-loaded siRNAs can be released into the cytoplasm, and this only occurs for a limited time after internalization Therefore, it is important for the development of siRNA drugs to further understand the escape mechanism of siRNA and how to improve the escape efficiency Recent studies have reported on the new endotheline mesh modification of hybrid nanoplexes (EhCv/siRNA NPs) The endoscoconinated hybrid nanoplexes showed higher RNAi activity both in the body and in vitro than the unmodified nanoplexes Functional proteins on the endothelial mesh play an important role in the intracellular transport of siRNA, helping siRNA reach the cytoplasm through the endosome-Gorky-ER pathway (rather than the endosomean-lysozyse pathway), thus avoiding the degradation of the siRNA lysosomes In addition, the electroperation can make s.