In 1998, American scientists Andrew Fire and Craig Mello published a groundbreaking paper in Nature  that determined that double-stranded RNA (dsRNA) is the cause of post-transcriptional gene silencing (PTGS) in nematodes. They call this phenomenon RNA interference (RNAi).
The discovery of RNAi (Figure 1) explains the confusing gene silencing in plants and fungi and triggers a biological Revolution, and finally, studies have shown that non-coding RNAs are the major regulators of gene expression in multicellular organisms. The RNAi pathway regulates mRNA stability and translation in almost all human cells.
Figure 1 RNAi pathway Early events discovered and clarified (Source: Nature Reviews Drug Discovery)
In 2001, a Nature published by Sayda M. Elbashir et al., and a PNAS published by Natasha J. Caplen et al.  confirmed that dsRNAs consisting of 21 and 22 nucleotides are capable of inducing RNAi silencing in mammalian cells without causing non-specific interferon responses. These small interfering RNAs (siRNAs) quickly became biological studies. Tools that are ubiquitous because they can pass A base sequence easily inhibits any gene.
The efficacy and versatility of siRNAs for drug developers. The prospect of inhibiting genes encoding proteins and the potential to become "programmable" drugs Very "tempting." By 2003, many companies had deployed RNAi therapy.
Unfortunately, the first clinical trials using unmodified siRNAs produced immune-related toxicity. And suspicious (uncertained) RNAi effects. The second wave of clinical trials used systemically administered siRNA nanoparticle formulations, although significant advances have been made (eg, for the first time, siRNA nanoparticle system administration produces RNAi in humans). Effects), but still exhibit significant dose-limiting toxicity and insufficient efficacy. These developmental exposures have caused most pharmaceutical companies to withdraw from the RNAi field in the early 2010s, which has brought a financial crisis to the development of RNAi drugs. /p>
However, despite these challenges, some small RNAi companies and academic researchers have not given up on this. They have learned the painful teachings of previous clinical trial failures. Adhere to trigger design. Sequence selection. Improvements in chemical modification and delivery mechanisms. Substantial advances in these areas, combined with more sensible disease indication options. Better validation interventions. More mature clinical development processes and Improved manufacturing capabilities have created a safer and more effective candidate RNAi drug pipeline.
Figure 2 patisiran treatment mechanism (Source: Nature Reviews Drug Discovery)
2018 On August 10, the US FDA approved Alnylam's siRNA drug, ONPATTRO (patisiran), for the treatment of hereditary transthyretin amyloidosis (hATTR)-induced neurological damage.
hATTR is a rare. Hereditary. Life-threatening neurodegenerative disease caused by deposition of transthyretin (TTR) amyloid in the peripheral gastrointestinal tract and other organs. Progressive neuropathy. Cardiomyopathy. Walking disorders and various other debilitating symptoms, the median survival after diagnosis is 5-15 years.
Most TTR is produced in the liver. >120 mutations can cause hATTR.Patisiran to reduce serum TTR protein levels by silencing wild-type and mutant TTR mRNAs in hepatocytes (Fig. 2). Patisiran's approval brings new hope to hATTR patients, allowing The systematic delivery of RNAi drugs to liver tissue has become a reality in the clinic, indicating that the field of RNAi therapy has entered a new era.
Source: Nature Reviews Drug Discovery
March 7, Three scientists from the Beckman Institute in the United States published an in-depth review in the journal Nature Reviews Drug Discovery  on "Key advances in RNAi drug design and development. Current state of clinical pipelines and future development prospects." /p>
The article introduces the mechanism of RNAi and the history of early discovery, summarizes the motifs currently used in the synthesis of RNAi triggers. Design rules and chemical modifications, discusses various drug delivery routes, and evaluates RNAi drugs. The current clinical status of the pipeline, comparing patisiran and subsequent drug candidates, and analyzing future opportunities and challenges in the RNAi field.
Drug Design and Development
Figure 3 Mammalian miRNA Creatures Occurs. Synthetic RNAi triggering process and RNAi silencing pathway. (Source: Nature Reviews Drug Discovery)
Efficient specific inhibition of putative therapeutic targets in order to utilize the mammalian RNAi pathway (Figure 3) , RNAi pharmaceutical preparations must overcome the challenges associated with pharmacodynamics (including targeting specificity. Off-target RNAi activity. Immunosensor-mediated cytotoxicity) and pharmacokinetics. In systemic circulation. Cell uptake and endosomal escape (The escape of siRNA from endosomes to the cytoplasm enhances the RNAi effect ). These challenges are through the structural motif of RNAi triggers. Sequence selection and chemical modification, as well as delivery routes and excipients (excipients) Selection and design to solve.
Although RNAi pathway enzymes have restrictive structural requirements for the compatibility of dsRNA molecules, scientists have developed a A series of synthetic RNAi flip-flops with different structural motifs and functional properties (Figure 4). Synthetic RNAi flip-flops are typically fully base paired dsRNAs or short hairpin RNAs (shRNAs) with a total length between 15 and 30 bp. 15 bp dsRNAs lose their ability to participate in the RNAi machinery, while dsRNAs longer than 30 bp can induce non-specific cytotoxicity by activating the PKR pathway.
Figure 4 Different types of synthetic RNAi triggers are representative of secondary Structural motifs and their main mechanisms of entry into the RNAi pathway. (Source: Nature Reviews Drug Discovery)
2016, 1 published in Cancer Gene Therapy  The review provides a list of software packages for siRNA design and recommends the use of protocols. By discussing some of the issues involved, the review points out that in the future, developers of RNAi drugs may need to be widely around reasonable targets. Screening of target sequences to determine the best drug candidate.
For RNAi drugs, chemical modification (except for tissue targeting ligands) has two basic functions. First, they greatly improve safety by attenuating the activation of endogenous immunosensors that detect dsRNA. Second, they greatly enhance the ability of dsRNA triggers to resist endogenous endonuclease and exonuclease degradation. Effective In addition to these functions, chemical modifications can improve sequence selectivity to reduce off-target RNAi activity, as well as alter physical and chemical properties to enhance delivery.
dsRNA Triggering Chemical modification of the device. Size. Hydrophilicity and charge are cyclic to the system. Extravasation. Tissue penetration. Cell uptake and endosomal escape constitute a major challenge. Many chemical excipients (auxiliaries) have been developed to overcome these obstacles. , including nanoparticles. Lipid nanoparticles (LNPs). Polymers. Dendrimers. Nucleic acid nanostructures. Exosomes and GalNAc-coupled melittin-like peptides (NAGMLPs). Common targeting ligands for siRNA include adaptation Antibody. Polypeptides and small molecules (such as GalNAc) (Table 1).
Table 1 RNAi drug delivery methods and excipients
Data Sources (Nature Reviews Drug Discovery)
In addition to excipients, the method of administration and site of administration also have profound effects on the bioavailability and biodistribution of RNAi drugs. RNAi in clinical development 1. The drug involved in the administration includes systemic administration by intravenous injection and subcutaneous injection. Local administration by inhalation (in the lungs) or injection at a specific location (eg intraocular cerebrospinal fluid).
At present, in addition to the patisiran that has been approved for marketing Multiple drug candidates for liver and kidney indications are undergoing Phase I.II and III clinical trials (Table 2). In addition, in the next two years, some targeted central nervous system (CNS) and Other non-liver tissue IND applications are “imminent.” In addition, new technologies for RNAi payload (specificity enhancement) and excipients are expected to lead to new indication breakthroughs in the next five years.
Of course, it should be pointed out that there is still much room for improvement in pharmacokinetics and pharmacodynamics and toxicity limitation strategies of RNAi drugs. The emergence of some new technologies is expected to achieve this goal, such as techniques to improve the escape of endosomes. Antibody Coupling Techniques for the Effectiveness and Safety of RNAi Therapy. Techniques for reducing the toxicity of LNPs. Techniques for improving delivery methods. Techniques for rapidly reversing RNAi activity (many RNAi drugs last for several weeks after one administration may cause toxic side effects) . Limit RNAi Techniques for the function of specific cells in specific cells. Enrich the technology of preclinical animal models.
In summary, however, current progress indicates that RNAi therapy has a lot to look forward to in the next 10 years.
Table 2 Partial RNAi Therapy Currently in Clinical Trial
Nature Reviews Drug Discovery
From 1998 The RNAi phenomenon was discovered in the year. By 2006, the two discoverers jointly won the Nobel Prize in Physiology or Medicine, and by 2018 the world's first RNAi drug was approved. This 20-year development history proves the strength of persistence. There is no doubt that With the continuous advancement of related technologies, future RNAi therapy will achieve more breakthroughs.
1]Andrew Fire et al. Potent and specificgenetic interference by double- Stranded RNA in Caenorhabditis elegans. Nature (1998).
2] Sayda M. Elbashir et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature (2001).
3]Natasha J. Caplen et al. Specificinhibition of gene expression by small double-stranded RNAs in invertebrate andvertebrate systems. PNAS (2001).
4]Ryan L. Setten et al. Thecurrent state and future directions of RNAi-based therapeutics. Nature ReviewsDrug Discovery(2019).
< p>5] Yang Jiangyong et al. Intracellular siRNA delivery: application of modified cell-penetrating peptides to escape from endosomes. International Journal of Pharmaceutical Research (2009).
6]E Fakhr et al. Precise and Effective siRNAdesign: a key point in competent gene silencing. Cancer Gene Therapy (2016).
1# A major milestone in RNAi therapy, the FDA approved the first hATTR drug Onpattro