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In 1998, American scientists Andrew Fire and Craig Mello published a groundbreaking paper in Nature, confirming 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 (Fig. 1) explains the confusing gene silencing in plants and fungi and triggers a revolution in biology. Finally, studies have shown that non-coding RNAs are the main regulator of gene expression in multicellular organisms. factor. The RNAi pathway regulates mRNA stability and translation in almost all human cells.

Figure 1 Early events found and elucidated by the RNAi pathway

In 2001, a Nature published by Sayda M. Elbashir et al., and a PNAS published by Natasha J. Caplen et al. demonstrated that dsRNAs consisting of 21 and 22 nucleotides were able to induce RNAi silencing in mammalian cells, and Can cause a non-specific interferon response. These small interfering RNAs (siRNAs) are quickly becoming ubiquitous tools in biological research because they can easily inhibit any gene through a single base sequence.


For drug developers, the potency and versatility of siRNAs, the prospect of inhibiting genes encoding proteins, and the potential to become “programmable” drugs are “attractive.” By 2003, many companies had deployed RNAi therapy.


Unfortunately, the first clinical trials using unmodified siRNAs produced immune-related toxicity and a suspected (indeterminate) RNAi effect. The second wave of clinical trials used systemically administered siRNA nanoparticle formulations, although significant advances have been made (eg, the first confirmed, siRNA nanoparticle system administration can produce RNAi effects in humans), but still exhibits significant doses. Limit toxicity and insufficient efficacy. The problems exposed in these developments led most pharmaceutical companies to withdraw from the RNAi field in the early 2010s, which brought a financial crisis to the development of RNAi drugs.


However, despite these challenges, some small RNAi companies and academic researchers have not given up on this. They learned from the painful lessons of previous clinical trial failures and insisted on improving trigger design, sequence selection, chemical modification and delivery mechanisms. Substantial progress in these areas, combined with more sensible disease indication options, better validation interventions, more mature clinical development processes, and improved manufacturing capabilities, has created a safer and more effective candidate RNAi drug pipeline.

Figure 2 The treatment mechanism of patisiran

On August 10, 2018, 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 transthyretin (TTR) amyloid deposition in the peripheral nervous system, heart, gastrointestinal tract, and other organs. The patient has 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 in the TTR can result in hATTR. Patisiran reduced serum TTR protein levels by silencing wild-type and mutant TTR mRNAs in hepatocytes (Figure 2). Patisiran’s approval has given new hope to hATTR patients, and 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.

On March 7th, 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.” .


The article introduces the mechanism of RNAi and the history of early discovery, summarizes the motifs, design rules and chemical modifications currently used in the synthesis of RNAi triggers, discusses various drug delivery routes, and evaluates the current clinical status of RNAi drug pipelines. Compare patisiran and subsequent drug candidates and analyze future opportunities and challenges in the RNAi field.


Drug design and development

Figure 3. Mammalian miRNA biogenesis, synthetic RNAi triggering, and RNAi silencing pathways.


In order to utilize the mammalian RNAi pathway (Figure 3) for effective specific inhibition of putative therapeutic targets, RNAi pharmaceutical preparations must overcome pharmacologically relevant challenges (including targeting specificity, off-target RNAi activity, immunosensor mapping). Guided cytotoxicity) and pharmacokinetics related challenges in systemic circulation, cellular uptake, and endosomal escape (siRNA can escape RNAi effects from endosomal escape to cytoplasm). These challenges are addressed by the structural motifs, sequence selection and chemical modification of RNAi triggers, as well as the choice and design of delivery routes and excipients (excipients).


Structural motif


Although RNAi pathway enzymes have restrictive structural requirements for the compatibility of dsRNA molecules, scientists have developed a series of synthetic RNAi flip-flops with different structural motifs and functional properties (Figure 4). Synthetic RNAi triggers are typically fully base paired dsRNAs or short hairpin RNAs (shRNAs) with a total length between 15 and 30 bp. DsRNAs shorter than 15 bp lose the ability to participate in the RNAi machinery, while dsRNAs longer than 30 bp can induce non-specific cytotoxicity by activating the PKR pathway.



Sequence selection


In 2016, a review published in Cancer Gene Therapy provided a list of software packages for siRNA design and recommended the use of protocols. Through a discussion of some of the issues involved, the review points out that in the future, developers of RNAi drugs may need to perform extensive target sequence screening around a reasonable target to determine the best drug candidate.


Chemical modification


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 efficacy by enhancing the ability of dsRNA triggers to resist endogenous endonuclease and exonuclease degradation. . In addition to these functions, chemical modifications can also improve sequence selectivity to reduce off-target RNAi activity, as well as alter physical and chemical properties to enhance delivery.


Delivery of excipients


The chemical modification, size, hydrophilicity, and charge of dsRNA triggers pose significant challenges to systemic circulation, extravasation, tissue penetration, cellular uptake, and endosomal escape. Many chemical excipients (auxiliaries) have been developed to overcome these barriers, including nanoparticles, lipid nanoparticles (LNPs), polymers, dendrimers, nucleic acid nanostructures, exosomes, and GalNAc-coupled melittin Peptides (NAGMLPs). Common targeting ligands for siRNA include aptamers, antibodies, polypeptides, and small molecules (such as GalNAc) (Table 1).


Table 1 Method for delivery of RNAi drugs and excipients


Dosing position


In addition to excipients, the method of administration and site of administration also have profound effects on the bioavailability and biodistribution of RNAi drugs. The administration of RNAi drugs in clinical development involves systemic administration by intravenous injection and subcutaneous injection, local administration by inhalation (in the lungs) or injection at a specific location (such as intraocular, cerebrospinal fluid).



Clinical product


Currently, in addition to patisiran, which has been approved for marketing, a number of drug candidates for liver, kidney, and ocular indications are undergoing Phase I, II, and III clinical trials (Table 2); in addition, over the next two years, Some IND applications targeting the central nervous system (CNS) and other non-liver tissues are “imminent.” In addition, new technologies for RNAi payloads (specific enhancement) and excipients are expected to bring new breakthroughs in the next five years.

Of course, it should be pointed out that there is still much room for improvement in the pharmacokinetic, pharmacodynamic and toxicity limiting strategies of RNAi drugs. The emergence of new technologies is expected to achieve this goal, such as techniques to improve endosomal escape, antibody coupling techniques that increase the effectiveness and safety of RNAi therapy, techniques to reduce the toxicity of LNPs, techniques to improve delivery, and rapid reversal of RNAi activity. Techniques (many RNAi drugs last for several weeks after one administration, may cause toxic side effects), techniques that limit the role of RNAi drugs in specific cells, and techniques that enrich preclinical animal models.

But overall, the current progress shows that in the next 10 years, RNAi therapy has a lot to look forward to.




From the discovery of RNAi in 1998, the two discoverers jointly won the Nobel Prize in Physiology or Medicine in 2006, and the world’s first RNAi drug was approved in 2018. This 20-year history of development proves the strength of persistence. Undoubtedly, with the continuous advancement of related technologies, future RNAi therapy will make more breakthroughs.

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