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RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion. It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality. After a two-decade journey from its discovery, two approvals of siRNA therapeutics, ONPATTRO® (patisiran) and GIVLAARI™ (givosiran), have been achieved by Alnylam Pharmaceuticals. Reviewing the long-term pharmaceutical history of human beings, siRNA therapy currently has set up an extraordinary milestone, as it has already changed and will continue to change the treatment and management of human diseases. It can be administered quarterly, even twice-yearly, to achieve therapeutic effects, which is not the case for small molecules and antibodies. The drug development process was extremely hard, aiming to surmount complex obstacles, such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity, stability, specificity and potential off-target effects. In this review, the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed. All clinically explored and commercialized siRNA delivery platforms, including the GalNAc (N-acetylgalactosamine)–siRNA conjugate, and their fundamental design principles are thoroughly discussed. The latest progress in siRNA therapeutic development is also summarized. This review provides a comprehensive view and roadmap for general readers working in the field.
Introduction
Gene therapy is a promising therapeutic platform because it targets disease-causing genes in a sequence-specific manner, which enables more precise and personalized treatment of diverse life-threatening diseases.1 By introducing a certain nucleic acid modality to the desired tissue of the patient, gene expression can be downregulated, augmented or corrected. Small interfering RNA (siRNA), microRNA (miRNA) and inhibitory antisense oligonucleotides (ASOs) are representative molecules used to trigger gene inhibition, whereas plasmid DNA, messenger RNA (mRNA), small activating RNA (saRNA), splicing-modulatory ASOs and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) systems are usually employed to increase or correct target gene expression.2,3,4 Currently, many therapeutic programs have been explored to treat certain diseases.
RNA interference (RNAi) is a natural defense mechanism for the invasion of exogenous genes.5,6 RNAi modalities, e.g., siRNA and miRNA, can knockdown the expression of target genes in a sequence-specific way (Fig. 1) by mediating targeted mRNA degradation (for siRNA and miRNA) or mRNA translation repression (for miRNA). As a result of the slight differences between siRNA and miRNA, siRNA can typically trigger more efficient and specific gene silencing than miRNA, whereas one miRNA may compromise the expression of several different target genes simultaneously. Hence, siRNA and miRNA have different roles in pharmaceutical practice.
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Review Article
Open Access
Published: 19 June 2020
Therapeutic siRNA: state of the art
Bo Hu,
Liping Zhong,
[…]
Xing-Jie Liang
Signal Transduction and Targeted Therapy volume 5, Article number: 101 (2020) Cite this article
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ABSTRACT
RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion. It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality. After a two-decade journey from its discovery, two approvals of siRNA therapeutics, ONPATTRO® (patisiran) and GIVLAARI™ (givosiran), have been achieved by Alnylam Pharmaceuticals. Reviewing the long-term pharmaceutical history of human beings, siRNA therapy currently has set up an extraordinary milestone, as it has already changed and will continue to change the treatment and management of human diseases. It can be administered quarterly, even twice-yearly, to achieve therapeutic effects, which is not the case for small molecules and antibodies. The drug development process was extremely hard, aiming to surmount complex obstacles, such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity, stability, specificity and potential off-target effects. In this review, the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed. All clinically explored and commercialized siRNA delivery platforms, including the GalNAc (N-acetylgalactosamine)–siRNA conjugate, and their fundamental design principles are thoroughly discussed. The latest progress in siRNA therapeutic development is also summarized. This review provides a comprehensive view and roadmap for general readers working in the field.
Download PDF
Introduction
Gene therapy is a promising therapeutic platform because it targets disease-causing genes in a sequence-specific manner, which enables more precise and personalized treatment of diverse life-threatening diseases.1 By introducing a certain nucleic acid modality to the desired tissue of the patient, gene expression can be downregulated, augmented or corrected. Small interfering RNA (siRNA), microRNA (miRNA) and inhibitory antisense oligonucleotides (ASOs) are representative molecules used to trigger gene inhibition, whereas plasmid DNA, messenger RNA (mRNA), small activating RNA (saRNA), splicing-modulatory ASOs and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) systems are usually employed to increase or correct target gene expression.2,3,4 Currently, many therapeutic programs have been explored to treat certain diseases.
RNA interference (RNAi) is a natural defense mechanism for the invasion of exogenous genes.5,6 RNAi modalities, e.g., siRNA and miRNA, can knockdown the expression of target genes in a sequence-specific way (Fig. 1) by mediating targeted mRNA degradation (for siRNA and miRNA) or mRNA translation repression (for miRNA). As a result of the slight differences between siRNA and miRNA, siRNA can typically trigger more efficient and specific gene silencing than miRNA, whereas one miRNA may compromise the expression of several different target genes simultaneously. Hence, siRNA and miRNA have different roles in pharmaceutical practice.
Fig. 1

Schematic illustrations of the working mechanisms of miRNA (a) and siRNA (b)
Full size image
Since the establishment of the RNAi concept in 1998, siRNA therapeutics have experienced many ups and downs. In 2001, Tuschl et al.7 successfully silenced the expression of a specific gene by introducing chemically synthesized siRNA into mammalian cells, leading to the emergence of a developmental upsurge. Although siRNA therapy once suffered due to the obstacles of its stability, specificity and delivery, advances in chemical modification and delivery brought the field to a robust and rapidly developing area of research again in recent years. After a 20-year journey, the United States Food and Drug Administration (FDA) and the European Commission (EC) approved ONPATTRO® (patisiran, ALN-TTR02) as the first commercial RNAi-based therapeutic for the treatment of hereditary amyloidogenic transthyretin (hATTR) amyloidosis with polyneuropathy in adults in 2018.2,8 Recently, the FDA-approved GIVLAARI™ (givosiran, ALN-AS1) for the treatment of adults with acute hepatic porphyria (AHP).9,10,11,12
siRNA has innate advantages over small molecular therapeutics and monoclonal antibody drugs because siRNA executes its function by complete Watson–Crick base pairing with mRNA, whereas small molecule and monoclonal antibody drugs need to recognize the complicated spatial conformation of certain proteins. As a result, there are many diseases that are not treatable by small molecule and monoclonal antibody drugs since a target molecule with high activity, affinity and specificity cannot be identified. In contrast, theoretically, any gene of interest can be targeted by siRNA since only the right nucleotide sequence along the targeting mRNA needs to be selected. This advantage confers the siRNA modality with a shorter research and development span and a wider therapeutic area than small molecule or antibody drugs, especially for those genes that are unfeasible for development with such strategies.
Although siRNA holds promising prospects in drug development, several intracellular and extracellular barriers limit its extensive clinical application. Naked and unmodified siRNA possesses some disadvantages, such as (1) unsatisfactory stability and poor pharmacokinetic behavior and (2) the possible induction of off-target effects. The phosphodiester bond of siRNA is vulnerable to RNases and phosphatases. Once it is systematically administered into circulation, endonucleases or exonucleases throughout the body will quickly degrade siRNA into fragments, thus preventing the accumulation of intact therapeutic siRNA in the intended tissue. In theory, siRNA only functions when its antisense strand is completely base-paired to the target mRNA. However, a few mismatches are tolerated by the RNA-induced silencing complex (RISC), which may lead to undesired silencing of those genes with a few nucleotide mismatches. In addition, the RISC-loaded sense strand of siRNA may also knockdown the expression of other irrelevant genes. Moreover, unformulated and unmodified siRNA may lead to the activation of Toll-like receptor 3 (TLR3) and adversely affect the blood and lymphatic systems.13 These discoveries have raised many concerns about the undesirable effects and pharmaceutical issues of siRNAs.
To maximize the treatment potency and reduce or avoid the side effects of siRNA, researchers have made great efforts to investigate various chemical modification geometries and to develop many different delivery systems. As a result, a series of modification patterns were proposed and evaluated preclinically and clinically with respect to their effects on activity, stability, specificity and biosafety. Delivery materials derived from lipids, lipid-like materials (lipidoids), polymers, peptides, exosomes, inorganic nanoparticles, etc., have been designed and investigated.2,8,14,15,16,17,18,19,20,21,22 As a result, several modification patterns and delivery platforms have been employed in clinical studies. Here, the detailed evolution and advances in the modification and delivery technologies of siRNA are comprehensively summarized and discussed. This review provides an overview and a handbook for reviewing siRNA therapeutic development.
siRNA modification
During the early stages of developing siRNA therapeutics, many agents were designed based on completely unmodified or slightly modified siRNA to arrive at appropriate tissue and then silence the target gene. These molecules can mediate gene silencing in vivo, especially in tissues that receive local drug administration, e.g., eyes. However, limited efficacy and potential off-target effects may be observed with these modalities. For example, Kleinman and colleagues23 observed that bevasiranib and AGN211745 triggered significant activation of toll-like receptor 3 (TLR3) and its adapter molecule TRIF, inducing the secretion of interleukin-12 and interferon-γ. Bevasiranib and AGN211745 are siRNA therapeutics developed for the treatment of age-related macular degeneration.24 The VEGFA-targeting bevasiranib25 and VEGFR1-targeting AGN21174526 are unmodified and slightly modified siRNAs, respectively. Moreover, Kleinman and colleagues further demonstrated that siRNA classes with sequences of 21 nucleotides or longer, regardless of which genes they target, can suppress CNV in mice compared to bevasiranib and AGN211745. These findings eventually led to the termination of the clinical investigation of bevasiranib in 2009.
Chemically modified siRNAs, such as siRNAs with substitution of the 2′-OH with a 2′-O-methyl (2′-OMe)27 or 2′-methoxyethyl (2′-MOE)28 group or the substitution of certain nucleotides with locked nucleic acid (LNA),29 unlocked nucleic acid (UNA)30 or glycol nucleic acid (GNA)31 (Fig. 2), can efficiently suppress immunostimulatory siRNA-driven innate immune activation, enhance activity and specificity, and reduce off-target-induced toxicity. To enhance the potency and reduce the potential toxicity of siRNA, numerous chemical modification geometries have been established and tested.32