The Growing Class of Novel RNAi Therapeutics

Abstract

The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran] were revealed to act on the 3′-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term “RNAi” therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action.

SIGNIFICANCE STATEMENT

The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term “RNAi” therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.

Introduction — RNAi Tools and Therapeutics

Since the discovery of functional, small noncoding RNAs, RNA interference (RNAi) has been revealed as a critical mechanism in cells to govern posttranscriptional gene regulation (Lee et al., 1993; Wightman et al., 1993; Fire et al., 1998; Hamilton and Baulcombe, 1999) that has provided researchers with the foundational means to selectively manipulate target gene expression and develop novel therapies (Sheng et al., 2020; Yu et al., 2020; Feng et al., 2021; Ning and Yu, 2021; Yu and Tu, 2022; Gogate et al., 2023; Traber and Yu, 2023). The major forms of RNAi molecules, genome-derived microRNAs (miRNA) or exogenous miRNA mimics or biosimilars and exogenously introduced small interfering RNAs (siRNAs), converge into the RNA-induced silencing complexes to achieve posttranscriptional gene regulation (Fig. 1A) (Gebert and MacRae, 2019; Shang et al., 2023; Traber and Yu, 2023). Endogenous miRNAs have been revealed to control target gene expression through the cleavage, degradation, and/or translational repression of the targeted mRNA that may be dependent on the extent of miRNA-mRNA sequence complementarity ranging from partial (e.g., only requiring 7–9 nt from the 5′ end of a 22-nt miRNA) to near complete base pairing (Yekta et al., 2004; Bazzini et al., 2012; Djuranovic et al., 2012; Hu et al., 2020; Hauptmann et al., 2022). By contrast, exogenous siRNAs are often designed to target a singular, specific mRNA sequence through complete base-pair complementarity to exert mRNA cleavage and degradation (Abifadel et al., 2003; Miller et al., 2003; Ui-Tei et al., 2004; Jagla et al., 2005; Birmingham et al., 2007; Naito and Ui-Tei, 2013; Jung et al., 2017; Friedrich and Aigner, 2022). In addition to double-stranded miRNAs and siRNAs, their respective precursors, namely single-strand pre-miRNAs and short hairpin RNAs, are also employed to achieve target gene regulation upon entering the RNAi machineries (Ketting et al., 2001; Brummelkamp et al., 2002; Siolas et al., 2005; Moore et al., 2010; Sheng et al., 2020; Zhang et al., 2021).

Fig 1.

RNAi mechanisms and molecular pharmacology of six FDA-approved RNAi therapeutics. (A) Mechanistic actions of the genome-derived miRNAs and exogenous RNAi agents. MiRNA genes are transcribed into primary miRNA transcripts and processed by the nuclear microprocessor, Drosha-DGCR8 complex, to release shorter precursor miRNAs and further by cytoplasmic Dicer/TRBP complex to offer miRNA duplexes. The guide or antisense strand (blue) of endogenous miRNA or exogenous siRNA and miRNA mimics is loaded into the RNA-induced silencing complex where canonical miRNAs partially bind to the 3′UTR of target transcripts, while siRNAs are typically designed to act on the CDS of target transcript via complete base pairing to induce posttranscriptional gene regulation. (B) Molecular pharmacology of six FDA-approved siRNA therapeutics (antisense in blue, and sense in red). Patisiran is formulated as a lipid nanoparticle for the treatment of hATTR by downregulating TTR. Givosiran, lumasiran, inclisiran, vutrisiran, and nedosiran are all conjugated with GalNAc moieties to improve hepatocyte targeting for the treatments of acute hepatic porphyria by downregulating ALAS1, PH1 by reducing HAO1, heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease by suppressing PCSK9, hATTR by downregulating TTR, and PH1 by silencing LDHA, respectively. Note that, while givosiran targets the CDS of ALAS1 mRNA with near-complete binding, patisiran, lumasiran, inclisiran, vutrisiran, and nedosiran are all designed to act on the 3′UTRs of target mRNAs via complete or near-complete binding to achieve gene silencing as endogenous miRNAs.

Furthermore, while it is well established that canonical miRNA-controlled gene regulation is enhanced when acting on the 3′-untranslated region (3′UTR) of target mRNAs (Grimson et al., 2007; Shang et al., 2023), some miRNAs exhibit non-canonical binding at the protein coding sequence (CDS) in the vicinity of rare codons or within CDS repeats (Gebert and MacRae, 2019; Shang et al., 2023). Even so, it should be noted that binding to the 3′UTR of target mRNA remains the main criterion for the prediction of miRNA target sites (Gebert and MacRae, 2019) and tend to be both more selective and effective approximately 15 nt downstream of the stop codon (Grimson et al., 2007). Controversially, there has yet to be a clear definition regarding the primary target binding sites for siRNAs, although siRNAs are traditionally and usually designed to target the CDS segments of their respective mRNAs (Naito and Ui-Tei, 2013; Fakhr et al., 2016; Yu et al., 2020; Friedrich and Aigner, 2022; Traber and Yu, 2023). Designing siRNA in this way is typically favored to take advantage of CDS harboring fewer polymorphisms and more conserved sequences compared with the UTRs (Jagla et al., 2005; Birmingham et al., 2007; Fakhr et al., 2016; Friedrich and Aigner, 2022). Rather, given their effectiveness in controlling target gene expression, RNAi molecules continue to be developed as novel therapeutics whose chemistry and clinical pharmacology are different from conventional small molecule and protein therapeutics (Diener et al., 2022; Yu and Tu, 2022; Gogate et al., 2023; Traber and Yu, 2023).

Since 2018, a total of six siRNA therapeutics have been approved by the United States Food and Drug Administration (FDA) for the treatment of various diseases through the RNAi regulatory mechanism (Yu and Tu, 2022; FDA, 2023; Gogate et al., 2023; Traber and Yu, 2023), in which nedosiran was approved most recently for the control of hyperoxaluria (Fig. 1B; Table 1) (FDA, 2023). The growing number and expanding disease indications of approved siRNA therapeutics provide incentives to develop more RNAi therapies. Interestingly, in-house sequence alignment analyses of vutrisiran and nedosiran antisense sequences with their mRNA targets, transthyretin (TTR) (FDA, 2022; Adams et al., 2023) and lactate dehydrogenase enzyme A (LDHA) (FDA, 2023), respectively, have revealed that both act on the 3′UTRs of their respective targets, following the canonical miRNA mechanistic action. Thus, vutrisiran and nedosiran follow similar mechanistic actions of patisiran, lumasiran, and inclisiran disclosed very recently (Yu and Tu, 2022; Traber and Yu, 2023). By contrast, only one (givosiran) of the six FDA-approved siRNA therapeutics is directed to interfere with the CDS of its target mRNA (Fig. 1B). In addition, the antisense strands of patisiran, givosiran, and nedosiran (O’Leary et al., 2016; FDA, 2018, 2019, 2023; Siramshetty et al., 2022) contain one or two mismatched nucleotides to their corresponding target mRNA sequences. Indeed, previous works have shown that both miRNAs and siRNAs share overlapping characteristics in base-pairing complementarity as well as regulatory mechanisms (Saxena et al., 2003; Martin and Caplen, 2006; Jung et al., 2017; Hauptmann et al., 2022), yet they still retain their respective nomenclatures. While the current drug labels are informative regarding the chemistries of individual siRNA therapeutics and their RNAi mechanisms of action, specifically naming each as an “siRNA” therapeutic might not fully recapitulate the pharmacology of this novel class of therapeutics nor embrace the growing number and forms of double- or single-stranded therapeutic RNAs, such as miRNA mimics, pre-miRNAs, and short hairpin RNAs, as the term “RNAi therapeutics” having been used in the fields (Adams et al., 2018; Balwani et al., 2020; Sheng et al., 2020; Yu et al., 2020; Garrelfs et al., 2021; Yu and Tu, 2022; Gogate et al., 2023; Traber and Yu, 2023).

TABLE 1.

Chemistries of individual FDA-approved RNAi therapeutics. Sense and antisense sequences were obtained from the FDA inserts. Interestingly, the sense strand of newly approved nedosiran is designed to form a hairpin or stem-loop structure in which the loop is comprised of four GalNAc aminosugar conjugated ribonucleosides. As another note, the guanosine at position 6 from the 5’ end of the nedosiran antisense strand is instead called a cytosine in an alternative source (Siramshetty et al., 2022) to the FDA label.

RNA Therapeutic	Chemistry	Year approved
Patisiran (Onpattro)	Sense: 5′-GUmAACmCmAAGAGUmAUmUmCmCmAUmdTdT-3′ Antisense: 3′-dTdTCAUmUGGUUCUCAUmAAGGUA-5′	2018
Givosiran (Givlaari)	Sense: 5′-CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm-L96-3′ Antisense: 3′-UmsGmsGmUfCmUfUmUfCmUfCmAfCmAfGmAfGmUfAmGfAfsAfsUm-5′	2019
Lumasiran (Oxlumo)	Sense: 5′-GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm-L96-3′ Antisense: 3′-AmsCmsCmUmGmAmAmAfGmUfAmGmGmAmCfCfUmUfUmAmUmsAfsUm-5′	2020
Inclisiran (Leqvio)	Sense: 5′-CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmUm-L96-3′ Antisense: 3′-AmsAmsGmAmUmCfUmGfGmAfCmAfAmAfAmCfGmAfAfAfAmsCfsAm-5′	2021
Vutrisiran (Amvuttra)	Sense: 5′-UmsGmsGmGmAmUmUfUmCfAfUfGmUmAmAmCmCmAmAmGmAm-L96-3′ Antisense: 3′-CmsUmsAmCmCmCmUmAfAmAfGmUmAmCmAfUmUmGfGmUmUmsCfsUm-5′	2022
Nedosiran (Rivfloza)	Sense: 5′-AmsUmGfUmUfGmUmCfCfUfUfUmUfUmAfUmCfUmGmAmGmCmAmGmCm -Cm-AdemG-AdemA-AdemA-AdemA-GmGmCmUmGmCm-3′ Antisense: 3′-GmsGmsUmAmCfAmAfCmAfGmGmAmAfAfAmAfGmAfGfsAfsCfsUm*-5′	2023

A, adenosine; AdemA, GalNAc aminosugar conjugated adenosine; AdemG, GalNAc aminosugar conjugated guanosine; Af, 2′-fluoroadenosine; Am, 2′-O-methyladenosine; C, cytidine; Cf, 2′-fluorocytidine; Cm, 2′-O-methylcytidine; dT, thymidine; G, guanosine; Gf, 2′-fluoroguanosine; Gm, 2′-O-methylguanosine; s, phosphorothioate; U, uridine; Uf, 2′-fluorouridine; Um, 2′-O-methyluridine; Um*, 2′-O-methyl-4’-O-((methoxy)phosphoryl)methyluridine.

In this minireview, we summarize the common and unique chemistry and molecular pharmacology of all six FDA-approved siRNA therapeutics and discuss the current and perplexing naming systems to support the use of a general and unified nomenclature of “RNAi” therapeutic to align with the current therapeutic nomenclature criteria in pharmacology based on mechanism of action.

Common and Unique Chemistry and Delivery of the FDA-Approved RNAi Therapeutics

As the primary ribonucleotide sequence of the siRNA antisense strand is designed for complete complementary base pairing with a target transcript, application of chemical modifications is expected to increase the metabolic stability and optimize target selectivity, potency, and safety of the RNAi molecules (Siolas et al., 2005; Birmingham et al., 2007; Naito and Ui-Tei, 2013; Fakhr et al., 2016; Hu et al., 2020; Sheng et al., 2020; Yu et al., 2020; Yu and Tu, 2022; Traber and Yu, 2023). The six FDA-approved siRNA therapeutics all contain extensive chemical modifications on the ribose subunit of individual ribonucleotides, including methylation of the 2′-hydroxyl group (2′-O-methyl) (2′-O-methyladenosine; Am) and substitution of the 2′-hydroxyl group with fluorine (2′-fluoro) (2′-fluoroadenosine; Af) (FDA, 2023; Gogate et al., 2023; Traber and Yu, 2023) (Table 1). Interestingly, the antisense strand of the newly approved nedosiran consists of a uniquely modified uridine at the 5′-end with conventional 2'-O-methyl and novel 4'-O-((methoxy)phosphoryl)methyl modifications of the ribose subunit (FDA, 2023). Moreover, the 3′-ends of both antisense and sense strands of the first siRNA medication (patisiran) include two thymidine nucleosides (dT) while maintaining internucleotide phosphonate linkages (Traber and Yu, 2023). By contrast, two phosphorothioate linkages are applied to the 3′-ends of antisense strands as well as 5′-ends of sense strands for all other FDA-approved siRNA therapeutics to improve their metabolic stability (FDA, 2023; Gogate et al., 2023; Traber and Yu, 2023).

In addition to extensive chemical modification, two other main techniques are employed to achieve optimal pharmacokinetic properties or delivery of current siRNA therapeutics, namely siRNA encapsulation within a lipid nanoparticle or conjugation to a ligand to enhance targeted tissue or organ distribution or selectivity (Fig. 1B; Table 1) (Yu et al., 2020; FDA, 2023; Gogate et al., 2023; Traber and Yu, 2023). Patisiran remains the only siRNA medication to employ lipid nanoparticle formulation, while all five remaining siRNA therapeutics incorporate N-acetylgalactosamine (GalNAc; L96) ligands to enhance hepatocyte uptake (FDA, 2023; Traber and Yu, 2023). Differing from all previous siRNA therapeutics, the newly approved nedosiran is comprised of a novel hairpin sense strand (FDA, 2023), which is likely important for its overall metabolic stability. Likewise, the four ribonucleotides that form this loop structure within nedosiran are all conjugated to similar GalNAc aminosugar ligands for hepatocyte targeted delivery and subsequent pharmacological action (Fig. 1B; Table 1) (FDA, 2023). Interestingly, while information regarding sequences, chemistries, and target mRNA binding locations of siRNA therapeutics currently in clinical trials may not yet be as transparent as those FDA-approved, it is of interest to note that currently two siRNA therapeutic candidates use a nanoparticle system like that of patisiran while the remaining majority employ GalNAc conjugation for siRNA-payload delivery (Ahn et al., 2023).

Common and Unique Molecular Pharmacology of the FDA-Approved RNAi Therapeutics

All FDA-approved siRNA therapeutics use endogenous RNAi machinery to achieve posttranscriptional gene regulation by mRNA cleavage and degradation for the treatment of respective diseases with differing therapeutic targets, as described in their FDA-approved prescribing information (Fig. 1B). Patisiran and vutrisiran are both indicated for the treatment of hereditary transthyretin-mediated amyloidosis (hATTR) (Adams et al., 2018; FDA, 2018, 2022; Adams et al., 2023; Traber and Yu, 2023), a disease characterized by the buildup of amyloid fibrils in the liver caused by the unregulated aggregation of TTR (Goldman and Schafer, 2016; Kristen et al., 2019). The patisiran and vutrisiran antisense strands target for cleavage and degradation the TTR mRNA at the 3′UTR (Table 2) that encodes the TTR protein important in amyloid fibril formation in several tissue types (Suhr et al., 2015; Adams et al., 2018; FDA, 2018, 2022; Kristen et al., 2019; Adams et al., 2023). Of particular note, the antisense stand of patisiran contains a two-nucleotide overhang at the 3′ end (Table 1) of which the second is mismatched to the target mRNA sequence (Table 2). The RNAi of TTR mRNA by patisiran and vutrisiran function to specifically reduce TTR protein production in liver and thus the abundance of both wild-type and mutant TTR in blood circulation and TTR deposits in tissues (FDA, 2018, 2022). More specifically, analyses from Phase 3 clinical trials showed that patients exhibited mean maximum reductions of 84.3% (sustained over 18 months) and 96% (sustained over 90 days) in serum TTR levels after treatment with patisiran and vutrisiran, respectively (Zhang et al., 2020; Habtemariam et al., 2021). Between the two, the more recently approved vutrisiran exhibits improved liver-specific delivery and may contribute to increased potency compared with its predecessor, patisiran (Foster et al., 2018; Weng et al., 2019; Habtemariam et al., 2021; Traber and Yu, 2023). Both patisiran and vutrisiran are available for prescription and administered either intravenously at a dose of 0.3 mg/kg every 3 weeks or by subcutaneous injection of 25 mg administered once every 3 months, respectively (FDA, 2018, 2022).

TABLE 2.

Base pairings between individual antisense strands of FDA-approved RNAi therapeutics and respective mRNA targets. Antisense sequences were obtained from the FDA inserts, and mRNA sequences were obtained from the National Center for Biotechnology Information. Nedosiran is the most recently approved siRNA therapeutics and harbors unique binding complementarity. Antisense nucleotides mismatched to their target mRNA sequences are lacking connecting lines, among them the guanosine at position 6 from the 5′ end of nedosiran antisense strand is instead called a modified cytosine in alternative source (Siramshetty et al., 2022) to the FDA therapeutic label.

Similarly, lumasiran and nedosiran are indicated to treat primary hyperoxaluria type 1 (PH1), an autosomal recessive disorder of the liver characterized by increased deposition of calcium oxalate crystals in the kidneys and urinary tract (Cochat and Rumsby, 2013; FDA, 2020, 2023; Garrelfs et al., 2021; Traber and Yu, 2023). Instead of targeting the same mRNA to treat PH1, the antisense strands of lumasiran and nedosiran instead target the 3′UTRs of hydroxyacid oxidase 1 (HAO1) and LDHA transcripts (Table 2), respectively, to achieve mRNA cleavage and degradation to decrease the production of their respective proteins in the liver and downstream calcium oxalate crystal formation and subsequent oxalate accumulation in the kidneys and urinary tract (FDA, 2020; Garrelfs et al., 2021; Sas et al., 2021; Scott and Keam, 2021; FDA, 2023; Traber and Yu, 2023). The HAO1 mRNA, targeted by lumasiran, encodes the glycolate oxidase enzyme that is upstream in the oxalate production pathway, while the LDHA mRNA, targeted by nedosiran, encodes the hepatic enzyme LDHA enzyme responsible for the final step in oxalate production by metabolizing glyoxylate to oxalate (Yang et al., 2020; Garrelfs et al., 2021; Sas et al., 2021; Scott and Keam, 2021; FDA, 2023). Specifically, analyses from pivotal clinical trials demonstrated mean maximum reduction in unitary oxalate excretion of 76% and 68% when treated with lumasiran and nedosiran, respectively (Garrelfs et al., 2021; Baum et al., 2023). With the recent approval of nedosiran, both siRNA therapeutics are available for prescription and are administered through subcutaneous injection. Lumasiran has a dosing regimen of either 6 mg/kg or 3 mg/kg once monthly for three doses for patients with body weights between 10–20 kg or 20 kg and above, respectively, with variable maintenance doses dependent on patient body weight (FDA, 2020). Alternatively, nedosiran has a dosing regimen for adults of either 160 mg or 128 mg monthly for patients with body weight greater or equal to 50 kg or less than 50 kg, respectively, while dosing for children follows the same weight distinctions of either 160 mg or 3.3 mg/kg monthly (FDA, 2023). Interestingly, the antisense strand of nedosiran is comprised of two mismatched nucleotides to the target LDHA mRNA sequence, one within the 5′ end “seed sequence” and another at the 3′ end (Table 2) (O’Leary et al., 2016; FDA, 2023). It should be noted that an alternative source (Siramshetty et al., 2022) to the FDA insert opposes the 5′ end “seed sequence” mismatch and instead place a modified cytosine in place of the modified guanosine (Table 2).

Moreover, inclisiran is the first non-statin, siRNA medication indicated to treat both heterozygous familial hypercholesterolemia as well as clinical atherosclerotic cardiovascular disease (FDA, 2021; Santulli et al., 2021; Traber and Yu, 2023), characterized by elevated blood circulation of low-density lipoprotein cholesterol (LDL-C) (McGowan et al., 2019; Bardolia et al., 2021; Traber and Yu, 2023). The antisense stand of inclisiran targets the 3′UTR of the proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA encoding the PCSK9 protein (Table 2). PCKS9 is a low abundant circulating protein responsible for decreasing the natural low-density lipoprotein cholesterol (LDL-C) receptor endocytosis and lysosomal degradation cycle, leading to increased circulating LDL-C and downstream incidence of heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease (Raal et al., 2020; FDA, 2021; Lamb, 2021; Wang et al., 2022; Traber and Yu, 2023). By reducing PCSK9 protein levels, inclisiran enhances LDL-C receptor recycling and decreases LDL-C levels in blood circulation (Raal et al., 2020; Lamb, 2021; Wang et al., 2022; Traber and Yu, 2023). More specially, analyses of a Phase 3 clinical trial resulted in a 52.6% reduction in circulating LDL-C levels after two injections of inclisiran over 180 days (Ray et al., 2020). Inclisiran is available and prescribed in combination with the maximally tolerated statins with an initial subcutaneous dose of 284 mg with subsequent maintenance doses every six months (FDA, 2021).

Finally, givosiran is indicated for the treatment of acute hepatic porphyria (FDA, 2019; Sardh and Harper, 2022; Traber and Yu, 2023). Acute hepatic porphyria is characterized by a dysfunction within the heme synthesis pathway that results in an increase in aminolevulinate synthase 1 (ALAS1) and downstream production of aminolaevulinic acid and porphobilinogen neurotoxic metabolites (Bissell et al., 2017; Anderson, 2019; Scott, 2020; Kothadia et al., 2022; Sardh and Harper, 2022; Traber and Yu, 2023). However, compared with the all FDA-approved siRNA therapeutics, the antisense strand of givosiran instead targets for cleavage and degradation the CDS of the ALAS1 mRNA (Table 2). Thus, givosiran functions to decrease the protein levels of ALAS1 responsible for the downstream production of neurotoxic metabolites, leading to the reduction of circulating neurotoxins (FDA, 2019; Agarwal et al., 2020; Balwani et al., 2020; Scott, 2020; Traber and Yu, 2023). Specifically, analysis of a Phase 3 clinical trial demonstrated that treatment with givosiran resulted in an 86% and 91% reduction in urinary aminolaevulinic acid and porphobilinogen neurotoxic metabolites, respectively, as a proxy for measuring siRNA-induced ALAS1 reduction (Balwani et al., 2020). Givosiran is available for prescription and administered subcutaneously at a dose of 2.5 mg/kg once a month (FDA, 2019). Of note, the antisense strand of givosiran contains a 5′ end mismatch to the targeted sequence on the ALAS1 mRNA (Table 2), a well-known characteristic of miRNA (O’Leary et al., 2016; FDA, 2019; Li et al., 2021).

Interestingly, while all FDA-approved siRNA therapeutics cleave and degrade target mRNAs, givosiran remains the only FDA-approved siRNA medication that follows the generally accepted siRNA mechanistic action by binding to the CDS segment, although with incomplete complementarity (Fig. 1B; Table 2). By contrast, the five remaining siRNA therapeutics all bind to the 3′UTRs of their respective mRNA targets with three (lumasiran, inclisiran, and vutrisiran) exhibiting complete base-pair complementarity (FDA, 2018, 2019, 2020, 2021, 2022) and two (patisiran and nedosiran) exhibiting miRNA-like, incomplete base-pair complementarity (Fig. 1; Table 2) (Siramshetty et al., 2022; FDA, 2023) to incite their mRNA cleavage and degrative activities. Therefore, these five “siRNA” therapeutics, especially the newly approved nedosiran, instead follow canonical miRNA mechanistic action, indicating that these “siRNA” therapeutics behave as “miRNAs” or at least “miRNA biosimilars” designed with complete or near complete complementarity to their target transcripts (Yu et al., 2020; Yu and Tu, 2022; Traber and Yu, 2023). In addition, this further suggests that targeting the 3′UTR might be more efficacious than the CDS as the 3′UTR is closely related to mRNA stability and miRNA function (Tian and Manley, 2017; Gebert and MacRae, 2019; Shang et al., 2023). Thus, this finding may also offer an insight into the strategy employed by these RNAi therapeutics to increase the effectiveness by more closely mimicking the functional and conserved miRNAs derived from the genomes within eukaryotes (Fig. 1A) (Tian and Manley, 2017; Gebert and MacRae, 2019; Shang et al., 2023; Traber and Yu, 2023), in support of the use of miRNA or RNAi molecules targeting the 3′UTRs in basic research and clinical therapy. Together, this inherent difference newly discovered within a common RNAi molecule class further supports the return to a more transparent, precise, and comprehensive nomenclature that embraces the growing number and forms of therapeutic RNAi molecules.

Today, there are tens of therapeutic RNAi molecules making their way through the clinical trial pipeline (Ahn et al., 2023; Gogate et al., 2023; Iacomino, 2023; Traber and Yu, 2023). In fact, there are eight RNAi therapeutics candidates named as “siRNAs” (fitusiran, tivanisiran, fazirsiran, olpasiran, belcesiran, cemdisiran, revusiran, and ARO-APOC3) that have recently completed or are currently in Phase 3 clinical trials, with several others in Phase 2 and earlier preclinical trials to treat infectious, hematologic, hereditary, fibrotic, and metabolic diseases as well as diseases of the eye, skin, lung, kidney, and brain (Ahn et al., 2023; Gogate et al., 2023; Traber and Yu, 2023). In comparison, there are three RNAi therapeutics candidates named as “miRNAs” or “miRNA mimics” (MRX34, TargomiRs, and Remlarsen) that entered clinical investigations, with several others that act as anti-miRNAs or antagomirs (Iacomino, 2023; Traber and Yu, 2023; Seyhan, 2024). Further, an additional therapeutic candidate termed “miRNA” (AMT-130) is currently in a Phase 1/2 clinical trial for Huntington’s disease and uses a modified viral vector to express its therapeutic miRNA in cells as compared with other synthetic miRNA mimic or siRNA candidates and approved RNAi therapeutics (Iacomino, 2023; Traber and Yu, 2023; Seyhan, 2024). However, it should be noted that, while most clinical emphases remains on “siRNA”-termed therapeutics, the use of miRNA therapy remains in its infancy with several candidates under preclinical investigation in several disease types, including malignant, neurologic, and cardiovascular diseases for known and putative roles in inflammation, proliferation, necrosis, apoptosis, and autophagy (Iacomino, 2023; Traber and Yu, 2023; Seyhan, 2024).

Unifying and Pharmacological Nomenclature of RNAi Therapeutics

While complete sequence complementarity between siRNA and target mRNA, and not mRNA target site, have been inherently used to classify siRNA and miRNA molecules or biosimilars, previous studies have revealed that “siRNAs” engineered with miRNA characteristics (i.e., imperfect (40–80%) complementarity or “bulge” binding) are able to effectively regulate target gene expression through mRNA cleavage and degradation (Saxena et al., 2003; Martin and Caplen, 2006; Hauptmann et al., 2022; FDA, 2023). Consequently, effective “siRNA” molecules can now be designed with sequences that exhibit incomplete complementary to their target mRNAs, as miRNAs are defined (Yekta et al., 2004; Bazzini et al., 2012; Djuranovic et al., 2012; Jung et al., 2017; Hu et al., 2020; Hauptmann et al., 2022), yet regulate target gene expression though mRNA cleavage, as both siRNAs and miRNAs are defined (Abifadel et al., 2003; Miller et al., 2003; Ui-Tei et al., 2004; Jagla et al., 2005; Birmingham et al., 2007; Naito and Ui-Tei, 2013; Friedrich and Aigner, 2022), all while retaining the nomenclature of “siRNA”. Further, it has been well documented that siRNAs may induce cleavage of target mRNAs with complete (Abifadel et al., 2003; Miller et al., 2003; Ui-Tei et al., 2004; Jagla et al., 2005; Birmingham et al., 2007; Naito and Ui-Tei, 2013; Friedrich and Aigner, 2022) or partial complementarity (Saxena et al., 2003; Lin et al., 2005; Jackson et al., 2006; Vickers et al., 2009; Jung et al., 2017), in direct contrast with the problematic distinguishment of miRNAs from siRNAs simply by partial or complete, respectively, sequence complementarity-dependent mRNA cleavage or translational repression (Hu et al., 2020; Traber and Yu, 2023). In fact, the antisense strands of patisiran, givosiran, and nedosiran exhibit this near complete complementarity with mismatched nucleotides (Table 2), yet called as “siRNA” therapeutics (O’Leary et al., 2016; FDA, 2018, 2019, 2023; Li et al., 2021; Siramshetty et al., 2022).

These findings, together with the implications that five siRNA therapeutics follow canonical miRNA mechanistic action by binding the 3′UTR, disrupt the traditional “siRNA” molecule nomenclature paradigm as the term “siRNA” therapeutic might not fully describe their complete pharmacology. Moreover, any attempt to alternatively distinguish therapeutic siRNAs and miRNAs based on the targeted mRNA sites would then require both siRNA and miRNA molecules to have a defined canonical binding location on target mRNAs (Yu et al., 2020; Yu and Tu, 2022; Traber and Yu, 2023). As a result, it is then conceivable that therapeutic “siRNAs” and siRNA molecules alike, might be seen as exogenous “miRNAs” designed to bind their target mRNA sequences with complete complementarity.

Instead, an alternative means of naming therapeutic siRNAs, miRNAs, and other forms of RNAi molecules being approved by the FDA and under clinical and preclinical development (Yu et al., 2020; Gogate et al., 2023; Traber and Yu, 2023) is to return to a nomenclature that embraces the growing number and various forms of therapeutic RNAi molecules and aligns with current pharmacological criteria, i.e., nomenclature by mechanism of action. Typically, therapeutics on the market are classified either by their disease indication (e.g., antidepressant therapeutics) or pharmacological action (e.g., selective serotonin reuptake inhibitors), while some are classified by their chemistry (e.g., sulfonamides). Hence, it would be advantageous for basic and translational scientists, clinicians, and educators to follow a known and more cohesive nomenclature with fewer confounding exceptions. In this way, all FDA-approved siRNA therapeutics and the growing number and forms of therapeutic RNAi molecule candidates are appropriately named according to their complete pharmacology, and not their chemistry or extent of sequence complementary.

Thus, the most clear, informative, transparent, and complete means of naming therapeutic siRNAs and miRNAs is under the simple and pharmacologically unified nomenclature of “RNAi” therapeutics having been previously used in the field (Adams et al., 2018; Balwani et al., 2020; Sheng et al., 2020; Yu et al., 2020; Garrelfs et al., 2021; Yu and Tu, 2022; Gogate et al., 2023; Traber and Yu, 2023). Accordingly, while, for example, givosiran and inclisiran target two different mRNAs at different binding locations, with variable degrees of binding complementarities, and for different disease indications, both would be recapitulated pharmacologically as RNAi therapeutics. Following this unified nomenclature leaves no room for error in naming current and future RNAi therapeutics and provides a clear and fully defined pharmacological action that is independent of unique chemistry, therapeutic targets, binding locations, sequence complementarity, and disease indications. Therefore, a return to the term “RNAi” therapeutics (Bumcrot et al., 2006; Adams et al., 2018; Balwani et al., 2020; Yu et al., 2020; Garrelfs et al., 2021) is in concordance with recognized criteria for therapeutic nomenclature in the fields of pharmacology and pharmacological sciences based on the common mechanism of action of all siRNA therapeutics being approved and the growing number and variable forms of therapeutic RNAi molecules under development.

Concluding Remarks

With the recent addition of nedosiran, there are now six FDA-approved siRNA therapeutics which share a common RNAi mechanism by acting on their specific therapeutic targets for the control of respective diseases. Notably, all siRNA therapeutics share characteristics of both siRNA and miRNA molecules by either exhibiting complete (lumasiran, inclisiran, and vutrisiran) or near complete (patisiran, givosiran, and nedosiran) base-pair complementarity with targeted mRNAs to incite cleavage and degradation via the RNAi pathway, in which five (patisiran, lumasiran, inclisiran, vutrisiran, and nedosiran) interfere with the 3′UTRs and only one (givosiran) acts on the CDS. Therefore, the term “RNAi” therapeutics coherently aligns the growing number and various forms of novel RNAi therapeutics based on common pharmacological action and is more informative for professionals and the general public.

Abbreviations

ALAS1

aminolevulinate synthase 1

CDS

protein coding sequence

FDA

United States Food and Drug Administration

GalNAc or L96

N-acetylgalactosamine

HAO1

hydroxyacid oxidase 1

hATTR

hereditary transthyretin-mediated amyloidosis

LDHA

lactate dehydrogenase enzyme A

LDL-C

low-density lipoprotein cholesterol

miRNA

microRNA

PCSK9

proprotein convertase subtilisin/kexin type 9

PH1

primary hyperoxaluria type 1

RNAi

RNA interference

siRNA

small interfering RNA

TTR

transthyretin

3′UTR

3′ untranslated region

Authorship Contributions

Participated in research design: Traber, Yu.

Performed data analysis: Traber, Yu.

Wrote or contributed to the writing of the manuscript: Traber, Yu.