Small interfering RNA: Discovery, pharmacology and clinical development—An introductory review

Post‐transcriptional gene silencing targets and degrades mRNA transcripts, silencing the expression of specific genes. RNA interference technology, using synthetic structurally well‐defined short double‐stranded RNA (small interfering RNA [siRNA]), has advanced rapidly in recent years. This introductory review describes the utility of siRNA, by exploring the underpinning biology, pharmacology, recent advances and clinical developments, alongside potential limitations and ongoing challenges. Mediated by the RNA‐induced silencing complex, siRNAs bind to specific complementary mRNAs, which are subsequently degraded. siRNA therapy offers advantages over other therapeutic approaches, including ability of specifically designed siRNAs to potentially target any mRNA and improved patient adherence through infrequent administration associated with a very long duration of action. Key pharmacokinetic and pharmacodynamic challenges include targeted administration, poor tissue penetration, nuclease inactivation, rapid renal elimination, immune activation and off‐target effects. These have been overcome by chemical modification of siRNA and/or by utilising a range of delivery systems, increasing bioavailability and stability to allow successful clinical translation. Patisiran (hereditary transthyretin‐mediated amyloidosis) was the first licensed siRNA, followed by givosiran (acute hepatic porphyria), lumasiran (primary hyperoxaluria type 1) and inclisiran (familial hypercholesterolaemia), which all use N‐acetylgalactosamine (GalNAc) linkage for effective liver‐directed delivery. Others are currently under development for indications varying from rare genetic diseases to common chronic non‐communicable diseases (hypertension, cancer). Technological advances are paving the way for broader clinical use. Ongoing challenges remain in targeting organs beyond the liver and reaching special sites (e.g., brain). By overcoming these barriers, siRNA therapy has the potential to substantially widen its therapeutic impact.

Miescher in 1869 (Dahm, 2005) and the subsequent characterisation of its complex, double-helical structure by Watson and Crick (1953), scientists have investigated the mechanisms by which genotypic expression can be altered to influence phenotype, leading to disease modification.This has driven the creation of several therapeutic modalities acting on different stages of genetic expression, affecting protein synthesis and delivering clinical benefits.Brenner et al. (1961) identified mRNA as being responsible for carrying genetic information from nuclear DNA to the cytoplasmic protein synthetic machinery.
Post-transcriptional gene silencing aims to target and degrade these mRNA transcripts and silence the expression of specific target genes (Vazquez et al., 2004).RNA interference (RNAi) using small interfering RNA (siRNA) is a post-transcriptional gene-silencing technology that is now delivering effective medicines (Zhang et al., 2021).Our introductory review aims to describe the utility of siRNA, by exploring the underpinning biology, pharmacology, recent advances and clinical developments, alongside potential limitations and ongoing challenges, focusing on non-communicable diseases.

| DISCOVERY OF siRNA
In most eukaryotic cells, RNAi is a regulatory mechanism controlling gene activity by silencing post-transcriptional expression of specific genes (Bartel, 2018).Napoli et al. (1990) reported a new RNAi-type phenomenon, while experimenting on pigmentation in petunia flowers (Figure 1).They attempted to overexpress chalcone synthase (CHS), an important enzyme in the anthocyanin synthetic pathway responsible for violet pigmentation, by introducing a chimeric petunia CHS F I G U R E 1 Small interfering RNA (siRNA) timeline-discovery to regulatory approval (preclinical milestones are depicted in yellow, and clinical milestones are in green).GalNAc, N-acetylgalactosamine; RISC, RNA-induced silencing complex; RNAi, RNA interference; US FDA, US Food and Drug Administration gene, via Agrobacterium, into the genotype.However, rather than heavier pigmentation, this unexpectedly resulted in white (unpigmented) flowers and associated loss of CHS mRNA.This led the authors to hypothesise that the introduced chimeric CHS gene inhibited anthocyanin biosynthesis.A similar occurrence was described in 1992 in Neurospora crassa (a filamentous fungus) and found to be spontaneously and progressively reversible (Romano & Macino, 1992).A further major insight was described by Fire et al. (1998), who coined the term 'RNA interference' to describe the process, for which they were subsequently awarded the Nobel Prize in Physiology or Medicine (2006).Working with the nematode Caenorhabditis elegans, they demonstrated that the most effective trigger for post-transcriptional gene silencing was a double-stranded RNA and that single-stranded RNA was 10-fold to 100-fold less effective at targeting mRNA and less specific (Fire et al., 1998).A small anti-sense double-stranded RNA was subsequently characterised in eukaryotic cells (Hamilton & Baulcombe, 1999), and siRNA is now recognised to be a naturally occurring gene-silencing pathway (Piatek & Werner, 2014).Thereafter, Bernstein et al. (2001) described the RNA-induced silencing complex (RISC), an essential component in this process, in Drosophila melanogaster.Subsequently, Elbashir et al. (2001) and Song et al. (2003) demonstrated RNAi in cultured mammalian cells and mice, respectively.Continued work enabled the development of targeted therapeutic siRNAs, which are the focus of our article.

| siRNA: MOLECULAR MECHANISM
Synthetic siRNAs have a distinct structure with a short doublestranded RNA sequence with sense (non-guide) and anti-sense (guide) strands composed of 20-24 bp (typically 21 bp), with phosphorylated 5 0 and hydroxylated 3 0 ends and two nucleotides overhanging on the 3 0 end of each strand (Figure 2a).It is now recognised that siRNA length is an important determinant of function, with longer sequences potentially inducing global gene silencing and cell death (Raja et al., 2019).The molecular mechanism by which synthetic siRNAs result in gene silencing is depicted in Figure 2b.The first step in the process is endosomal entry of synthetic siRNA into the cell and subsequent endosomal escape.Subsequently, siRNA binds to the RISC, and a member of the Argonaute (AGO) protein family recognises and selects one strand of the duplex as the guide (anti-sense) strand, whereas the non-guide (sense) strand is degraded (Sheu-Gruttadauria & MacRae, 2017).The selection of the guide and nonguide strands within the RISC is determined by the relative thermodynamic stability in base pairing at the 5 0 end, where the strand whose 5 0 end is less stably base paired being identified as the guide strand (Iwakawa & Tomari, 2022).The AGO protein consists of three domains that are important in its function, namely, the PAZ, MID and PIWI domains.The MID and PAZ domains each forms pockets that recognise and anchor the 5 0 and 3 0 ends of the guide strand, respectively, whereas the PIWI domain is important for the nuclease activity and cleavage of complementary target mRNA (Iwakawa & Tomari, 2022).In humans, only Argonaute 2 (AGO2) is catalytically active, although four AGO proteins have been characterised (Iwakawa & Tomari, 2022).It is important to appreciate that the interactions between the siRNA guide strand and the AGO protein are mediated by the sugar-phosphate backbone, whereas the bases remain free for complementary base pairing with the target mRNA.In addition to AGO proteins, the assembly and proper functioning of the RISC requires other key proteins (Wu et al., 2020).
The resultant single-stranded RNA remains bound to the RISC and guides it to recognise and bind to complementary target mRNA, which results in subsequent cleavage of mRNA, preventing it from acting as a translational template for protein synthesis.In turn, this causes post-transcriptional silencing of the effector gene that reduces synthesis of its target protein (Svoboda, 2020).This mechanism has been further characterised by the recognition that, after formation of the RISC, and the separation of the non-guide strand, the remaining guide strand can be functionally divided in to four domains: (a) seed region (nucleotides 2-8)-is crucial for target recognition; (b) central region (nucleotides 9-12)-is important for cleavage of target mRNA; (c) 3 0 supplementary region (nucleotides 13-17)-stabilises the bound target mRNA; and (d) tail region (nucleotides 18-3 0 end)-regulates the recruitment of additional factors required for RISC function (Iwakawa & Tomari, 2022).Interactions between siRNA and RISC, and subsequent interaction and cleavage of mRNA are facilitated by a wide variety of other molecules and processes, which interact to enable specific post-transcriptional gene silencing.Detailed description of these molecules and their interactions have been well characterised previously (Iwakawa & Tomari, 2022).Once this mechanism of sequence-specific RNAi and gene silencing using siRNAs was identified, it was quickly adopted as a tool to silence targeted gene expression and develop effective therapeutic strategies to tackle a wide array of diseases.
First discovered in 1993, microRNAs (miRNAs) are endogenous RNAs synthesised in the nucleus as precursor molecules (pre-miRNA) and subsequently cleaved to produce mature duplex miRNA of 18-25 nucleotides (stem-loop structure) (Lam et al., 2015).Like siRNA, miRNA produces post-transcriptional gene silencing through RNAi, where silencing occurs through translational repression, or degradation by de-adenylation, de-capping or exonuclease action (see Section 4.2) (Nobrega et al., 2020).Since their initial discovery, many different miRNAs have been characterised, with some being highly biologically conserved across different species, including humans (O'Brien et al., 2018).

| COMPARISON OF siRNA THERAPY WITH OTHER THERAPEUTIC APPROACHES
siRNA therapy provides novel opportunities and some advantages compared with other therapeutic approaches, which justify the substantial recent investment in this field.Technological advances are now overcoming pharmacological barriers, paving the way for broader clinical use.However, some limitations and challenges remain to be overcome.

| Comparison with existing medications
One of the key advantages of siRNA therapeutics is their ability to potentially target the expression of almost any single gene/protein of interest, addressing targets that might be undruggable using traditional small-molecule approaches.For example, it is estimated that only 20% of the proteome can be targeted by commonly used medications (Finan et al., 2017).Knowing the target gene sequence, matching siRNAs can be developed, with rapid lead optimisation and manufacture.The challenge of tailoring this naked siRNA to develop a therapeutic agent with a suitable pharmacological profile is now well on the way to being solved.This contrasts with small-molecule drugs acting at protein-level targets such as receptors, enzymes and channels that require complex development processes to ensure selectivity as well as suitable pharmacology.In addition, although only 1-2% of siRNA entering cells escapes the endosome and enters the cytosol, once F I G U R E 2 (a) Small interfering RNA (siRNA) structure and (b) siRNA molecular mechanism.AGO, Argonaute protein; dsRNA, double-stranded RNA; OC, other components of RISC; RISC, RNA-induced silencing complex; ssRNA, singlestranded RNA bound to the RISC, it is protected from nuclease degradation, allowing prolonged action (Gilleron et al., 2013).This can translate into a prolonged therapeutic effect, with approved siRNA medications like inclisiran showing clinical benefit for up to 6 months after a single s.c.administration (Fitzgerald et al., 2017).This gives a distinct advantage in the treatment of chronic diseases requiring regular medications, and the potential to substantially improve medication adherence, leading to better disease control.Furthermore, siRNAbased therapies have a high degree of selectivity, and although certain protein-based medications such as antibodies have similar selectivity, they are more expensive to synthesise and manufacture, giving siRNA-based therapies a distinct advantage (Sheridan, 2019).Beyond this, siRNAs do not permanently modify the genome, allowing discontinuation of therapy and control, which is an important safety consideration (Resnier et al., 2013).Also, gene mutations can result in cancers, that are initially sensitive to conventional chemotherapy, developing tolerance to treatment, whereas with siRNA, there is a potential to adapt the product (Gottesman, 2002).In addition, siRNAs provide an opportunity to target rare diseases that currently lack effective therapies, as evident from their early success in the treatment of genetic disorders.
However, concerns around safety remain and cost-effectiveness may need to be considered, especially for chronic diseases where existing effective medications are available.Theoretical concern also exists regarding siRNA therapies due to their long durations of action and the difficulty in reversing adverse events.Therefore, siRNA therapeutics may benefit from technologies that can reverse target silencing activity, mitigating such concerns.The demonstration in vivo that an oligonucleotide complementary to the siRNA anti-sense strand can reverse GalNAc-conjugated siRNA (GalNAc-siRNA)-mediated RNAi experimentally supports this possibility (Zlatev et al., 2018).Importantly, failure in Phase II and III trials may reflect insufficient knockdown of the target protein or that the target chosen is not critical to the pathogenesis of the disease process.

| Comparison with other RNAi therapeutics
miRNA was the first post-transcriptional gene-silencing pathway identified, and mechanistically, the miRNA and siRNA pathways converge, with miRNA binding to RISC and forming a complex called miRISC (Lam et al., 2015).However, an important distinction exists between how miRNAs and siRNAs recognise mRNA targets.Although siRNAs are entirely complementary to the target mRNA, miRNAs are only partially complementary, usually in the 3 0 UTR region (Nobrega et al., 2020).Due to this partial complementarity, the AGO2 activation and cleavage within the RISC does not occur, unlike with siRNAs (Nobrega et al., 2020).Instead, silencing occurs through translational repression, or degradation by de-adenylation, de-capping or exonuclease action (Huntzinger & Izaurralde, 2011).This partial pairing also results in miRNAs being able to target multiple mRNAs.For example, miRNA-124, expressed in brain tissue, can suppress 174 annotated genes (Lim et al., 2005).In addition to facing challenges in delivery similar to siRNA therapeutics, miRNAs also have a higher likelihood of off-target effects and toxicity due the targeting of multiple mRNAs and silencing the expression of many genes compared with the very high degree of selectivity for their target of siRNAs (Lam et al., 2015).Furthermore, it is important to highlight that miRNA-based therapeutics are yet to receive regulatory approval, although some are in clinical development.

| Comparison with other gene-silencing therapies
RNAi and anti-sense oligonucleotides are to date the two most widely used methods of therapeutic gene silencing (Nobrega et al., 2020).
Anti-sense oligonucleotides are synthetic, single-stranded molecules (8-50 nucleotides), which bind to complementary specific mRNAs through base pairing (Nobrega et al., 2020).They result in posttranscriptional gene silencing either via base pairing and cleavage of the specific mRNA by RNAse H or simply by occupancy of target mRNA and blocking interaction with RNA binding proteins and thereby mRNA translation (Nobrega et al., 2020).As single-stranded molecules, anti-sense oligonucleotides are less stable than siRNAs (Watts & Corey, 2012).Furthermore, emerging evidence suggests the development of tolerance to certain anti-sense oligonucleotides, due to increased transcription and production of pre-mRNAs, subsequent to anti-sense oligonucleotide binding with target mRNA.This shortens their duration of activity and necessitates more frequent administration (Liang et al., 2020).However, due to their structure, anti-sense oligonucleotides enter cells more effectively than siRNAs.At present, it is not clear whether one gene-silencing method is superior to the other, and both remain in parallel development to overcome challenges to their success.
Table S1 provides a summary comparing siRNA therapy with existing medications, other RNAi therapeutics and gene-silencing therapies.

| PHARMACOLOGICAL CHALLENGE-KINETICS AND DYNAMICS
Despite the promise of wide therapeutic application, numerous challenges had to be overcome prior to siRNA technology successfully translating into clinical use (Figure 3).

| Absorption
Oral administration is generally considered the ideal route for drug administration, owing to convenience and cost-effectiveness.Nonetheless, the harsh environment in the gastrointestinal tract, and siR-NAs being negatively charged, leads to rapid degradation and poor absorption (Akhtar, 2009).This remains a serious challenge, so currently approved therapies for systemic targets require administration via i.v. or s.c.routes (Balwani et al., 2020;Raal et al., 2020).Other methods of siRNA delivery include local administration, where they are directly applied to the target organ or tissues, such as the eyes, mucous membranes and skin (Figure 3a) (Grzelinski et al., 2006;Palliser et al., 2006).This avoids barriers to systemic administration and can achieve good target bioavailability.However, few conditions lend themselves to local administration.

| Distribution
After absorption, siRNAs normally enter the circulation rapidly (Huang et al., 2011).Their intracellular site of action necessitates penetration of the vascular endothelium, limited by their anionic charge.In general, penetrating the endothelium is dependent on the size of the capillary pores, which is the maximum particle size limit (Figure 3b) (Lau et al., 2012).The liver comprises sinusoidal (discontinuous) capillaries, allowing exchange of macromolecules and hence easier access for siR-NAs (Lau et al., 2012).siRNAs can also accumulate at other sites with fenestrated 'leaky' capillaries, such as tumours, in which rapid angiogenesis and endothelial discontinuity favour penetration.It is estimated that siRNAs can accumulate at 20-40% higher concentrations at tumour sites than in normal tissue (Leng et al., 2017).Hence, to date, most successful siRNA therapies have targeted the liver (Balwani et al., 2020;Raal et al., 2020) and malignant tumours.Biodistribution studies in mice using radiolabelled siRNA nanoparticles have shown rapid clearance from the blood and accumulation in the liver (23% of the injected dose at 60 min) and renal excretion with subsequent accumulation in the bladder (73% at 60 min) (Park et al., 2016).
Further improvements are needed to effectively target non-liver, nontumour sites.It is important to recognise that accumulation of siRNAs in the liver and other sites with fenestrated capillaries could result in inadvertent toxic effects, when other systemic sites are the therapeutic target (Sun et al., 2017).
The next challenge to overcome is penetration of the cell membrane, composed of a negatively charged phospholipid bilayer and functional proteins.This is achieved by endocytosis, which necessitates engulfed siRNA to escape the endosome to bind with the RISC in the cytoplasm (Figure 3b) (Meade & Dowdy, 2007), before the endosome fuses with, and is digested by, lysosomes.Studies have shown that only 1-2% of siRNA entering cells via endosomes reaches the cytosol (Gilleron et al., 2013).Nevertheless, siRNAs do achieve measurable and beneficial clinical effects, and further improvements in endosomal penetration might have deleterious effects as described in Section 5.4.

| Metabolism and elimination
Naked unmodified siRNAs are rapidly cleared from the blood, with studies showing that their half-life within the systemic circulation is only 5-10 min (Gao et al., 2009).Their low molecular weight ($13 kDa) and small size ($7 nm) facilitate glomerular filtration (pore size $8 nm) (Figure 3c) (Tatiparti et al., 2017).In addition, naked siRNA is susceptibility to rapid degradation by a host of ubiquitous exonucleases and endonucleases within the plasma, tissues and cytoplasm within a few minutes, limiting the accumulation of intact siRNA at its intended target site (Hu et al., 2020) and therapeutic potential (Volkov et al., 2009).Furthermore, circulating naked siRNA can become protein coated, facilitating macrophage degradation, further reducing plasma and biological half-life (Sajid et al., 2020).
Therefore, several methods to protect siRNAs from degradation and systemic elimination have been developed (as described in Section 6), prolonging their half-life and achieving adequate target concentrations.

| Immune activation and off-target effects
Key safety concerns regarding siRNA therapy include off-target sequence-specific gene silencing and sequence-independent initiation of immune responses leading to toxicity.Different methods are employed to screen for sequence-dependent off-target effects, including the use of cell models and bioinformatic algorithms (Jackson & Linsley, 2010).Induction and activation of the immune system must be minimised to avoid reduced therapeutic efficacy and causing undesired off-target effects.siRNAs can activate immune responses and stimulate the production of cytokines such as IFN-13α (Figure 3d) (Judge et al., 2005), which occurs via toll-like receptors (TLRs), which can recognise foreign pathogen-associated molecular patterns, as well as by TLR-independent pathways (Meng & Lu, 2017).siRNA length is very important, with longer double-stranded RNA (>30 nucleotides) rapidly inducing IFNs, with subsequent degradation (Subhan & Torchilin, 2020).siRNA could also suppress non-target genes, causing potentially serious consequences.
Two mechanisms are postulated to be responsible: (a) siRNAs are able to tolerate a few mismatches on the target mRNA, forming incomplete base pairs with resultant gene silencing, and (b) saturation of RISCs and competition with endogenous miRNAs could limit the activity of endogenous RNAi pathways (Raja et al., 2019).In addition, sequence-independent accumulation in cells, such as those of the proximal renal tubule, could result in toxicity (van de Water et al., 2006).Therefore, technological advances to improve stability and specificity have become an integral part of the preclinical development of siRNAs, with the aim of improving pharmacokinetic, pharmacodynamic and safety profiles.Furthermore, it is important to highlight that the pharmacokinetic and pharmacodynamic profiles of newer siRNAs are enhanced by the resultant chemical modifications and delivery methods.

| TECHNOLOGICAL ADVANCES
To provide a suitable pharmacological profile for systemic therapy, and maximise efficacy, it is necessary to modify naked siRNAs.This has been achieved either by chemical modification or by the utilisation of a range of delivery systems to increase siRNA stability and bioavailability.

| Chemical modification
Over time, approaches to enhance the efficacy of siRNAs have included the chemical modification of sugars, phosphate backbone and the bases of oligoribonucleotides, as well as modification to the termini and duplex structure (Figure 4).Some of these modifications, after demonstrating potential utility in vitro, have successfully made their way into in vivo siRNA therapies in clinical use and in clinical trials (Table 1).

| Phosphate backbone modifications
The inter-nucleotide phosphodiester linkages of unmodified siRNAs are negatively charged and can be easily cleaved by nucleases (Selvam et al., 2017).Substitution of this backbone can generate higher potency with better resistance to degradation by RNAses (Hall et al., 2006).Examples include usage of amide linkages and triazole dimers, as well as substitution with boranophosphate, phosphorodithioate and phosphorothioate.It is important to recognise, though, that overuse of modifications may reduce efficacy and increase toxicity (Amarzguioui et al., 2003).Therefore, careful consideration regarding not only the type of chemical modification but also its site(s) and extent is important to maximise efficacy and minimise toxicity.

| Sugar modifications
Recognition that the 2 0 -OH in the ribose sugar is not essential for the action of siRNAs and that the 2 0 -OH group is known to participate in their degradation via nucleases has led to modifications that can confer a higher degree of stability and enhance binding affinity, while reducing immunogenicity and immune-mediated off-target effects.
The most widely used sugar modification at this site includes the usage of 2 0 -O-methyl, 2 0 -O-methoxyethyl and 2 0 -O-fluorine groups.
However, extensive use of a single modification can result in a proportional decrease in silencing activity (Wu et al., 2014).In contrast, alternating different 2 0 -OH modifications, such as the use of alternative 2 0 -O-methyl and 2 0 -O-fluorine modifications, gives better results, enhances stability against enzymatic activity and retains therapeutic potential (Lam et al., 2015b), a successful approach in designing siRNA therapeutics that has made its way into clinical trials.

| Base modifications
Modifications include the substitution of nucleotide bases by 5 0 -bromouracil, pseudouracil, 2 0 -thiouracil, 5 0 -iodouracil, dihydrouracil and hypoxanthine, introduced to improve thermal stability, by enhancing the formation of hydrogen bonds between complementary nucleotides in vitro (Chernikov et al., 2019).There is evidence that a small number of base modifications (≤10%) can minimise stimulation of the immune system and enhance in vitro thermal stability (Anderson et al., 2010).Although phosphate backbone and sugar modifications have proven rather effective (Table 1), base modification is yet to be widely used in development of therapeutic siRNAs.

| Modification to the termini and duplex structure
The terminal ends of the siRNA can be modified in various ways to enhance stability and efficacy.In addition, as discussed later, conjugation with other larger molecules is utilised in the targeted delivery of siRNAs.Modification to the termini of the non-coding strand doubles binding to the RISC and increases the accumulation and stability of siRNAs in organs (Chernikov et al., 2019).Conjugation with aromatic compounds, such as hydroxyphenyl, naphthyl, phenyl and pyrenyl derivatives, helps to protect siRNAs from enzymatic degradation and improves thermal stability and membrane permeability (Selvam et al., 2017).Modifications to the usual double-stranded structure of native siRNA have also been evaluated, including use of a small internally segmented interfering RNA (sisiRNA), with three strands (two coding and one non-coding), which has shown better gene-silencing potency and reduced off-target effects in vitro (Bramsen et al., 2007).
A wide variety of chemical modifications have been evaluated to enhance the stability and efficacy of native siRNAs.However, their utility varies and most of the successful therapeutic siRNAs employ a combination of chemical modifications to achieve pharmacological properties best suited to clinical use (Table 1).In addition, it has been proposed that the increased half-life of chemically stabilised siRNA in acidic intracellular compartments (e.g., lysosomes) contributes towards its extended duration of action, with resultant slow release to the cytosol enabling continuous loading of RISC and prolonged target silencing (Brown et al., 2020).

| siRNA delivery systems
To be effective, siRNAs need to evade clearance by non-target organs and tissues, have the ability to penetrate the desired target tissues and cells and interact with them without eliciting harmful immune responses or other adverse effects (Cheng et al., 2015).Different delivery systems have been devised to accomplish these objectives, with varying degrees of success and clinical utility (Figure 5).They are broadly divided into viral and non-viral delivery systems, with the latter currently more successful.
F I G U R E 4 Chemical modifications of small interfering RNA (siRNA) (modifications present in siRNA therapies defined in Table 1 (Tatiparti et al., 2017).However, their effectiveness, and clinical utility, is limited by pre-existing recipient immunity to the viral vectors (Aronson et al., 2019), the risk they will initiate an immune response in the host (leading to loss of efficacy during subsequent dosing and adverse outcomes [Bryson et al., 2017]), the possibility of unwanted incorporation into the host DNA (Nguyen et al., 2021) and the costs involved in the production of safe vectors for human use.With these limitations, more attention is currently being given to non-viral delivery.

| Non-viral delivery systems
The two non-viral delivery methods currently used in siRNA therapy are conjugation with ligands that better target delivery into desired tissues and cells, and the encapsulation of siRNA into vesicles.

Conjugation with ligands
Conjugates couple siRNA molecules to compounds that may enhance their pharmacological properties.The use of precise ligands that bind to specific cellular receptors enables the delivery of siRNA molecules to specific target cells/tissues in the body, with cellular entry being facilitated by receptor-mediated endocytosis.Also, conjugation with substances that use natural cellular transport mechanisms (such as for cholesterol) helps siRNAs to enter cells.In addition, targeted delivery helps to minimise off-target effects, and due to their low molecular size, most bioconjugates are inherently less immunogenic (Chernikov et al., 2019).An important requirement for optimal biological activity is the ability to cleave the linker molecule once inside the cell, thereby enabling efficient formation of the RISC.Examples of commonly studied bioconjugates include lipophilic molecules, peptides, singlestranded oligonucleotides (aptamers) and antibodies (Chernikov et al., 2019).Peptide conjugates have the advantage of targeted delivery being able to bind with specific proteins on the cellular membrane (Liu et al., 2014).
An example of a clinically successful bioconjugate with receptor specificity is the glycoprotein N-acetylgalactosamine (GalNAc) with high specificity and binding affinity to the asialoglycoprotein receptor (ASGPR), specifically and abundantly expressed in hepatocyte membranes (Cedillo et al., 2017).Nair et al. (2014) first demonstrated that GalNAc-siRNA facilitates targeted delivery of siRNA to hepatocytes in vitro and in vivo (in mice) (Figure 1).Since then, the method has been used successfully to deliver several licensed siRNA Lipids and cholesterol conjugates help siRNAs interact with the cell membrane and facilitate biodistribution by forming complexes with low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles (Wolfrum et al., 2007).In vivo studies have shown that cholesterol conjugates are more efficiently retained ($60%) than other lipophilics, with efficient bioaccumulation in liver, adrenals and spleen (Biscans et al., 2019).Conjugation with aptamers (synthetic oligonucleotides) is another approach (Catuogno et al., 2018), which may have an added advantage if able to penetrate the blood-brain barrier (Esposito et al., 2018).However, their clinical utility is limited at present due to their susceptibility to nucleases, renal clearance and immunogenicity (Chernikov et al., 2019).Antibody-siRNA conjugates can also be used to target specific cell receptor antigens, with an advantage over other similar ligands of higher binding efficiency and prolonged presence in blood due to a higher molecular weight, but immunogenicity and low endosomal escape are limitations to be overcome (Svoboda, 2020).It is evident that bioconjugates offer a successful strategy to deliver siRNA therapeutics, with the advantages of targeted delivery and lower toxicity, especially as they require fewer excipients than vesicle-based delivery methods.
However, certain conjugate molecules require further optimisation to enhance clinical utility.

Encapsulation
Extracellular vesicles are effective endogenous vehicles for transferring miRNA between cells (Buck, 2022), an approach that has been successfully adopted for siRNA therapy, including the use of liposomes, nano-emulsions, dendrimers and synthetic polymers, as well as nanoparticles derived from metals, metal oxides and carbon-based materials (Tatiparti et al., 2017).Nanoparticles help to shield siRNAs from nucleases and exposure to the host immune system, reduce renal clearance and facilitate endocytosis-mediated cellular uptake, with the ability for targeted delivery by the conjugation of the nanoparticle with specific ligands (Tatiparti et al., 2017).This can improve pharmacokinetic profile and bioavailability, allowing for lower siRNA doses (Zhang et al., 2020).Currently, lipid-based nanoparticles are the clinically most successful of these, with siRNA encapsulated by a bilayer of lipids carrying a positive surface charge, with molecules such as cholesterol as helper lipids, which enhances siRNA packaging and stability and eases penetration through the phospholipid bilayer of cell membranes (Hu et al., 2019).In addition, PEGylation of the surface of lipid-based nanoparticles reduces opsonisation and clearance by the reticuloendothelial system (Sun et al., 2017).Furthermore, the lipid bilayer facilitates endosomal escape (Kim, 2020).However, the larger size of nanoparticles ($100 nm) may limit vascular penetration, so they are most suited for sites with fenestrated endothelium or leaky vasculature, such as the liver or cancer tissue (Sun et al., 2017).
Other disadvantages of lipid-based nanoparticles include toxicity due to excipients and the requirement for i.v.therapy, which involves supervision by trained clinical staff (Crooke et al., 2018).Cationic polymers with a branched/linear structure can also be used as effective delivery vehicles, with the capability to condense large nucleic acids into stable nanoparticles that stimulate endocytosis and endosomal escape (Putnam, 2006).However, significant concerns still remain with regard to their toxicity, especially at higher doses (Kichler, 2004).
Currently, bioconjugates remain at the forefront, whereas methods are being developed to overcome shortcomings in nanoparticle-based delivery.

| siRNA UTILITY IN TREATMENT AND PREVENTION OF DISEASES
The beauty of siRNA therapy is that any disease linked to a responsible protein can potentially be targeted, generating great enthusiasm for developing siRNA-based therapies for a wide range of disease conditions.Patisiran (Alnylam Pharmaceuticals), used in the management of hereditary transthyretin-mediated amyloidosis, was the first siRNA to receive approval by the US Food and Drug Administration (FDA) in August 2018 (Kristen et al., 2018).Three other siRNA therapies have received the US FDA approval since then, including givosiran (acute hepatic porphyria) in November 2019, lumasiran (primary hyperoxaluria type 1) in November 2020 and inclisiran (heterozygous familial hypercholesterolaemia) in December 2021 (Zhang et al., 2021).We describe below the currently available evidence on these siRNA therapies and others in various stages of clinical development (Table 1), evolving from the treatment of rare genetic conditions to use in common chronic non-communicable diseases and beyond.siRNA therapies are also currently being evaluated for communicable diseases (Levanova & Poranen, 2018;Mehta et al., 2021), but are not covered in our review.

| Familial transthyretin amyloidosis
Familial transthyretin amyloidosis is a rare (prevalence 1 in 100,000 USA) autosomal dominant disorder of the transthyretin gene leading to abnormal tissue accumulation of amyloid proteins, with resultant organ dysfunction and premature mortality (Sekijima, 2001).Patisiran, the first commercialised siRNA therapy, targets both wild-type and mutant transthyretin and is currently indicated for i.v.administration in polyneuropathy (Stage 1 or 2) in patients with familial transthyretin amyloidosis (300 μgÁkg À1 every 3 weeks; max 30 mg per dose).Evidence from Phase IIII trials showed an improvement (vs.placebo) in polyneuropathy disability score (8% vs. none), quality of life (51% vs. 10%), gait speed (53% vs. 13%) and motor strength (40% vs. 1%) (Adams et al., 2018).This was a significant advance for a disease with a poor prognosis, for which early liver transplantation offers the opti- Results are currently awaited (Alnylam Pharmaceuticals, 2022a, 2022b;Habtemariam et al., 2020).

| Acute hepatic porphyria
Acute hepatic porphyrias are a group of rare diseases (prevalence 0.5-10 per 100,000) resulting from mutations in enzymes involved in haem biosynthesis (Syed, 2021).Up-regulation of hepatic δ-aminolevulinic acid synthase 1 (ALAS1) leads to build-up of toxic haem intermediates, porphobilinogen and δ-aminolevulinic acid (δ-ALA).Givosiran inhibits hepatic synthesis of ALAS1, reducing porphobilinogen and δ-ALA concentrations (Syed, 2021).Evidence from the Phase III randomised controlled trial (ENVISION; n = 94) showed a 74% decrease in acute attack rate with treatment in comparison with placebo, with sustained lowering of urinary δ-ALA and porphobilinogen, reduced pain score and better quality of life, during the 6 months of the study (Balwani et al., 2020).These promising findings culminated in givosiran being licensed by the US FDA as an orphan medication with breakthrough therapy designation.However, concerns regarding adverse effects remain, with elevations in serum aminotransferase and creatinine concentrations, and reduction of estimated glomerular filtration rate, seen in the givosiran group (Balwani et al., 2020).The mechanisms responsible for these changes are yet to be identified, with hypotheses for change in renal function including inhibition of ALAS1 affecting renal tubular cell homeostasis and reduced δ-ALA (potent vasoconstrictor) affecting renal perfusion, toxicities secondary to gene-silencing on-target activity (Lazareth et al., 2021).Therefore, long-term safety with givosiran needs to be carefully monitored.

| Primary hyperoxaluria
Primary hyperoxaluria type 1 (the commonest form; prevalence 1-3 per 1,000,000) is a rare disorder characterised by increased hepatic production of oxalate, which deposits as crystals in the kidney and other organs causing organ damage and dysfunction, due to a mutation in the AGT gene encoding an enzyme metabolising glyoxylate to pyruvate and glycine, with resultant glyoxylate accumulation (Forbes et al., 2021).Lumasiran reduces the synthesis of the hepatic glycolate oxidase enzyme, with subsequent reduction in oxalate synthesis (Garrelfs et al., 2021).Evidence from a Phase III randomised controlled trial (ILLUMINATE-A, n = 39) demonstrated a 65% decrease in 24 h urinary oxalate excretion in the lumasiran group, which was seen as early as the first month (Garrelfs et al., 2021), with the majority of participants attaining normal/near-normal concentrations at 6 months.
However, longer term studies are needed to confirm clinical benefits of delaying the onset and progression of renal disease.Mild transient injection-site reactions were the commonest adverse effect in the lumasiran group.These promising results led to lumasiran being granted a licence as an orphan drug and a breakthrough treatment.
However, it is important to acknowledge the limited safety data for siRNA therapies in rare genetic diseases, due to smaller sample sizes and the short duration of trials.Nedosiran is in Phase III development for primary hyperoxaluria, suppressing liver LDH, the main enzyme generating oxalate from glyoxylate (Forbes et al., 2021).Inhibition of LDH is postulated to be effective for all three primary hyperoxaluria subtypes, as it regulates the final common step in the biosynthesis of oxalate.Results from Phase III trials are awaited.

| Haemophilia
Haemophilia A and B are X-linked recessive disorders (prevalence 1 in 5000 male births), characterised by the deficiency of coagulation factors, leading to bleeding tendencies.Fitusiran is a therapy targeting the production of antithrombin in the liver, a natural anticoagulant, with its reduction aiming to restore haemostasis in patients with haemophilia (Pasi et al., 2021).Evidence from Phase I studies shows that monthly s.c.fitusiran was well tolerated and able to lower antithrombin concentrations from baseline in a dose-dependent manner, with resultant improved thrombin generation (Pasi et al., 2021).A Phase II open-label study among patients with haemophilia showed sustained decrease in antithrombin concentrations of $75%, and the majority did not experience spontaneous bleeds during the study period.However, a patient's death, as a result of a severe thromboembolic event, led to the suspension of the study in September 2017, later being lifted as new risk mitigation measures were introduced, with no further events.Published data from ongoing Phase III trials (ATLAS) of this promising therapy are awaited (Zhang et al., 2021).

| Alpha-1 antitrypsin (A1AT) deficiency
A1AT deficiency is a rare disease (1 in 3500 of European descent) related to defective production of A1AT protein, which protects the body from the neutrophil elastase enzyme (Meseeha & Attia, 2022).
Reduced A1AT activity causes lung damage, and excessive deposition of abnormal A1AT protein in the hepatocytes results in liver damage (Meseeha & Attia, 2022).ARO-AAT targets production of the defective A1AT protein, decreasing its accumulation in the liver and reduc-

| Hypercholesterolaemia
Hypercholesterolaemia (increased LDL cholesterol) is an important risk factor responsible for the formation of atherosclerotic plaques and subsequent atherosclerotic cardiovascular disease (AsCVD), a leading cause of morbidity and mortality among adults worldwide (Pirillo et al., 2021).Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease expressed mainly in the liver, responsible for the degradation of LDL receptors, thereby playing a crucial role in determining circulating concentrations of LDL cholesterol (Blanchard et al., 2019).Inclisiran reduces the synthesis of PCSK9 in the liver (Ray et al., 2020).After successful completion of Phase I and II testing (Fitzgerald et al., 2017;Ray et al., 2017), it was evaluated in Phase III randomised controlled trials among patients with AsCVD (ORION-10 trial; n = 1561) and patients with AsCVD or an AsCVD risk equivalent (ORION-11 trial; n = 1617), who had increased LDL-cholesterol concentration in spite of receiving statins at the highest tolerated dose (Ray et al., 2020).In these two trials, patients received either s.c.inclisiran (284 mg) or placebo (1:1 allocation) on Day 1, Day 90 and every 6 months thereafter over a total duration of 540 days.
Results showed that at Day 510, inclisiran significantly decreased LDL cholesterol by 52% in ORION-10 and by 50% in ORION-11, in comparison to placebo.Occurrence of adverse events was comparable between groups in both trials, though injection-site reactions were more frequent in the inclisiran group (2.6% vs 0.9% ORION-10 and 4.7% vs. 0.5% in , but all mild and non-persistent.The occurrence of serious adverse events and deaths was similar between study groups in both trials, with no adverse signals from laboratory results.These trials (Ray et al., 2020) demonstrated the ability of inclisiran given at infrequent intervals (every 6 months) to achieve a sustained and substantial reduction in serum LDL cholesterol (≈50%), among patients treated with other lipid-lowering therapies.Similar results have been observed in adults with heterozygous familial hypercholesterolaemia, a genetic disorder that affects 1 in 250 persons globally (Raal et al., 2020).A Phase III randomised controlled trial (n = 482) in adults with heterozygous familial hypercholesterolaemia receiving s.c.inclisiran (300 mg) or placebo (Days 1, 90, 270 and 450) showed a 40% reduction of LDL cholesterol in the treatment group compared to an 8% increase in the placebo group.LDL-cholesterol reduction was observed irrespective of familial hypercholesterolaemia genotype, and adverse events (both total and serious) were comparable between groups.The convenience of the treatment regimen will have a positive impact on long-term adherence, thereby improving disease control.Although not seen, a theoretical concern for such treatments with long durations of action is the possibility of irreversible adverse events (Ray et al., 2020).Excellence, 2021).In addition to injection-site reactions, the other commonly reported adverse effects were headache (18%), back pain (5%), diarrhoea (5%) and nasopharyngitis (12%), all of which were mild to moderate in severity (Hardy et al., 2021).Other approaches to PCSK9 inhibition include the use of monoclonal antibodies (e.g., alirocumab and evolocumab) that bind circulating PCSK9.Studies have shown that they are both safe and efficacious in reducing LDL cholesterol, although the higher cost and the need for biweekly/monthly injections are important concerns in clinical use (Sabatine, 2019).However, it is important to appreciate that only emerging evidence from longer term follow-up studies will demonstrate the true impact of inclisiran on changes in the occurrence of AsCVD and associated mortality and identify adverse effects that are rare or associated with long-term use.

Successful conclusion of
For example, initial trials of evolocumab, a monoclonal antibody PCSK9 inhibitor, have not shown improvement in cardiovascular mortality, despite reduction of LDL cholesterol, raising the question whether reduction in PCKS9 activity alone reduces mortality (Bamji, 2020).The

| Hypertension
Hypertension is a primary risk factor for cardiovascular disease and chronic kidney disease, and non-optimal blood pressure (BP) is the biggest single risk factor contributing to the global burden of disease and to global all-cause mortality, leading to 9.4 million deaths and 212 million lost healthy life years (8.5% of the global total) each year (Forouzanfar et al., 2016).Also, one in four adults globally are affected by hypertension, making it one of the most common conditions in humans, with a rapid increase in prevalence during the last few decades (Forouzanfar et al., 2017).In addition, satisfactory BP control remains a challenge, even in developed countries.A major reason for failure is poor adherence to long-term daily treatment (Burnier & Egan, 2019), and siRNAs offer a unique solution, helping patients achieve optimal BP control and reduced risk of cardiovascular events.
Zilebesiran is currently being developed as a treatment for hypertension (Huang et al., 2020).It is a GalNAc conjugate delivered by s.c.injection, targeting liver angiotensinogen, thereby suppressing angiotensin I and II concentrations, with resultant lowering of BP.Interim results from a Phase I trial among patients (n = 84) with mild to moderate hypertension showed that a single dose of zilebesiran resulted in a dose-related decrease in serum angiotensinogen (>90% sustained up to 3 months at higher doses) and BP (>15 mmHg reduction of 24 h systolic BP) over 8 weeks without hypotension (Huang et al., 2021).The evidence supports infrequent dosing intervals of 3-6 months, offering a potential breakthrough addition to the existing arsenal of antihypertensive medications.At present, zilebesiran is in several Phase II trials among patients with mild-moderate hypertension (KARDIA-1) and with BP poorly controlled on standard antihypertensive medications, and results are currently awaited.

| Cancer
Cancer is a complex set of diseases with multiple genetic and cellular abnormalities, occurring as a result of both environmental and hereditary risk factors.siRNA therapy offers a novel approach to cancer therapy, and there is, understandably, much activity in this area.ALN-VSP02 is a lipid nanoparticle-based therapy that contains two active siRNAs, targeting the expression of vascular endothelial growth factor A (VEGF-A) and kinesin spindle protein (KSP), being evaluated in patients with advanced solid tumours.Phase I studies showed good tolerability and antitumour activity (Cervantes et al., 2011).siG12D (targeting the KRAS oncogene) was developed for pancreatic cancer, with a novel delivery system composed of a small-scale biodegradable polymeric matrix called LOcal Drug EluteR, which is implanted into the tumour under ultrasound guidance and releases the drug locally within the tumour over an extended period of 12-16 weeks.In a Phase I/IIa trial, in combination with gemcitabine, siG12D was tolerated well and safe and demonstrated a potential efficacy in patients with locally advanced pancreatic cancer (Varghese et al., 2020).Another Phase II trial in a similar cohort of patients is currently underway.
Atu027 targets PK N3 in the vascular endothelium, improving vascular endothelial integrity, thereby potentially inhibiting local tumour invasion and metastasis.A Phase I trial demonstrated adequate safety among patients with advanced solid tumours, with 41% having stable disease for ≥8 weeks (Schultheis et al., 2014).Evidence from Phase II trials among patients with metastatic and locally advanced pancreatic cancer, where Atu027 was used in combination with gemcitabine, showed a beneficial effect on progression-free survival (dose dependent), warranting further investigation (Schultheis et al., 2016).TKM-PLK1 is another therapy that has completed Phase II trials, targeting polo-like kinase protein (PLK-1), regulating cellular proliferation.TKM-PLK1 was generally well tolerated but had only limited efficacy in a Phase I trial among patients with hepatocellular carcinoma (el Dika et al., 2019).A Phase II trial also showed satisfactory safety, with preliminary efficacy in patients with adrenocortical cancer (Demeure et al., 2016).EphA2 siRNA is currently being evaluated in a Phase I trial in patients with advanced or recurrent solid tumours.It targets EphA2, a receptor TK, overexpressed in these forms of cancer, for which results are awaited (Oner et al., 2021).
Familial adenomatous polyposis is an inherited disease (autosomal dominant) leading to the development of precancerous polyps in the large intestine (Waller et al., 2016).CEQ508, targeting β-catenin, is delivered orally into the intestinal mucosa via a live-attenuated Escherichia coli.In a Phase I trial, it showed satisfactory safety and a significant decrease in β-catenin expression among patients with familial adenomatous polyposis (Trieu et al., 2017).Evidence for further clini- show a significant benefit in the primary endpoints (change in ocular pain scores, total corneal staining and conjunctival hyperaemia) (Zhang et al., 2021).A sub-population of participants with dry eye syndrome secondary to Sjogren's syndrome showed improvements in pain scores, total corneal staining and quality of life, making it a potential candidate in this population (Zhang et al., 2021).Cosdosiran (intravitreal injection) targets caspase-2, an enzyme involved in apoptosis.
It was being developed as a neuroprotectant therapy for acute nonarteritic anterior ischaemic optic neuropathy, because apoptosis is considered to be the primary cause of retinal ganglion cell death, leading to progressive loss of vision (Solano et al., 2014).Despite promising results in Phase I, the Phase II/III study in patients with non-arteritic anterior ischaemic optic neuropathy was prematurely terminated as an interim analysis did not warrant continuation (Quark Pharmaceuticals, 2020).Bamosiran, targeting β 2 -adrenoceptors, was effective in reducing ocular pressure in animals and showed safety in Phase I trials (Martínez et al., 2014;Moreno-Montañés et al., 2014).Data from Phase II trials showed that only one of the tested doses (1.125%) was comparable to standard treatment with timolol (0.5%), with non-inferiority where baseline ocular pressure was >25 mmHg.Further results are awaited (Jayanetti et al., 2020).
Several therapies for age-related macular degeneration and diabetic macular oedema targeting the angiogenic cytokine, VEGF, have been studied in different clinical trials.Bevasiranib, administered as an intravitreal injection, was the first to reach Phase III studies, although development was discontinued as it was unlikely to meet its endpoint (Garba & Mousa, 2010).Sirna-027, a therapy that targets the VEGF receptor, was terminated in Phase II development after failing to meet its efficacy endpoint (Kaiser et al., 2010).PF-04523655, targeting RTP801 in patients with diabetic macular oedema, was terminated early in Phase II development, based on predetermined futility criteria and discontinuation rates (Nguyen et al., 2012)  Hepcidin, a liver peptide, plays a key role in iron homeostasis, deficiency of which is implicated in the hyperabsorption of iron in hereditary haemochromatosis, beta-thalassaemia and other iron-loading anaemias (Ganz, 2016).SLN124 aims to increase hepatic hepcidin synthesis by silencing the expression of its repressor transmembrane serine protease 6 (TMPRSS6) (Porter et al., 2021).Phase I trials showed that SLN124 reduced serum iron concentration and had satisfactory safety and tolerability (Porter et al., 2021).It is currently under evaluation in other Phase I trials among patients with alpha/beta-thalassaemia and myelodysplastic syndrome (Silence Therapeutics, 2020).

| Diseases of the kidney
Acute kidney injury (AKI) is an abrupt decrease in kidney function, which encompasses both injury (structural damage) and impairment (loss of function) (Makris & Spanou, 2016).Due to the accompanying fluid, electrolyte and acid-base imbalances, AKI carries a significant morbidity and mortality.AKI is predictable in some situations, such as cardiovascular surgery and renal transplantation, allowing the opportunity to instigate prophylactic treatment.p53 (DNA-binding transcription factor) activates genes regulating cell death on exposure to ischaemic stress (Ranjan & Iwakuma, 2016), so teprasiran, targeting p53, has been developed for this purpose.A naked siRNA, with minimal modification, is internalised and cleared by the kidney following systemic administration.In a Phase II trial among high-risk patients undergoing cardiac surgery, it significantly decreased the occurrence, severity and duration of early AKI (Thielmann et al., 2021).It also underwent evaluation for delayed graft failure prophylaxis among patients undergoing cadaveric donor renal transplantation, with teprasiran-treated patients having a significantly better estimated glomerular filtration rate (GFR) at Day 30 (Gallagher et al., 2017).It is currently in Phase III evaluation in those at high risk for AKI after cardiovascular surgery and among cadaveric renal transplant recipients.

| Disease of the liver
Liver fibrosis occurs as a result of excess deposition of extracellular matrix proteins, such as collagen, with resultant cirrhosis of the liver often requiring liver transplantation (Bataller & Brenner, 2005).Heat shock protein 47 (HSP47) is a collagen-specific molecule important for the accurate folding of procollagen in the endoplasmic reticulum and overexpressed in fibrotic diseases (Ruigrok et al., 2021

| Diseases of the lung
Idiopathic pulmonary fibrosis is an interstitial lung disease leading to chronic, progressive lung fibrosis (Barratt et al., 2018).As with its role in hepatic fibrosis, HSP47 is implicated here.Based on encouraging early signs of effectiveness in advanced liver fibrosis, a Phase II study of ND-L02-s0201 in idiopathic pulmonary fibrosis was initiated in 2018 (JUNIPER trial) (Bristol-Myers Squibb, 2017).Excellair, an inhaled therapy targeting Syk kinase in allergic asthma, showed promising symptomatic improvement in Phase I studies (Fujita et al., 2013).However, development of Excellair has since been discontinued in Phase II.(COX-2) (Zhou et al., 2017), and is currently in Phase II development.
Based on its mechanism of action, it is also being evaluated for in situ squamous cell carcinoma (Phase II), basal cell carcinoma (Phase II) and cholangiocarcinoma/hepatocellular carcinoma/liver metastases among patients with advanced solid tumours (Phase I) (Sirnaomics, 2021a(Sirnaomics, , 2021b(Sirnaomics, , 2021c)).RXI-109, targeting connective tissue growth factor, has been effective in decreasing the recurrence of scar formation after elective revision in Phase II trials (Pavco et al., 2015).BMT101 and OLX10010 also target connective tissue growth factor and are currently in Phase II trials for hypertrophic scars and keloids (Hugel Pharmaceuticals, 2021;Olix Pharmaceuticals, 2021).

| CHALLENGES AND LIMITATIONS
Although poised to transform treatment of some conditions, siRNAbased therapeutics face challenges that limit their full clinical potential.There are several pharmacological challenges to overcome to achieve a better pharmacokinetic/pharmacodynamic profile for human use.These include limitations in route of administration, sitespecific delivery, rapid renal elimination and inactivation by nucleases, poor vascular and cellular penetration, and challenges with endosomal escape.Also, immune activation and the potential for off-target effects create safety concerns.In addition, the use of higher doses to achieve sufficient target-site concentrations may increase the likelihood of non-specific off-target effects and toxicity.Already, though, what has been learnt about useful chemical modifications, and the development of suitable carriers, is helping to overcome these challenges.Constant innovation in the siRNA field continues to identify potential solutions for existing challenges, including chemical modifications helping to reduce dosage requirements (milligrams to micrograms) and increase biological half-life (from minutes to months) (Hu et al., 2020).However, it is important to appreciate that extensive chemical modification must be balanced against loss of efficacy and increasing toxicity.Furthermore, carrier molecules and their excipients can be responsible for adverse effects.Therefore, careful consideration needs to be given to balancing benefits and risks.Overall, the huge strides being made in bio-technological engineering will likely soon help siRNA therapeutics to realise their full potential.

| CONCLUSIONS AND FUTURE DIRECTION
Since the recognition and understanding of RNAi in the 1990s, efforts have been underway to exploit its potential as a novel  Alexander, Christopoulos et al., 2021;Alexander, Fabbro et al., 2021a, 2021b;Alexander, Kelly et al., 2021;Alexander, Mathie et al., 2021).

AUTHOR CONTRIBUTIONS
PR and DJW made substantial contributions to the conception, interpretation and presentation of information in the manuscript and were involved in drafting of the manuscript and revising it critically for important intellectual content.JWD and MA revised the manuscript critically for important intellectual content.

CONFLICTS OF INTEREST
K E Y W O R D S medication adherence, post-transcriptional gene silencing, RNA interference, siRNA, therapeutics 1 | INTRODUCTION Ever since the discovery of DNA by Swiss physician Friedrich

F
I G U R E 3 Pharmacological challenges in small interfering RNA (siRNA) therapeutics.(a) Routes of administration, (b) distribution (poor penetration of vascular endothelium and cell membrane, increased accumulation in sites with 'leaky' capillaries and poor endosomal escape), (c) metabolism and elimination (rapid renal clearance and degradation by nucleases) and (d) immune activation (toll-like receptor stimulation and cytokine production) and off-target effects (incomplete pairing).* indicates poor systemic bioavailability.HPTS, hypertrophic scars; i.l., intralesional; i.v.t., intravitreal; MΦ, macrophages therapeutics and many others in development.The exclusive expression and abundance of ASGPRs in hepatocytes and their rapid recycling contributes to enhance GalNAc-siRNA delivery.Binding of the GalNAc-siRNA conjugates to the ASGPR results in rapid internalisation via receptor-mediated endocytosis.The subsequent drop in endosomal pH causes the separation of the GalNAc-siRNA from ASGPR, enabling it to be rapidly recycled to the cell surface, facilitating further internalisation of GalNAc-siRNA conjugates(Prakash et al., 2014).The GalNAc conjugate is cleaved within the endosome by glycosidases.Their ease of synthesis and refinement, rapid absorption via the s.c.route, long half-life and lower toxicity are among the many reasons for their increasing use(Springer & Dowdy, 2018).The development of this technology has greatly advanced siRNA therapeutics, enabling its use for systemic delivery targeting proteins synthesised in the liver.Other similar ligands are in development to target pancreatic beta cells with glucagon-like peptide-1, skeletal/cardiac muscle with transferrin receptor protein-1 and cancer cell receptors with folate(Zhang et al., 2021).
mal outcome.Risks associated with patisiran include infusion-related reactions, which are minimised by premedication with corticosteroids and antihistamines, and reduced vitamin A levels (due to reduced transthyretin), which are responsive to supplementation (Urits et al., 2020).Other common adverse effects include arthralgia, dyspepsia, dyspnoea, increased risk of infection, muscle spasms, peripheral oedema and vertigo (Urits et al., 2020).Vutrisiran is another therapy for familial transthyretin amyloidosis currently in late-stage development (Phase III, HELIOS-A and HELIOS-B trials) with orphan drug designation by the US FDA (Fast Track designation) and the European Medicines Agency.It has a more convenient administration method (s.c.) with less frequent dosing (3-monthly) than patisiran.
ing liver fibrosis.A Phase I study among healthy volunteers evaluating the safety, tolerability and the effect of ARO-AAT on serum A1AT concentrations showed an average A1AT knockdown of 87%(Arrowhead Pharmaceuticals, 2020).Phase II trials are currently underway, evaluating ARO-AAT in patients with A1AT deficiency and those with A1AT deficiency-associated liver disease, with promising interim results (ArrowheadPharmaceuticals, 2020Pharmaceuticals, , 2021)).Belcesiran is a further therapy in Phase II development for A1AT deficiencyrelated liver disease, with evidence from Phase I showing a significant reduction in A1AT concentrations(Dicerna Pharmaceuticals, 2021).
the above studies led to the approval on inclisiran first by the European Medicines Agency (December 2020), followed by approval in the United Kingdom (August 2021) and the United States (December 2021).At present, inclisiran (284 mg, s.c.injection, Day 0, and at 3 months and 6 months thereafter) is indicated for the treatment of primary hypercholesterolaemia (heterozygous familial and non-familial) or mixed dyslipidaemia, in combination with a statin, or with a statin and other lipid-lowering therapies, or with other lipid-lowering therapies or alone if a statin is contraindicated or not tolerated.However, as per National Institute of Health and Care Excellence (NICE) guidelines, it is recommended in those with the above indications only if there is a history of any of the following cardiovascular events: acute coronary syndrome, coronary or other arterial revascularisation procedures, coronary heart disease, ischaemic stroke or peripheral arterial disease, and LDL-cholesterol concentrations are persistently 2.6 mmolÁL À1 or more, despite maximum tolerated lipidlowering therapy (National Institute of Health and Care ongoing ORION-4 study (n = 15,000; 5 year median follow-up), a Phase III randomised controlled trial among participants with preexisting AsCVD, evaluating primary clinical outcomes with inclisiran (300 mg) versus placebo therapy, will shed further light on this question (University of Oxford, 2022).Nonetheless, inclisiran remains the most promising siRNA therapeutic agent to date, due to its potential influence on a very prevalent indication associated with major adverse clinical outcomes.Lipoprotein(a) is a lipoprotein particle synthesised in the liver, which is strongly linked with AsCVD.It is a cholesterol-rich particle similar to LDL, together with two attached apolipoproteins (apo (a) and apo B-100)(Schmidt et al., 2016).Apo(a) is encoded by the LpA gene, and olpasiran reduces apo(A) production, thereby reducing lipoprotein(a) concentrations.Preliminary data from an ongoing Phase I study show a 71-97% decrease in plasma lipoprotein(a) concentration sustained for several months, with good tolerability(Koren et al., 2022).However, further evidence regarding efficacy and safety is required before concluding on clinical utility.PRO-040201 is a therapy aiming to reduce serum LDL cholesterol by targeting apolipoprotein B (Apo(b)).It showed promising results in a Phase I trial, with a significant decrease of serum LDL (16%) and Apo(b) (21%) (Arbutus Biopharma Corporation, 2010).However, the trial was prematurely stopped as the result of an unwanted immune response occurring in a few participants receiving the highest dose, highlighting challenges to siRNA development and the key need for pharmacovigilance.
cal development is awaited.Other anti-cancer therapies that were evaluated in human trials have been terminated, at different stages of development, due to safety concerns or lack of efficacy.These include CALAA-01 (Phase I) targeting ribonucleotide reductase, the first systemically delivered siRNA with a targeted delivery system going into clinical trials (Figure 1) (Zuckerman et al., 2014) and DCR-MYC (Phase II) targeting the c-MYC oncogene.7.3 | Other diseases 7.3.1 | Diseases of the eye Dry eye syndrome (loss of tear film, with resultant inflammation and neurosensory abnormalities, including ocular pain, itching and burning) (Shimazaki, 2018) is a condition in which stimulation of transient receptor potential cation channel subfamily V member 1 (TRPV1) mediates ocular pain transmission, fibrogenesis and the stress response.It is targeted by tivanisiran (topical eye drop) (Moreno-Montañés et al., 2018), using naked siRNA as a local therapy.After success in Phases I and II, a Phase III trial (HELIX) of daily treatment with tivanisiran in comparison with placebo (artificial tears) did not 7.3.2| Haematological and immune-mediated diseasesParoxysmal nocturnal haemoglobinuria, atypical haemolytic uraemic syndrome and immunoglobulin A (IgA) nephropathy are examples of rare life-threatening complement-mediated diseases(Noris & Remuzzi, 2013).Terminal complement component 5 (C5), synthesised mainly in the liver, plays an important role in their pathogenesis.Cemdisiran, which reduces C5 production, showed promising results in Phase I studies in healthy volunteers and those with Paroxysmal nocturnal haemoglobinuria, with rapid and sustained liver C5 suppression over >12 months after single and multiple doses, and was generally well tolerated(Badri et al., 2021).It is now in Phase II development among patients with IgA nephropathy and atypical Haemolytic uraemic syndrome.
7.3.6| Diseases of the skinConnective tissue growth factor is a downstream regulator of the TGFβ1 signalling pathway and involved in the pathogenesis of hypertrophic scars and keloid formation(Zhu et al., 2016), and several programmes are targeting this pathway.Cotsiranib uniquely combines two siRNAs, targeting the expression of TGFβ1 and cyclooxygenase-2 therapeutic modality, with the earliest clinical studies launched around 15-20 years ago.The ability to specifically target key proteins and the potential for improved adherence with infrequent administration offer siRNA therapy a distinct advantage over most existing therapeutic strategies.Initially developed siRNAs were limited to local applications (e.g., eyes and skin) due to technological challenges and pharmacological barriers of the internal human biological environment.Advances in molecular biotechnology have enabled the development of chemically modified siRNA therapies with novel delivery strategies to overcome these challenges.Further advances have been aided by the ready availability of siRNA reagent kits and bioinformatics software, and whole-genome mapping, which has enabled greater precision, while helping to reduce undesirable offtarget effects (Dominska & Dykxhoorn, 2010).As a result, in recent years, we have seen the approval of several novel systemic therapies, focused on liver proteins.Research is now moving from evaluation in rare genetic diseases to common chronic conditions, such as hyperlipidaemia and hypertension, therapies that will arguably lead to substantially greater numbers of patients being treated with siRNA therapy.However, challenges still remain in selecting the right protein target, achieving sufficient knockdown, and especially in developing siRNA therapies for non-liver targets and the penetration of special target sites such as the blood-brain barrier.With this background, the submission of a clinical trial authorisation application by Alnylam Pharmaceuticals for ALN-APP, targeting amyloid precursor protein for the treatment of cerebral amyloid angiopathy and Alzheimer's disease, is encouraging (Alnylam Pharmaceuticals, 2021).Assuming these barriers can be overcome, siRNA therapy looks to have the potential to offer new treatments for many challenging diseases over the next few decades.9.1 | Nomenclature of targets and ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (( DJW has received non-personal support for research and consultancy from AbbVie, Actelion, AstraZeneca, Idorsia, Johnson & Johnson and Novartis and funding for research in hypertension from the British Heart Foundation.The University of Edinburgh/British Heart Foundation Centre for Cardiovascular Science received funding from Alnylam Pharmaceuticals for Phase I trials with zilebesiran.PR is a coinvestigator in zilebesiran Phase I trials.