Small Interfering RNAs (siRNAs)

Small (or short) interfering RNA (siRNA) is a double-stranded RNA molecule that affects gene expression by silencing genes.[2] They are naturally occurring double-stranded RNA molecules typically 20-25 nucleotides in length but can also be synthetically manufactured using modified nucleotides.[2] One of the most important advances in biology has been the discovery that siRNA (small interfering RNA) is able to regulate the expression of genes, by a phenomenon known as RNAi (RNA interference).[1]

Mechanism of Action

siRNA plays a major role in post-transcriptional gene silencing (PTGS) by binding to a homologous messenger RNA (mRNA) and triggering its destruction, thereby preventing it from being translated into protein.[2] Upon entry, the dsRNA is recognized and processed by Dicer, which cleaves the dsRNA into smaller fragments, typically around 20 nucleotides in length. These fragments are then loaded onto a protein complex known as the RISC.[3]

The strand that is retained as a functional component of the RNA Induced Silencing Complex (RISC) is referred to as the guide strand, whereas the other strand, which will be rapidly degraded by exonucleases, is known as the passenger strand. The guide strand recruits the RISC to the surface of mRNAs that are homologous to the siRNA sequence.[5] siRNA recognition of the target mRNA is conferred by the "seed region", a six nucleotide stretch corresponding to positions 2-7 on the antisense siRNA strand. After the siRNA seed region anneals, the catalytic RNase H domain of Argonaute then subjects perfectly complementary mRNA sequences 10 nucleotides from the 5' end of the incorporated siRNA strand to nucleolytic degradation, resulting in the translational inhibition of the target mRNA.[5]

Biogenesis and Processing

The cascade leading to the generation of mature siRNA begins with transcription by RNA polymerase II (in animals), RNA polymerase III (from a shRNA template), or RNA polymerase IV (in plants), forming double stranded RNA (dsRNA). These dsRNA are recognized by the RNA binding domain (RBD) of the Dicer complex, which contains a pair of RNaseIII type endonuclease catalytic domains. The dimeric Dicer complex then cleaves the dsRNA into ~21-28 nucleotide siRNA duplexes containing 2-nucleotide 3' overhangs with 5' phosphate and 3' hydroxyl termini, which are bound by the Dicer PAZ (Piwi Argonaute Zwille) domain.[5]

Their structure has hydroxylated 3' and phosphorylated 5' ends. siRNA production is catalyzed by an enzyme known as the Dicer enzyme.[6] siRNAs have very tight target specificity as they cleave the mRNA before translation, compared to the similar miRNA which silences genes by repressing translation. siRNA has 100% complementarity to its target mRNA.[6]

Historical Development

Although the broader phenomenon of RNA interference (RNAi) was first elucidated by Andrew Fire and Craig C. Mello in C. elegans in 1998, discovery of siRNA as a specific endogenous effector molecule capable of RNA interference is generally credited to David Baulcombe and Andrew Hamilton, who reported siRNA as a novel small antisense RNA underlying a posttrascriptional gene silencing (PTSG) mechanism in plants.[5] Another major milestone, which highlighted the potential applications of siRNA, was the 2001 paper by Thomas Tuschl and colleagues demonstrating the capability of synthetic siRNAs to mediate RNA interference in mammalian cells. This achievement led to the widespread use of synthetic siRNAs as a laboratory tool to selectively knock-down the activity of specific genes.[5]

Delivery Systems

Lipid Nanoparticles (LNPs)

Lipid nanoparticles (LNPs) are chemically synthesized multicomponent lipid formulations (~100 nm in size) encapsulating siRNA for delivery to the target tissue.[21] The prototypical LNP is a four-lipid quaternary system—Ionizable lipid, phospholipid, cholesterol and PEGylated lipid—but the combinatorial chemical space encompasses >105 possible formulations.[23] Overall, the LNP delivery system is currently one of the most effective siRNA delivery methods and is mainly used for intravenous administration. During the transport of LNP-encapsulated siRNA into the body, LNP first fuses with the lipid bilayer of the cell membrane and then releases siRNA into the cell, allowing systemic siRNA administration.[22]

GalNAc Conjugates

GalNAc, or N-acetylgalactosamine, is a sugar molecule that can recognize and bind to a cell surface protein, the asialoglycoprotein receptor (ASGPR), which is abundantly expressed on liver cells (hepatocytes). The binding affinity to the receptor increases exponentially if several GalNAc units are combined into a multivalent ligand. Our GalNAc-conjugated siRNA are trivalent, meaning that three GalNAc molecules are clustered and conjugated to one siRNA molecule.[21] GalNAc conjugates are minimal particles that forgo complex architectures by directly attaching a tri-valent GalNAc moiety to the oligonucleotide, thus achieving highly specific liver targeting via the asialoglycoprotein receptor.[23]

Direct comparison studies show that LNP delivery provides more rapid onset of action with shorter duration, while GalNAc conjugates exhibit delayed onset but prolonged effect due to slow release from endosomal compartments.[27] The predominance of GalNAc conjugates reflects their advantages for liver targeting: simplified synthesis, defined chemical composition, subcutaneous administration, and reduced immunogenicity compared to complex LNP formulations.[27]

Clinical Applications and Approved Drugs

To date, 6 agents (patisiran, givosiran, lumasiran, inclisiran, nedosiran, and vutisiran) are FDA-approved for managing adult patients with hATTR, AHP, reducing LDL-C in subjects with HeFH or ASCVD, and PH1 in adults and pediatric patients.[11] Patisiran, branded as ONPATTRO by Alnylam Pharmaceuticals, is the first commercialized siRNA therapeutic drug. Approved by the FDA on August 10, 2018, it treats peripheral nerve disease secondary to hereditary transthyretin-mediated amyloidosis (hATTR).[12]

Lumasiran, branded by Alnylam Pharmaceuticals as OXLUMO, was the third (and most recent) FDA-approved siRNA therapeutic. On November 23, 2020, it became the first FDA-approved treatment for the orphan disease primary hyperoxaluria type 1 (PH1).[12] Since then, additional siRNA drugs have been approved: givosiran for acute hepatic porphyria, lumasiran for primary hyperoxaluria type 1, and inclisiran for hypercholesterolemia management.[16]

Pharmacokinetics and Safety

Clinical ADA incidence rates are as follows: patisiran (3.6%), givosiran (0.9%), lumasiran (6%), inclisiran (1.7%), vutrisiran (2.5%), and nedosiran (0%). Furthermore, ADA formation has not affected the pharmacokinetics (PK), pharmacodynamics (PD), efficacy, or safety of these drugs.[14] With the exception of nedosiran, the elimination half-life of the other siRNAs is under 10 hours, significantly shorter than the dosing interval. This is a typical feature of siRNA drugs—dissociation between PK and PD.[14]

Therapeutic Advantages

siRNA therapeutics have several distinct advantages over traditional pharmaceutical drugs. RNAi is an endogenous biological process, so almost all genes can be potently suppressed by siRNA. The identification of highly selective and inhibitory sequences is much faster than the discovery of new chemicals, and it is relatively easy to synthesize and manufacture siRNA on a large scale.[1]

siRNAs of 20 nucleotides in length can recognize any target gene with high specificity and minimal off-target effects due to the base complement pairing recognition mechanism. Second, the exceptional safety profile. siRNAs exert their post-transcriptional gene silencing effects exclusively in the cytoplasm, preventing nuclear entry and genome integration, thus minimizing the risk of host gene mutations. Third, the remarkable efficiency.[10]

Challenges and Limitations

However, many challenges, including rapid degradation, poor cellular uptake, and off-target effects, need to be addressed in order to carry these molecules into clinical trials.[1] However, there are challenges associated with the use of siRNA. For example, sometimes cleaving is not achieved due to mismatches between the siRNA and areas of the target mRNA near the cleaving site. There are other nonspecific effects when using siRNA. RNAi intersects with other pathways, leading to the occasional triggering of these nonspecific effects.[6]

Besides, siRNA delivery to the target site without toxicity is a big challenge for researchers, and naked-siRNA delivery possesses several challenges, including membrane impermeability, enzymatic degradation, mononuclear phagocyte system (MPS) entrapment, fast renal excretion, endosomal escape, and off-target effects.[18]

Future Perspectives

It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality.[7] As noted in Figure 3, the most-explored targets include cancer and cardiovascular disease, but there are additional areas of interest: Cancer: Preclinical studies and early-phase clinical trials have explored targets like KRAS, VEGF, EGFR, HER2, and c-MYC in various cancers. These targets are key to tumor growth, angiogenesis and immune evasion. Early findings suggest that when these drivers are downregulated, there is a notable effect on tumor regression and metastasis.[16]

"Although only three siRNA therapeutics have been approved by the FDA to date, more are sure to follow in the coming years," the authors wrote. They highlighted the benefits of siRNAs over other novel therapeutic classes. For instance, siRNAs are relatively less expensive to synthesize and manufacture, and they may be administered as infrequently as biannually, as well as have the potential to be self-administrable.[20]