Therapeutic siRNA: state of the art

Abstract

RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion. It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality. After a two-decade journey from its discovery, two approvals of siRNA therapeutics, ONPATTRO^® (patisiran) and GIVLAARI™ (givosiran), have been achieved by Alnylam Pharmaceuticals. Reviewing the long-term pharmaceutical history of human beings, siRNA therapy currently has set up an extraordinary milestone, as it has already changed and will continue to change the treatment and management of human diseases. It can be administered quarterly, even twice-yearly, to achieve therapeutic effects, which is not the case for small molecules and antibodies. The drug development process was extremely hard, aiming to surmount complex obstacles, such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity, stability, specificity and potential off-target effects. In this review, the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed. All clinically explored and commercialized siRNA delivery platforms, including the GalNAc (N-acetylgalactosamine)-siRNA conjugate, and their fundamental design principles are thoroughly discussed. The latest progress in siRNA therapeutic development is also summarized. This review provides a comprehensive view and roadmap for general readers working in the field.

Fig. 1

Schematic illustrations of the working mechanisms of miRNA (a) and siRNA (b)

Fig. 2

Structures of chemical modifications and analogs used for siRNA and ASO decoration. According to the modification site in the nucleotide acid, these structures can be divided into three classes: phosphonate modification, ribose modification and base modification, which are marked in red, purple and blue, respectively. R = H or OH, for RNA or DNA, respectively. (S)-cEt-BNA (S)-constrained ethyl bicyclic nucleic acid, PMO phosphorodiamidate morpholino oligomer

Fig. 3

Representative designs for the chemical modification of siRNA. The sequences and modification details for ONPATTRO^®, QPI-1007, GIVLAARI™ and inclisiran are included. The representative siRNA modification patterns developed by Alnylam (STC, ESC, advanced ESC and ESC+) and arrowhead (AD1-3 and AD5) are shown. Dicerna developed four GalNAc moieties that can be positioned at the unpaired G–A–A–A nucleotides of the DsiRNA structure. 2′-OMe 2′-methoxy, 2′-F 2′-fluoro, GNA glycol nucleic acid, UNA unlocked nucleic acid, SS sense strand, AS antisense strand

Fig. 4

Preclinical and clinical performance of siRNAs with comprehensive modification chemistries. a Sense and antisense strand optimization by using transthyretin (Ttr)-targeting siRNAs and the combinatorial designs explored across 10 siRNAs. The effect relative to the parent is described as the model-adjusted mean difference in the activity of the design variant (DV) compared to the parent. b–d The liver exposure, RISC loading, and silencing activities of the parent, DV18, and fully 2′-OMe conjugates. Data are shown as the mean ± SD. e Gene-silencing duration of antithrombin-targeted siRNAs modified with the designs of patent and DV22 evaluated in Cynomolgus monkeys (n = 3 per group). Asterisks indicate that a significant difference was observed between the parent and DV22. Error is shown as the SD. f C5 knockdown profile after applying a single dose of Cemdisiran that employs ESC modification in a phase 1/2 clinical study. Error is represented as the SEM. a–e Copyright of Elsevier Inc., 2018. f Copyright of Alnylam Pharmaceuticals, 2016

Fig. 5

Schemes of representative clinically studied siRNA formulations and their pharmacodynamic performance. a–g Schemes of a lipid nanoparticles, b DPC™ or EX-1™, c TRiM™, d GalNAc–siRNA conjugates, e LODER™, f iExososme and g GalXC™. h Efficacy of ONPATTRO^® (a liposome formulation) in healthy volunteers, in which siRNA targeting PCSK9 is formulated in lipid nanoparticles. Copyright Massachusetts Medical Society, 2013. i Levels of urinary aminolevulinic acid (ALA) and porphobilinogen (PBG) after once-monthly and once quarterly doses of GIVLAARI™ in acute hepatic porphyria (AHP) patients. Copyright Alnylam Pharmaceuticals, 2019. j Reduction of plasma PCSK9 after a single increasing dose of inclisiran. Copyright Massachusetts Medical Society, 2017. k Serum HBsAg reduction in hepatitis B patients who received the treatment of a single dose of ARC-520 (1–4 mg/kg) (formulated with DPC2.0) on a background of daily oral NUCs. PBO, patients on NUC therapy receiving placebo injection. NUCs nucleos(t)ide viral replication inhibitors. The error is shown as the SEM. Copyright American Association for the Advancement of Science, 2017. l Antitumor effect of siG12D-LODER™ combined with chemotherapy in locally advanced inoperable pancreatic cancer in a patient. A computed tomography (CT) scan was performed before and after (nine months later) the administration of siG12D-LODER™. The tumor measured 35.42 and 26.16 mm in the longest diameter, respectively. m PANC-1 tumor growth in animals receiving iExosomes or other control formulations.N = 3 mice per group. Copyright Macmillan Publishers Limited, part of Springer Nature, 2017. n The 24-h Uox (urinary oxalate) values of healthy volunteers and patients treated with a single dose of Nedosiran (DCR-PHXC, a GalXC™ therapeutic) (3 and 6 mg/kg). Copyright Dicerna Pharmaceuticals, 2020. o Reduction of serum AAT in volunteers receiving a single dose of ARO-AAT (a TRiM™ therapeutic, 35–300 mg). The error bars show the SEM. Copyright Arrowhead Pharmaceuticals, 2019

Fig. 6

siRNA delivery platforms that have been evaluated preclinically and clinically. Varieties of lipids or lipidoids, siRNA conjugates, peptides, polymers, exosomes, dendrimers, etc. have been explored and employed for siRNA therapeutic development by biotech companies or institutes. The chemical structures of the key component(s) of the discussed delivery platforms, including Dlin-DMA, Dlin-MC3-DMA, C12-200, cKK-E12, GalNAc–siRNA conjugates, MLP-based DPC2.0 (EX-1), PNP, PEI, PLGA-based LODER, PTMS, GDDC4, PAsp(DET), cyclodextrin-based RONDEL™ and dendrimer generation 3 are shown. DLin-DMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLin-MC3-DMA (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate, DPC Dynamic PolyConjugates, MLP membrane-lytic peptide, CDM carboxylated dimethyl maleic acid, PEG polyethylene glycol, NAG N-acetylgalactosamine, PNP polypeptide nanoparticle, PEI poly(ethyleneimine), LODER LOcal Drug EluteR, PLGA poly(lactic-co-glycolic) acid, PTMS PEG-PTTMA-P(GMA-S-DMA) poly(ethylene glycol)-co-poly[(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl))] ethane methacrylate-co-poly(dimethylamino glycidyl methacrylate), GDDC4 PG-P(DPAx-co-DMAEMAy)-PCB, where PG is guanidinated poly(aminoethyl methacrylate) PCB is poly(carboxybetaine) and P(DPAx-co-DMAEMAy) is poly(dimethylaminoethyl methacrylate-co-diisopropylethyl methacrylate), PEG-PAsp(DET) polyethylene glycol-b-poly(N′-(N-(2-aminoethyl)-2-aminoethyl) aspartamide), PBAVE polymer composed of butyl and amino vinyl ether, RONDEL™ RNAi/oligonucleotide nanoparticle delivery

Fig. 7

Tissues targeted by siRNA and miRNA therapeutics currently being investigated at the clinical stage. The corresponding therapeutic names are shown beside the tissues