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.34 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.38 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.264 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.238N = 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).265 Copyright Dicerna Pharmaceuticals, 2020. o Reduction of serum AAT in volunteers receiving a single dose of ARO-AAT (a TRiM™ therapeutic, 35–300 mg).266 The error bars show the SEM. Copyright Arrowhead Pharmaceuticals, 2019
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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
The relatively high molecular weight (~13–16 kD) and net negative charge prevent artificial siRNA from crossing the cell membrane. Hence, we attempted to determine whether any cell can internalize siRNA without a carrier, leading to the conclusion that naked siRNA can only be taken up by a few cell types, e.g., retinal ganglion cells (RGCs) and neurons. In addition, researchers have also been devoted to identifying various carriers to achieve efficient transmembrane delivery. As a result, cationic cell-penetrating peptides (CPPs) become a choice at the early stage. CPPs, typically tailed with arginine-rich sequences, can form bidentate bonds by the interaction between the guanidinium groups and the negative phosphates, sulfates and carboxylates on the cell surface.113 This interaction causes membrane pore formation, leading to the cellular uptake of siRNA. As another strategy, the negative charge of siRNA can be neutralized by positively charged lipids or polymers, conferring siRNA the ability to more readily bind to the membrane and become easily internalized via adsorptive pinocytosis.
Finally, siRNAs must effectively escape from endosomes and lysosomes to the cytoplasm, where antisense strands of siRNAs need to be loaded into RISCs. Many delivery systems employ a pH-sensitive unit to respond to pH changes in the endosome and lysosome,114,115 where they will absorb H+, presenting a positive charge on the surface. Then, the osmotic pressure will increase in the endosome or lysosome, resulting in the internal flow of Cl− and H2O. Finally, these changes may cause membrane disruption and siRNA release to the cytoplasm. This so-called 'proton sponge effect' or 'colloid osmotic pressure effect' results in membrane destabilization116,117,118,119 or membrane swelling,120,121 respectively. However, the underlying mechanism of endosomal release remains to be further illuminated. Only 1–2% of internalized LNP-loaded siRNAs were released into the cytoplasm, and this only occurred within a limited time frame after internalization.122,123 Hence, further understanding the escape mechanism and how to enhance the escape efficiency is of great importance for siRNA drug development. Recently, Wang and colleagues124 developed novel endoplasmic reticulum (ER) membrane-modified hybrid nanoplexes (EhCv/siRNA NPs). Compared with unmodified nanoplexes, they showed much higher RNAi activity in vitro and in vivo. The functional proteins on the ER membrane have an important role in intracellular trafficking of siRNA, helping siRNA reach the cytoplasm through the endosome–Golgi–ER pathway instead of the endosome–lysosome pathway, thereby avoiding the lysosomal degradation of siRNA. In addition, electroporation enables siRNA to directly cross the cell membrane, which also constitutes an effective approach to circumvent the endosomal escape issue.125,126,127,128,129,130,131
To date, two RNAi therapeutics, ONPATTRO® and GIVLAARI™, have been approved for commercial application, and two siRNAs, lumasiran (ALN-GO1) and inclisiran, have been submitted for new drug application (NDA) to the FDA. Seven siRNAs are undergoing phase 3 clinical studies, and more candidates are in the early developmental stage. Various delivery systems, e.g., LNPs, DPC™, TRiM™, GalNAc–siRNA conjugates, LODER™ polymers, exosomes and polypeptide nanoparticles (PNPs), have been explored (Figs. 5, 6). Based on these systems, plentiful drug pipelines have been established. We discuss this information in the following sections.
Naked siRNA-based therapeutics
Naked siRNA can be defined as a system that contains no delivery system that is associated with siRNA either covalently or noncovalently.40 Because no protective effect is offered by the delivery vehicle, siRNA should be modified carefully to be resistant to enzyme degradation and extend its circulation time in the bloodstream. As siRNA is naturally filtered to the kidney, it can be used to silence kidney-expressed genes and treat renal diseases.132 Alternatively, a feasible strategy for naked siRNA drug development is the local injection of this molecule into specific organs that are relatively closed off and contain few nucleases, e.g., the eye.
QPI-1002 (I5NP)132,133 and QPI-100736,134 were developed by Quark Pharmaceuticals (Table 1). QPI-1002 is a 19-base pair, 2′-O-methylated, blunt and naked siRNA135 targeting p53 for treating acute kidney injury (phase 2) and delayed graft function (phase 3). The sequence of the sense strand is 5′-GAAGAAAATTTCCGCAAAA-3′.135 Because of their small sizes, most siRNAs accumulate in the kidney after being intravenously administered, achieving concentrations 40 times greater than those in other organs, followed by rapid entry into proximal tubule cells. An in vitro study determined that QPI-1002 elicited near-complete p53 mRNA elimination at a concentration of ~1 nM, with an IC50 of ~0.23 nM. This molecule also effectively inhibited p53 protein expression, even at a transfection concentration of 0.5 nM. Furthermore, bilateral renal-clamp studies were conducted to identify the effect of siRNA on the preservation of kidney function. Compared with PBS-treated animals, siRNA-treated animals showed ischemia with decreased serum creatinine levels from 3.7 mg/dL to 1.9 mg/dL. Adverse effects (AEs) were found only when the dose was higher than 1000 mg/kg in nonhuman primates and higher than 800 mg/kg in rats.133 A phase 1 clinical study (NCT00554359) of QPI-1002 revealed an ideal safety profile.36,132
Preclinical investigations have validated the potent gene silencing of QPI-1007 in cells and have demonstrated its curative effects on optic nerve-damaged animal models. Data136 have shown that QPI-1007 triggered over 80% gene suppression in HeLa cells and exhibited an IC50 of ~0.8 nM against human caspase-2 mRNA. In animal models, eyes treated with siRNA showed a dose-dependent increase in RGC survival from 5 to 20 μg. In particular, in animals dosed with 20 and 35 μg QPI-1007, RGC densities in the injured eye recovered to close to healthy levels (~98%). Furthermore, a phase 1/2a clinical trial (NCT01064505) reported that most common AEs, such as conjunctival hemorrhage, conjunctival edema, eye irritation, and eye pain, were typical of intravitreal injection, and no serious adverse effects (SAEs) were observed. By comparing the best-corrected visual acuity (BCVA) following a single dose of QPI-1007 with natural history historical controls from the Ischemic Optic Neuropathy Decompression Trial (IONDT, 1998), which is the most comprehensive survey of the disease and serves as the gold standard in the field, it was concluded that QPI-1007 significantly protected the optic nerve as the proportions of subjects who lost 3 lines of visual acuity were markedly decreased. The proportions were 0%, 0% and 3.6% at months 3, 6 and 12, respectively, following a single dose of QPI-1007. In contrast, the proportions for natural history controls were 9%, 15% and 16% at months 3, 6 and 12, respectively. Currently, an international multi-centered phase 2b/3 clinical trial is being conducted in the United States, China, Israel and other countries and regions.
ALN-RSV01 (asvasiran sodium), a naked siRNA that targets the respiratory syncytial virus (RSV) nucleocapsid (N) gene and inhibits viral replication, was explored for the potential treatment or prevention of RSV infection. The sequences of ALN-RSV01 are as follows: sense strand (5′–3′): GGCUCUUAGCAAAGUCAAGdTdT; and antisense strand (5′–3′): CUUGACUUUGCUAAGAGCCdTdT.137 ALN-RSV01 was administered via inhalation with an investigational electronic nebulizer. Although the clinical study of ALN-RSV01 has been terminated, clinical data indicate that it is well tolerated in vivo138 and may have beneficial effects on long-term allograft function in lung transplant patients infected with RSV (NCT00658086).139,140 Another clinical result reported in 2016 showed that ALN-RSV01 may help prevent bronchiolitis obliterans syndrome (BOS) after lung syncytial virus infection in lung transplant recipients.141
According to the different charge properties of siRNA-binding lipids under neutral conditions, LNPs may be divided into several classes: ionizable LNPs, cationic LNPs and neutral LNPs. Ionizable LNPs are nearly uncharged during circulation but become protonated in a low pH environment, e.g., in the endosomes and lysosomes. These molecules may interact with apolipoprotein E3 (ApoE3), which transports lipids to hepatocytes. Cationic LNPs exhibit a constitutive positive charge in blood circulation and in endosomes or lysosomes. Cationic LNPs also primarily accumulate in hepatocytes; however, this is independent of the ApoE3 interaction. Electrostatic-induced nonspecific binding with plasma protein and relatively higher immunogenicity may cause cationic LNPs to be less efficacious146 and more toxic in vivo than ionizable LNPs;147 as a consequence, most pharmaceutical firms and institutes have made efforts to develop novel ionizable lipids to achieve efficient hepatocyte-targeted delivery of siRNA with minimal side effects. For example, research teams from Arbutus Biopharma, Alnylam Pharmaceuticals and the Massachusetts Institute of Technology continue to establish lipid-based delivery systems. Three generations of lipid delivery systems have been developed by Arbutus and Alnylam (Fig. 6). They employed DLin-DMA (1,2-dilinoleyloxy-3-dimethylaminopropane),148 DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate)107 and L319 (di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino) butanoyl) oxy) heptadecanedioate)149 as the key lipids and exhibited median effective doses (ED50s) of 1, 0.005 and <0.01 mg/kg, respectively, when loaded with anti-factor VII siRNA and injected via the tail vein. DLin-DMA was used to develop TKM-080301,150 ALN-VSP151 and ALN-TTR01,34 whereas DLin-MC3-DMA was employed in ALN-TTR0234 and ALN-PCS152 development. L319 was derived from DLin-MC3-DMA by incorporating a biocleavable ester linkage within hydrophobic alkyl chains.149 It is readily degraded in vivo, and the metabolite of L319 is a potential substrate for the β-oxidation pathway of fatty acids. L319 is rapidly eliminated from intracellular compartments (plasma, liver and spleen) and is excreted in vivo, which suggests high tolerance of L319-LNPs throughout the body.
Anderson and colleagues153 synthesized and screened thousands of lipidoids through a combinatorial chemistry strategy. Consequently, three generations of lipidoid materials were selected from these libraries: 98N12-5(I)-based,154 C12-200-based155 and cKK-E12-based153 LNPs (Fig. 6). In particular, the ED50 of LNPs containing cKK-E12 is as low as 0.002 mg/kg, which is the lowest ED50 in the liver for all reported LNPs currently.153 In addition, many firms, including Dicerna Pharmaceuticals (Dicerna),156 Silence Therapeutics,157 Sirna Therapeutics,158 Nitto Denko Corporation,159 Life Technologies160 and Suzhou Ribo Life Science,49,142 have explored proprietary delivery technologies for preclinical and clinical investigations.