Carbon GAmes -A

G-A-A

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

In addition, 2′-C, 4′-C, and even the whole sugar ring can also undergo modifications, resulting in molecules including UNAs, LNAs, GNAs, (S)-cEt-BNAs, tricyclo-DNA (tcDNA) and phosphorodiamidate morpholino oligomers (PMOs) (Fig. 2). UNA, with higher flexibility and thermal destabilization than the unmodified product due to unconnected 2′ and 3′ carbons, can block the entry of passenger strands and promote the RISC loading of the guide strand by introducing chemical asymmetry into duplex siRNAs. Similarly, glycol nucleic acid (GNA),31 another thermally destabilizing nucleotide, can be used to erase off-target effect-induced hepatotoxicity by including it in the seed region of the siRNA guide strand. LNA is a bicyclic structure that contains a bridge between the 2′ oxygen and the 4′ carbon. It "locks" the ribose into its preferred C3′-endo conformation and significantly increases the affinity of base pairing.40 Data have shown that the incorporation of LNAs increases the DNA melting temperature up to 8 °C per LNA.73 Given the successes of LNAs, many more bicyclic and even tricyclic analogs, including ethyl-bridged nucleic acids (ENAs),74 constrained ethyl (cEt) nucleic acids75 and tricyclo-DNA,76 have been engineered and incorporated into RNA or DNA strands in succession. Moreover, PMOs77 do not look like classic nucleotides, as the ribose subunit has been substituted with a morpholine subunit. PMOs are uncharged at physiological pH and are not substrates of RNase H. Therefore, they are mainly used to block RNA splicing or translation.78

Base modification

Base replacement is of great benefit to nucleic acid-based drug development. For instance, the substitution of pseudouridine,79 2-thiouridine,80 N6-methyladenosine,80 5-methylcytidine81 (Fig. 2) or other base analogs of uridine and cytidine residues can reduce innate immune recognition while making ASOs more resistant to nucleases. However, the artificial base substitution of ASOs, similar to siRNA, is basically at the stage of research and development. Pharmaceutical corporations still hold a prudent attitude toward these molecules. Instead, these companies prefer to use naturally occurring base structures, e.g., 5mC and 6 mA, to modify certain base(s), probably because of concerns about the safety of the metabolized unnatural residues that potentially might be incorporated into the genome.

N-ethylpiperidine triazole-modified adenosine analogs with Hoogsteen or Watson–Crick (WC) facial localization have also been used to modify siRNA,82,83,84,85 which can disrupt nucleotide/TLR8 interactions and therefore reduce the immunogenicity of siRNA. N-ethylpiperidine 7-EAA triazole (7-EAA, 7-ethynyl-8-aza-7-deazaadenosine) can pair well with uridine and form an A-form helix structure. This molecule will be recognized as adenosine by Avian myeloblastosis virus reverse transcriptase in RNA strands.83

In addition, 6′-phenylpyrrolocytosine (PhpC) is a cytosine mimic showing excellent base pairing fidelity, thermal stability and high fluorescence.86 PhpC-containing siRNA shows gene-silencing activity comparable to that of the parent molecule, and its fluorescent properties make it useful for fluorescence-based detection or monitoring or for exploring the cellular uptake and trafficking of siRNA in cells.

The internal uridine substitution of 2,4-difluorotolylribonucleoside (rF) is well tolerated by siRNA activity.87 The base pairing between rF and adenosine (A) is relatively unstable compared to the uridine–adenosine pair because the fluorine atom is more hydrophobic than uridine and cannot serve as a hydrogen bond acceptor.

Zhang and colleagues88 reported that a 5-nitroindole modification at position 15 of the siRNA passenger strand greatly reduced the activity of the passenger strand, whereas the effectiveness of the siRNA guide strand was barely affected. This modification provided a practical strategy to reduce the passenger strand-mediated off-target effects.

siRNAs containing 5-fluoro-2′-

Moreover, many other less common base analogs have also been used in siRNA modifications. Their application helps researchers better understand the mechanism of gene silencing and helps develop new methods to mitigate off-target effects caused by harmful protein binding or undesired targeting of mRNA.90

Modification patterns used in clinical studies or for commercial siRNA

Early siRNA therapeutic programs typically employ partial or slight modifications by using 2′-OMe, 2′-F or PS,91,92 e.g., alternative 2′-OMe modifications were placed in the antisense strand of QPI-1007, whereas just one L-DNA was used in the sense strand. After making great efforts to understand the effects of various modifications on siRNA activity, specificity and toxicity, researchers have realized that heavy modification of siRNA, even full-length embellishment, probably do not affect siRNA activity. Furthermore, these judicious modification strategies can significantly enhance siRNA stability or biocompatibility. As a result, several different, interrelated modification patterns have been proposed and validated preclinically and clinically.

Standard template chemistry (STC) (Fig. 3) is a universal modification pattern established by Alnylam Pharmaceuticals. When the lengths of sense and antisense siRNA are 21 and 23 nt, respectively (referred to as "S21nt" and "AS23nt", respectively, in the following description), 2 PS linkages are placed at the 3′ terminus of the antisense strand, three consecutive 2′-F moieties are incorporated at positions 9, 10 and 11 of the sense strand from the 5′-end, and consecutive 2′-OMe modifications are placed at positions 11, 12 and 13 of the antisense strand from the 5′-end. Moreover, alternative 2′-OMe and 2′-F moieties are employed at other positions in both strands. It is worth mentioning that 2′-OMe and 2′-F are complementarily used for all positions of both strands of siRNA (Fig. 3). It is considered that placing three consecutive 2′-F moieties, instead of larger groups, at the middle of the sense strand aims to facilitate the cleavage and removal of the sense strand in RISC, as release of the sense strand is the prerequisite of RISC activation. PS linkages and OMe positioned at the terminals are used to enhance the capability of siRNA to resist degrading enzyme attack.

The STC design (e.g., as used in revusiran) has been proven to remarkably enhance siRNA stability and affinity without compromising intrinsic RNAi activity.93 ALN-AT3, another STC-modified RNAi therapeutic, also showed potent and durable gene silencing after single subcutaneous (s.c.) administration.94 However, the clinical investigation of revusiran was discontinued due to unbalanced death between revusiran- and placebo-treated groups. Although the detailed investigation report by Alnylam illustrated that the deaths of revusiran-treated patients were not correlated with the test drug, there are still strong concerns regarding STC-modified siRNA therapeutics. Hence, the next generation of modification patterns, called enhanced stabilization chemistry (ESC) (Fig. 3), was proposed. This design includes four more PS linkages at the 5′-end of the antisense strand and the 3′-end of the sense strand. Another important change was the reduction in 2′-F substitutions, probably because heavy use of 2′-F may magnify toxicity.95 These changes markedly enhanced siRNA potency and duration compared with the changes inherent to the STC design.9,96,97 This advancement allowed us to achieve the desired pharmacodynamic effect at a significantly lower dose, with a markedly reduced dosing frequency.98 Cemdisiran (ALN-CC5) achieved potent gene silencing as long as more than 1 year after receipt of a single dose of the testing therapeutic in a phase 1/2 clinical study (Fig. 4f), representing an unprecedented milestone in pharmaceutical history. Moreover, the newly approved GIVLAARI™ is an siRNA therapeutic based on the ESC design (Fig. 3).99

In addition, Alnylam comprehensively optimized the modification design variants (DVs) of the ESC strategy across multiple siRNAs with an iterative screening approach to further enhance stability without compromising intrinsic RNAi activity. As a result, advanced ESC designs, e.g., DV18 and DV22, were proposed (Figs. 3 and 4a–e).96 Compared to the former scheme, the advanced strategy maintains six PS linkages at three strand terminals; however, this modification scheme significantly reduces the proportion of 2′-F moieties. Only 10 and 8 2′-F substitutions are used in two strands for the advanced ESC designs of DV18 and DV22, respectively. For DV18, the 2′-F modifications are positioned at sites 7, 9, 10 and 11 in the sense strand and sites 2, 6, 8, 9, 14 and 16 in the antisense strand (all from the 5′-ends of the strands). Compared with DV18, DV22 has the 2′-F modifications at 8 and 9 in the antisense strand further replaced with 2′-OMe (Figs. 3 and 4a). Both the DV18 and DV22 designs can achieve significantly higher liver exposure and RISC loading and more potent and durable gene silencing than the parent design in preclinical species, including nonhuman primates (Fig. 4b–e). The underlying mechanisms were not fully unveiled in Alnylam's literature. However, Zheng et al.100 demonstrated that modification of the 14th position of the siRNA guide strand could eliminate its gene-silencing activity by reducing RISC loading and target degradation, and the larger the modification group used was, the higher the reduction efficiency. Song et al.28 further proved that decorations at positions 9 and 10 (for siRNA with a 19 nt/19 nt structure) markedly reduced the activity of the unmodified strand of siRNA without disturbing the potency of the modified strand. In light of this, the specificity and activity of siRNAs could be improved by introducing 2′-MOE at the cleavage site of the siRNA. These observations are in line with the rationales of advanced ESC designs.

Furthermore, investigators from Alnylam have disclosed that the hepatotoxicity of GalNAc (N-acetylgalactosamine)-siRNA conjugates is attributed to off-target gene silencing mediated by miRNA-like recognition between siRNA and a mistargeted RNA.31 Researchers have demonstrated that disorganizing the nucleotides of the seed region without changing the 2′-OMe, 2′-F or PS content or placing a GNA at position 7 of the siRNA antisense strand can markedly alleviate off-target effects and mitigate hepatotoxicity because these modifications affect the binding of siRNA with undesired target mRNA in a seed region-specific manner. The employment of GNA in the siRNA seed region is the main technical characteristic for the ESC+ design compared to advanced ESC designs (Fig. 3). Several investigational siRNA therapeutics of Alnylam, e.g., ALN-AAT02, ALN-HBV02, and ALN-AGT, utilize the ESC+ design as the modification pattern.

In addition to Alnylam, Arrowhead Pharmaceuticals (Arrowhead) proposed a series of new modification designs (Fig. 3) by combining the modification patterns (e.g., inverted bases) of siRNAs with 2′-OMe/2′-F decorations similar to the aforementioned STC and ESC modification patterns. Typically, the modification strategies proposed by Arrowhead are characterized by placing inverted deoxythymine (idT) at the strand terminus and/or termini, including UNA and X (without nucleoside base), flanking the UAU or UAUAU motif(s), conjugating the siRNA with cholesterol or other hydrophobic substrates, etc. These designs are currently being investigated in clinical trials.101 Dicerna Pharmaceuticals (Dicerna)102 employed their proprietary dicer-substrate siRNA (DsiRNA) technology to develop RNAi therapeutics. In addition, these researchers also established a series of DsiRNA modification chemistries. For instance, three consecutive 2′-F moieties may be placed at sites 9, 10, and 11 in the sense strand of siRNA from the 5′-end, and the other sites may use alternative 2′-OMe and 2′-F moieties in the flank sequence. One example of a constant flank sequence is 'GCAGCCGAAAGGUGC', which contains inner complementary pairing motifs of 'GCAGCC' and 'GGCUGC'. Consecutive 2′-OMe moieties may be used to modify these motifs, and consecutive RNA or DNA nucleosides without additional 2′ modifications may be employed for the bubble motif of GAAA. Moreover, DNA nucleosides can also be applied at sites 2, 12, 16, 18, 20 and 21 in the antisense strand from the 5′-end. PS linkages may be incorporated into the antisense strand at specific sites, as well as within the constant flank sequence (Fig. 3). The ligands may be placed at the bubble sequence. Silence Therapeutics103 (Fig. 3) and other RNAi-based biotech companies, such as Arbutus Biopharma, OliX Pharmaceuticals and Suzhou Ribo Life Science, have all been devoted to developing state-of-the-art siRNA modification chemistries to establish fruitful drug development pipelines.2