Acyclic (S)-glycol nucleic acid (S-GNA) modification of siRNAs improves the safety of RNAi therapeutics while maintaining potency

Abstract

Glycol nucleic acid (GNA) is an acyclic nucleic acid analog connected via phosphodiester bonds. Crystal structures of RNA-GNA chimeric duplexes indicated that nucleotides of the right-handed (S)-GNA were better accommodated in the right-handed RNA duplex than were the left-handed (R)-isomers. GNA nucleotides adopt a rotated nucleobase orientation within all duplex contexts, pairing with complementary RNA in a reverse Watson-Crick mode, which explains the inabilities of GNA C and G to form strong base pairs with complementary nucleotides. Transposition of the hydrogen bond donor and acceptor pairs using novel (S)-GNA isocytidine and isoguanosine nucleotides resulted in stable base-pairing with the complementary G and C ribonucleotides, respectively. GNA nucleotide or dinucleotide incorporation into an oligonucleotide increased resistance against 3'-exonuclease-mediated degradation. Consistent with the structural observations, small interfering RNAs (siRNAs) modified with (S)-GNA had greater in vitro potencies than identical sequences containing (R)-GNA. (S)-GNA is well tolerated in the seed regions of antisense and sense strands of a GalNAc-conjugated siRNA in vitro. The siRNAs containing a GNA base pair in the seed region had in vivo potency when subcutaneously injected into mice. Importantly, seed pairing destabilization resulting from a single GNA nucleotide at position 7 of the antisense strand mitigated RNAi-mediated off-target effects in a rodent model. Two GNA-modified siRNAs have shown an improved safety profile in humans compared with their non-GNA-modified counterparts, and several additional siRNAs containing the GNA modification are currently in clinical development.

Keywords: GNA; RNA therapeutics; RNAi therapeutics; glycol nucleic acid; oligonucleotide therapeutics; siRNAs.

FIGURE 1.

(A) Structures of building blocks used in nucleic acid therapeutics. (B) Schematics of lipid nanoparticle (LNP) formulations and GalNAc conjugate.

FIGURE 2.

Structures of XNAs with potential for use in nucleic acid–based therapeutics.

FIGURE 3.

Structure, conformations, base-pairing, and Ago2 binding of (S)-GNA. (A) RNA-U and (S)-GNA-T nucleotides. (B) GNA nucleotides adopt both gauche and anticonformations when incorporated into RNA. (C) Example of an (S)-GNA-T:RNA-A base pair showing a rotated nucleobase conformation for the GNA nucleotide (arrow). (D) An (S)-GNA-T residue can seamlessly and with optimal geometry replace an RNA nucleotide at position 7 of the antisense strand RNA bound to human Ago2. The RNA strand assumes a kink at that site that is associated with Ile-365 of Ago2 and results in unstacking of bases of nucleotides 6 and 7.

SCHEME 1.

General procedures for the synthesis of (S)-GNA (top) and (R)-GNA (middle) phosphoramidites starting from enantiopure glycidol. Various bases and base analogs (B) can be used to functionalize GNA (bottom). Detailed protocols for syntheses have been reported (Zhang et al. 2005, 2006; Schlegel and Meggers 2009; Schlegel et al. 2017, 2021).

FIGURE 4.

Crystal structures of (S)-GNA octamer duplexes, PDB ID 2JJA (left) and PDB ID 2XC6 (right), viewed across the two grooves (top) and along the helical axis (bottom).

FIGURE 5.

Various base-pairing modes between GNA and RNA contrasted with standard Watson–Crick base pairs.

FIGURE 6.

(A) Superimposed (S)-GNA T and TNA T inside RNA, demonstrating that GNA with the thymine base flipped (i.e., matching the TNA base orientation) results in a clash with the adjacent base (suboptimal stacking). (B) Crystal structure of RNA with an incorporated (S)-GNA T residue that displays the rotated orientation of the thymine base.

FIGURE 7.

Position-dependent effect of (S)-GNA on in vitro potency of siRNA-mediated silencing of the Ttr gene in an in vitro assay; antisense (guide) strand on left and sense (passenger) strand on right.

FIGURE 8.

View of the crystal structure of Ago2 bound to an RNA duplex (PDB ID 4W5T) kinked between positions 6 and 7 of the antisense (guide) strand (asterisk). Ago2 domains are highlighted and labeled, and the siRNA (antisense and sense) strands are colored in red and blue, respectively. Antisense strand residues 1 to 12 are numbered, and the 5′-phosphate group is highlighted in black and ball-and-stick mode.