Chemistry, structure and function of approved oligonucleotide therapeutics

Abstract

Eighteen nucleic acid therapeutics have been approved for treatment of various diseases in the last 25 years. Their modes of action include antisense oligonucleotides (ASOs), splice-switching oligonucleotides (SSOs), RNA interference (RNAi) and an RNA aptamer against a protein. Among the diseases targeted by this new class of drugs are homozygous familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, hereditary transthyretin-mediated amyloidosis, familial chylomicronemia syndrome, acute hepatic porphyria, and primary hyperoxaluria. Chemical modification of DNA and RNA was central to making drugs out of oligonucleotides. Oligonucleotide therapeutics brought to market thus far contain just a handful of first- and second-generation modifications, among them 2'-fluoro-RNA, 2'-O-methyl RNA and the phosphorothioates that were introduced over 50 years ago. Two other privileged chemistries are 2'-O-(2-methoxyethyl)-RNA (MOE) and the phosphorodiamidate morpholinos (PMO). Given their importance in imparting oligonucleotides with high target affinity, metabolic stability and favorable pharmacokinetic and -dynamic properties, this article provides a review of these chemistries and their use in nucleic acid therapeutics. Breakthroughs in lipid formulation and GalNAc conjugation of modified oligonucleotides have paved the way to efficient delivery and robust, long-lasting silencing of genes. This review provides an account of the state-of-the-art of targeted oligo delivery to hepatocytes.

Figure 1.

Timeline of the development of chemical modifications (magenta), oligonucleotide synthetic approaches (black), interference in biological information transfer (green), structures of DNA and RNA (blue), and FDA-approved oligo therapeutics (orange).

Figure 2.

(A) Native DNA, RNA and chemical modifications that are approved in therapeutics so far. (B) Different delivery systems; LNP = lipid nanoparticle.

Figure 3.

Currently approved oligonucleotide drugs and mRNA vaccines, year of approval (between 1998 and 2022), and indication. From top left to bottom right: VITRAVENE^®, MACUGEN^®, KYNAMRO^®, EXONDYS 51^®, VYONDYS 53^®, SPINRAZA^®, HEPLISAV-B^®, ONPATTRO^®, TEGSEDI^®, WAYLIVRA^®, GIVLAARI^®, OXLUMO^®, VILTEPSO^®, AMONDYS 45^®, LEQVIO^®, AMVUTTRA^®, COMIRNATY^® and SPIKEVAX^®. The mechanisms of action are: antisense oligonucleotide (ASO), aptamer, splice-switching oligonucleotide (SSO), small interfering RNA (siRNA) and mRNA (vaccine).

Figure 4.

Structures and physicochemical properties of single-stranded DNA (top; 21mer PS-DNA based on the VITRAVENE sequence with random phosphorothioate stereochemistry) and double-stranded siRNA (bottom; 21mer based on the ONPATTRO sequence and modification chemistry). Nucleobase C, O and N atoms are highlighted in green and backbone carbon, oxygen, hydrogen, phosphorus and sulfur atoms are colored in gray, red, white, orange, and yellow, respectively. In the RNA duplex, residues of the sense strand are colored in magenta. In the antisense strand, carbon atoms of the 3′-terminal dTdT overhang and 2′-O-methyl modifications are highlighted in black and cyan, respectively. In the single stranded ASO bases are exposed. In contrast, bases are paired and buried inside the duplex in the double-stranded siRNA.

Figure 5.

Cartoon of combined ASO mechanisms of action vis-à-vis an mRNA target, either in the nucleus or cytoplasm of the cell: (1) Eliciting RNase H with subsequent degradation of the target, (2) translation arrest, i.e. steric blockage and (3) modulation of splicing. mRNA regions include 5′-cap and 5′-untranslated region (5′-UTR), coding region, 3′-UTR and poly-A tail.

Figure 6.

VITRAVENE chemistry and mode(s) of action. Top: ASO sequence and chemical modification; yellow bars indicate phosphorothioate moieties and CpG steps are marked with a red bar. Bottom: individual steps in the ASO’s mechanism of action from cellular uptake to target degradation.

Figure 7.

(A) Phosphorothioates are first-generation ASO modifications. Structures of the phosphate (left), Sp-phosphorothioate (Sp-PS, center) and Rp-phosphorothioate (Rp-PS, right) backbones. R = H (DNA), R = OH (RNA), R = F (2′-F RNA), and R = OMe (2′-O-Me RNA). (B) First- and second-generation RNA modifications. PS (X = S) is important both for ASOs and SSOs and can be combined with 2′-modifications. PS, 2′-O-Me and 2′-F are initial RNAi modifications, and 2'-O-MOE is prominently represented in ASOs and SSOs. Advantages of RNA 2′-modifications are affinity, nuclease resistance and chemical and metabolic stability, and modulation of hydration and protein binding.

Figure 8.

(A) Phosphodiester cleavage reaction catalyzed by RNase H and Ago2. (B) Overlay of the crystal structures of B. halodurans (Bh) RNase H (light-blue ribbon cartoon) bound to a DNA (light-blue carbon atoms)-RNA (pink carbon atoms) hybrid (PDB ID 1zbi), and human Ago2 (Piwi domain; tan ribbon cartoon) bound to a passenger strand (tan carbon atoms)-guide strand (brown carbon atoms) duplex (PDB ID 4w5t). Mg²⁺ ions seen in RNase H complex are green spheres and 2′-OH oxygens are red spheres. Only protein atoms were used to superimpose the two complexes: RNase H E66–G194; Piwi Q589–Y790. (C) Overlay of the crystal structures of BhRNase H (light-blue ribbon cartoon) bound to a DNA (light-blue carbon atoms)–RNA (pink carbon atoms) hybrid (PDB ID 1zbi) and BhRNase H bound to DNA dodecamer duplex (tan ribbon cartoon; PDB ID 3d0p). Mg²⁺ ions seen in the complex with the hybrid duplex are green spheres. Only protein atoms were used to superimpose the two complexes.

Figure 9.

Conformational preorganization by additive gauche effects in 2′-O-[(2-methoxy)ethyl]-RNA (MOE RNA).

Figure 10.

2′-O-modifications with zwitterionic substituents, 2′-O-(3-aminopropyl) (AP), 2′-O-[2-(guanidinium)ethyl] (GE) and 2′-O-2-[2-(N,N-dimethylamino)ethoxy]ethyl] (DMAEOE). 2′-O-[2-(methylamino)-2-oxyethyl] (NMA).

Figure 11.

Sequences of three approved gapmer ASO therapeutics, KYNAMRO, TEGSEDI and WAYLIVRA. All elicit RNase H and encompass a 5–10–5 motif with PS/MOE–RNA wings (green) and a central PS–DNA window (blue).

Figure 12.

Schematic representation of the SMN2 exon 7 (capital font)/intron 7 (small font) junction. SPINRAZA blocks the intronic splicing silencer N1 (ISS-N1) located within intron 7 of SMN2 pre-mRNA (purple arrow), thereby preventing the skipping of exon 7 and promoting production of stable SMN2 protein. SMN2 intron 7 contains several U-rich clusters that are contacted by glutamine-rich RNA binding protein TIA1 to stimulate exon inclusion. The 3′-terminal end of exon 7, ISS-N1 and TIA1 binding site are highlighted with boxes and intron GU dinucleotides are colored in red. The SPINRAZA SSO contains 5-methyl-C (C*) and is fully PS and 2′-O-MOE modified.

Figure 13.

Phosphorodiamidate morpholino (PMO) SSOs that mediate skipping of additional exons for treatment of two types of Duchenne muscular dystrophy (DMD). (A) PMO structure. (B) Model of PMO conformation in a stretch of A-form RNA. Nucleotide sequence, 5′-modification and mode of action of PMO-SSOs (C) EXONDYS 51 and (D) VYONDYS 53.

Figure 14.

Structures of locked nucleic acid (LNA), bridged nucleic acids (BNAs), bicyclo-DNA (bcDNA), tricylco-DNA (tc-DNA) and North and South methanocarba nucleic acids (N-MC and S-MC, respectively). The preferred sugar puckers of the RNA and DNA analogs are given below the structural formulas.

Figure 15.

(A) Crystal structure at 2.5 Å resolution of human Ago2 in complex with a guide (red)-passenger (blue) strand siRNA duplex (PDB ID 4w5t) (217). Individual enzyme domains are colored differently and are labeled; N-terminal and linker domains are colored in different shades of gray. The MID domain harbors the binding site for the 5′-phosphate group of the guide strand that is colored in black, highlighted in ball-and-stick mode and marked with a red P. The PIWI RNA endonuclease domain (‘slicer’) cleaves the targeted mRNA opposite guide siRNA. The PAZ domain binds to the 3′-terminal overhang of the guide strand. Dashed lines indicate missing portions of either protein or RNA, e.g. nucleotides 15 to 19 of the guide strand. The arrow points to the sharp kink between base pairs arising from AS6 and AS7. (B) Cryo EM structure at 3.3 Å resolution of Drosophila Dicer-2:R2D2 heterodimer in complex with a guide (red)-passenger (blue) strand siRNA duplex and a piece of double-stranded RNA, dsRNA (PDB ID 7v6c) (290). Individual Dicer-2 and R2D2 domains are colored differently and are labeled (R2D2 RBD, RNA binding domain and CTD, C-terminal domain). Regions in Dicer-2 outside the helicase, RNase III and platform-PAZ domains are colored in light gray, and dashed lines indicate missing portions in the protein chains.

Figure 16.

(A) Individual steps of RNA interference directed by exogenously delivered siRNA: (1) RISC loading, (2) passenger strand removal, (3) binding of mRNA target, (4) target cleavage, and (5) release of cleavage products and pairing with the next target to begin a new round of cleavage. Guide siRNA, passenger siRNA, and mRNA target are colored in red, blue and black, respectively. The Ago2 MID domain is highlighted in green and red and gray dots mark the 5′-terminal phosphate and 3′-terminal TT overhang, respectively. (B) The phosphodiester cleavage reaction catalyzed by Ago2.

Figure 17.

Effects of chemical modification on the water structure around RNA. Minor groove hydration in (A) RNA and (B) 2′-F-RNA. RNA 2′-hydroxyl groups serve as bridge heads for tandem water bridges across the minor groove (89). Introduction of fluorine (highlighted in green) abolishes H-bonds between 2′-substituent and water, as apparent by altered distances and shifted water molecules. (291,294). Water molecules are gray spheres, H-bonds are thin solid lines and selected distances are indicated by dashed lines.

Figure 18.

Guide and passenger strand sequences and modifications in the five RNAi therapeutics that have received market approval to date. (A) ONPATTRO, (B) GIVLAARI, (C) OXLUMO, (D) LEQVIO and (E) AMVUTTRA. Ribo-, 2′-deoxyribo-, 2′-O-Me- and 2′-F-nucleotides are depicted as circles colored in red, blue, black and green, respectively. GIVLAARI, OXLUMO, LEQVIO and AMVUTTRA feature PS modifications (orange bars) and a triantennary GalNAc conjugate at the 3′-terminal end of the passenger strand.

Figure 19.

Structures of the phosphate analog E-vinylphosphonate (E-VP, left), (S)-GNA (center), and the amide backbone linkage (AM1, right).

Figure 20.

Platforms for delivery of functional siRNAs to liver: LNPs vs. GalNAc-siRNA conjugates.

Figure 21.

(A) LNP multi-component formulation for efficient systemic delivery of the RNAi therapeutic ONPATTRO to hepatocytes. (B) LNP multi-component formulation for intramuscular administration of mRNA vaccines COMIRNATY and SPIKEVAX.

Figure 22.

GalNAc conjugates for optimal hepatic delivery. The cartoon in the center shows siRNA–GalNAc conjugate binding and internalization by ASGPR, RISC loading and formation of functional RISC, mRNA target recognition and catalytic cleavage, and initiation of a new cycle. Panels around the simplified schematic of a hepatocyte depict, from the upper right and moving in a counterclockwise fashion: (i) The triantennary GalNAc conjugate L96. (ii) The crystal structure of ASGPR-H1-CRD (PDB ID 1DV8 (342)); Ca²⁺ ions are green, three disulfide bridges are highlighted in yellow, and the blue arrow points to the GalNAc binding site. (iii) The crystal structure of mannose binding protein (MBP) QPDWGH mutant bound to GalNAC (PDB ID 1BCH (343)); GalNAc carbon atoms are black and the ASGPR-H1-CRD is superimposed on one of the MBP-CRDs; note the central coiled-coil stalk that stabilizes the trimer. (iv) A model of GalNAc bound to ASGPR-H1-CRD based on the structure of the complex with lactose (PDB ID 5JPV (323)); the GalNAc oxygen attached to the L96 spacer is highlighted in magenta.

Figure 23.

Aptamers that target VEGF. (A) Secondary structure and chemical modification of MACUGEN for treatment of wet age-related macular degeneration (AMD). Ribo-, 2′-deoxyribo-, 2′-O-Me- and 2′-F-nucleotides are depicted as circles colored in pink, black, blue and green, respectively. (B) NMR solution structure of the VEGF heparin binding domain (HBD) comprising Ala-111 to Arg-165 (PDB ID 1KMX (372)). The backbone is traced with a ribbon colored in gray, and disulfide bridges as well as side chains that show the largest chemical shifts upon aptamer binding (373) are depicted in ball-and-stick mode and are colored in yellow and cyan, respectively. Red arrows point to residues U14 and Cys-137 that could be photo-crosslinked in the RNA aptamer-VEGF₁₆₅ complex (355). (C) Secondary structure of an all-2′-O-Me modified VEGF aptamer (361). Incorporation of a PS2 modification (yellow sphere; structure shown on the right) at either site boosts the K_d 1000-fold to ca. 1 pM (368). Both aptamers bind exclusively to the VEGF-HBD.

Figure 24.

Structural basis of CpG recognition by TLR9 in the crystal structure of the DNA-receptor complex (PDB ID 3wpe (412)). (A) Close-up view of the interaction between the CpG step and TLR9. The C5-methyl of cytosine (carbon highlighted in yellow) is not present in the crystal structure. It was added to show contacts with neighboring polar moieties (thin solid lines) that reduce the affinity of an antagonist, i.e. an ^mCpG-containing oligo, by ca. threefold relative to the corresponding CpG-containing agonist oligo. Views of the DNA-TLR9 complex with the molecular surface of the receptor colored according to (B) Coulombic potential (blue, positively polarized, and red, negatively polarized) and (C) hydrophobicity potential (orange, most hydrophobic and purple, least hydrophobic).