The success of small interfering RNAs (siRNAs) therapeutics in treating rare genetic liver disorders has spurred significant investment to expand these drugs to more common conditions, from hypertension to hepatitis B.1
The growth in the field has only been possible thanks to alterations to the oligonucleotides that enhance siRNA stability and efficacy. These include nucleotide modifications, backbone substitutions and conjugation with a liver-targeting ligand.
Givosiran (Givlaari®) was the first commercialised siRNA drug to feature these innovations when it was approved in 2019. This blog post reviews the chemical modifications that made givosiran an effective treatment in vivo and set the stage for the siRNA drugs that followed.
Modifications in givosiran and other siRNAs
siRNAs are double-stranded oligonucleotides designed to silence a specific gene. In the case of givosiran, the antisense strand guides the RNA-induced silencing complex (RISC) to the mRNA for 5'-aminolevulinate synthase 1 (ALAS1) and degrades it.
Givosiran consists of 21 bases on the sense strand and 23 bases on the antisense strand. Without modifications, givosiran or any other oligonucleotide therapeutic would be rapidly broken down by nucleases and be ineffective as a therapy.
Like pegaptanib, the first therapeutic aptamer, givosiran carries modifications in the 2'-OH position of its nucleotides. All the ribose rings have either a 2'-fluoro or a 2'-O-methyl group in this position (figure 1), increasing resistance to nucleases and promoting affinity for the target mRNA.2 The 2' modifications can also reduce immunogenicity, a significant hurdle for siRNAs.3
Figure 1. The 2'-fluoro and 2'-O-methyl modifications are common among nucleic acid therapeutics to protect against nucleases and improve target binding.
Givosiran also contains phosphorothioate bonds at the ends of the oligo strands, which provide additional protection against nucleases.
These modifications are collectively known as enhanced stabilisation chemistry and led to improvements in the toxicity and efficacy of a new generation of siRNAs.4 Since then, these modifications have continued to be refined, including reducing the number of 2'-fluoro substitutions.
GalNAc conjugation drives liver-targeted drug delivery
The main innovation for givosiran was the introduction of N-acetylgalactosamine (GalNAc). The only previously approved siRNA, patisiran (Onpattro®), used a lipid nanoparticle delivery system to reach the liver intact. Givosiran is covalently attached to GalNAc, which allows the oligo to specifically target hepatocytes without additional components.5
GalNAc binds to the asialoglycoprotein receptor (ASGPR), which is mainly found on the surface of liver cells. The oligo appears to enter the cell during ASPGR recycling (figure 2), when it is engulfed into an endosome.5 There, the low pH releases the GalNAc-siRNA from ASPGR, and glycosidases cleave the siRNA from the GalNAc. The free siRNA leaves the endosome and is free to engage with the target and induce an RNAi response.
Figure 2. GalNAc-siRNA conjugates may enter the cell via endocytosis of the GalNAc-siRNA-ASPGR complex.
The conjugated GalNAc ligand delivers a 10-fold increase in delivery to the liver in mouse models compared to the unmodified oligo.6
Clinical impact of siRNAs
Givosiran was developed to treat acute hepatic porphyria (AHP), a rare genetic disorder that affects the production of haem – the molecule responsible for carrying oxygen in the blood.7 By inhibiting ALAS1 expression, givosiran prevents toxic by-products from accumulating.
Most siRNA therapies approved after givosiran are used to treat other rare diseases causing liver dysfunction, including primary hyperoxaluria type 1 and transthyretin-related amyloidosis. However, the siRNA drug inclisiran (Leqvio®) is used to lower LDL cholesterol, representing a significant expansion in the number of patients who could benefit from this class of therapeutics.
Inclisiran demonstrates the potential for effective siRNA molecules to become blockbuster drugs. There are clinical trials to use candidate therapies to treat highly common conditions like hypertension or atherosclerosis.1 Many of these drugs under development continue to rely on GalNAc conjugation to target the liver and include the same nucleotide modifications as givosiran to improve their stability and effectiveness.
LGC Biosearch Technologies offers a wide range of solid supports, phosphoramidites and reagents to help therapeutic developers incorporate:
- GalNAc and other targeting ligands
- 2'-fluoro and 2'-O-methyl nucleotides
- Phosphorothioate linkages
- Other advanced modifications for developing new siRNAs.
Contact us or explore our oligo synthesis products to discover how we can help accelerate the development of your liver-targeted therapeutics.
Related Posts
- The mods that made pegaptanib, the first therapeutic aptamer
- Nucleic Acid Therapeutics Toolbox: Mipomersen, the first gapmer
- A knack for getting to the liver: GalNAc enables liver-targeted oligonucleotide therapeutics
- phosphorothioate bonds
References
- Guo S, Zhang M and Huang Y (2024) Three ‘E’ challenges for siRNA drug development. Trends in Molecular Medicine 30(1):13-24. doi: 10.1016/j.molmed.2023.10.005
- Mohammed AA et al. (2024) Oligonucleotides: evolution and innovation. Medicinal Chemistry Research 33:2204-2220. doi: 10.1007/s00044-024-03352-7
- Kenski KM et al. (2012) siRNA-optimized Modifications for Enhanced In Vivo Activity. Molecular Therapy Nucleic Acids 1:e5. doi: 10.1038/mtna.2011.4
- Hu B et al. (2020) Therapeutic siRNA: state of the art. Sig Transduct Target Ther 5, 101. doi: 10.1038/s41392-020-0207-x
- Springer AD, Dowdy SF (2018) GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics. Nucleic Acid Ther. 28(3):109-118. doi:10.1089/nat.2018.0736
- Prakash TP et al. (2014) Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 42(13):8796-8807. doi:10.1093/nar/gku531
- Balwani M et al. (2020) Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria. New England Journal of Medicine 382:2289-2301. doi:10.1056/NEJMoa1913147
