The success of a nucleic acid therapeutic is highly dependent on the modifications made to the nucleotides. These mods play a key role in providing the stability and efficacy in vivo needed for clinical impact. In this instalment of our Toolbox blog series, we take a closer look at mipomersen (Kynamro®) – the first gapmer therapeutic approved by the FDA. 
Mipomersen was developed to treat homozygous familial hypercholesterolemia, a genetic disorder that causes very high cholesterol levels. It is an antisense oligonucleotide (ASO), which binds to the messenger RNA coding for the ApoB-100 protein.
The aim of mipomersen is to prevent the mRNA being translated into protein. It achieves this by activating the cell’s in-built mechanism to degrade RNA:DNA complexes. When the ASO’s DNA bases bind to the complementary mRNA, it triggers RNase H to break down the mRNA. This results in the desired silencing.
Unlike the first ASO, fomivirsen, which consisted only of DNA, mipomersen is a gapmer, which has a central region of DNA with stretches of RNA at either end. This helps to protect the oligo from nucleases and improve binding affinity to the mRNA.
As the first approved gapmer, mipomersen was a pioneering drug. However, it also introduced other innovations, as we will see.
2'-O-methoxyethyl modification in RNA
All of the RNA bases in mipomersen have a 2'-O-methoxyethyl (2'-MOE) modification. Since it was introduced in this drug, several later antisense oligonucleotides have carried this mod.
2'-MOE offers several advantages that improve the characteristics of these therapeutics. Oligos carrying 2'-MOE modifications have greater nuclease resistance compared to unmodified and 2'-OMe-modified oligonucleotides.1
The 2'-MOE moiety increases the preference for the 3'-endo pucker conformation of the ribose ring, which favours duplex formation.2 This improves binding affinity, with a ΔTm of 0.9 to 1.6 °C per modification.
The specificity of oligonucleotides also appears to improve with the addition of 2'-MOE. These modified oligos show a greater fall in affinity when there are mismatches compared to unmodified deoxyoligonucleotides.3
In addition, the 2'-MOE group increases the lipophilicity of the molecule, improving its pharmacokinetic properties and delivering it to its target.
Methylated nucleobases
All the cytosine and uracil nucleobases in mipomersen carry a 5-methyl group. Each additional hydrophobic
group raises the Tm of hybridisation by 0.5 °C by excluding water molecules as the duplex forms.4
This was the first time that methylated nucleobases were included in an approved nucleic acid therapeutic, and they have continued to be used in many later ASOs.
One important benefit of methylation is to reduce the immune response to the oligos in vivo. CpG motifs are commonly found in microbial genomes and so their presence in ASOs can cause the immune system to expect an infection. However, the methylated cytosine reduces this stimulation.5,6
Phosphorothioate backbone
Mipomersen also has a phosphorothioate backbone. This was previously seen in fomiversen, the first nucleic acid therapeutic, and continues to be widely used. Phosphorothioate is unlike other modifications, which change a group on the nucleoside, instead it alters the linkages between bases by replacing one of the non-bridging oxygen atoms with a sulphur.
Like 2'-MOE, the phosphorothioate bonds protect against nucleases and improve the stability of the oligo in vivo.7 Phosphorothioate bonds also strengthen binding to proteins in the cell and plasma, which can enable entry into tissue and slow clearance from the body.8,9
However, hybridisation can suffer by around 0.5 °C lower Tm per phosphorothioate bond.4 Despite this, the advantages outweigh the potentially lower affinity and phosphorothioate backbones have been used in many approved ASOs and siRNAs.Legacy of mipomersen
Despite its innovations, mipomersen was not a commercial success. Its side effects affecting the liver meant that it was not approved by the European Medicines Agency and it faced competition from new drugs that had greater tolerability.10
However, it has had an important legacy. Mipomersen was only the second ASO approved by the FDA, almost 15 years after fomiversen. Since then, there has been a steady stream of new ASOs reaching the market. This includes four further gapmers: inotersen (Te gsedi®), tofersen (Qalsody®), eplontersen (Wainua®) and olezarsen (Tryngolza®). These all rely on the same pattern of DNA bases flanked by RNA as well as the 2'-MOE, methylated bases and phosphorothioate modifications.
These modifications played a crucial role in bringing the next generation of nucleic acid therapeutics to patients. LGC Biosearch Technologies™ offers a wide variety of nucleotide modifications to support therapeutic development. Our range of products feature many of the commonly used modifications in existing drugs and the latest mods being explored for the next generation of therapies.
Related Posts
- Pegaptanib, the first approved aptamer
- Phosphorothioate bonds
- 2'-O-methoxyethyl (2'-MOE)
- 5-Nitroindole – a universal base oligo modification
- Thio C6 linker
- 3' Spacer C3
- Locked nucleic acids
References
- Martin P (1995) Ein neuer Zugang zu 2′-O-Alkylribonucleosiden und Eigenschaften deren Oligonucleotide. Helvetica Chimica Acta 78, 486–504. doi: 10.1002/hlca.19950780219
- Manoharan M (1999) 2′-Carbohydrate Modifications in Antisense Oligonucleotide Therapy: Importance of Conformation, Configuration and Conjugation. Biochimica Et Biophysica Acta (BBA) - Gene Structure and Expression 1489: 117-130. doi:10.1016/S0167-4781(99)00138-4
- Hill AC and Hall J (2023) The MOE Modification of RNA: Origins and Widescale Impact on the Oligonucleotide Therapeutics Field. Helvetica Chimica Acta 106, no. 3: e202200169. doi: 10.1002/hlca.202200169
- Freier SM and Altmann KH (1997) The ups and downs of nucleic acid duplex stability: Structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Research 25(22):4429–4443. doi: 10.1093/nar/25.22.4429
- Henry S et al. (2000) Chemically modified oligonucleotides exhibit decreased immune stimulation in mice. J Pharmacol Exp Ther 292(2):468–479. doi: 10.1016/S0022-3565(24)35315-7
- Karikó K et al. (2005) Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity 23(2):165-175. doi:10.1016/j.immuni.2005.06.008
- Vosberg HP and Eckstein F (1982) Effect of deoxynucleoside phosphorothioates incorporated in DNA on cleavage by restriction enzymes. J Biol Chem 257(11):6595-6599. doi: 10.1016/S0021-9258(20)65184-5
- Crooke ST, Vickers TA and Liang XH (2020) Phosphorothioate modified oligonucleotide-protein interactions. Nucleic Acids Research 48(10):5235-5253. doi:10.1093/nar/gkaa299
- Crooke ST et al. (2020) The Interaction of Phosphorothioate-Containing RNA Targeted Drugs with Proteins Is a Critical Determinant of the Therapeutic Effects of These Agents. J Am Chem Soc. 142(35):14754-14771. doi:10.1021/jacs.0c04928
- Parham JS and Goldberg AC (2019) Mipomersen and its use in familial hypercholesterolemia. Expert Opin Pharmacother. 20(2):127-131. doi: 10.108 0/14656566.2018.1550071
