The core part of oligonucleotide therapeutics is the carefully refined sequence of nucleotides that precisely binds the intended target. However, beyond this, there are a range of modifications and additions that are essential for the oligo to be effective in vivo. From getting where it’s needed to surviving ubiquitous nucleases, there are several challenges that a new therapeutic must overcome to successfully reach market.
In our new Toolbox blog series, we take a look at some of the key elements that were crucial to pioneering therapeutics as well as up-and-coming modifications that could be used in the drugs of the future.
Pegaptanib (sold as Macugen) became the first approved aptamer drug in 2004. Aptamers are oligos that fold into specific structures that help them bind to their target, like a chemical antibody. By contrast to antibody proteins, aptamers are much smaller and cheaper to synthesise.1
Pegaptanib binds with very high affinity to VEGF, a protein that encourages new blood vessels to form and causes leakage from blood vessels. Pegaptanib blocks the activity of VEGF, making it an effective treatment for wet age-related macular degeneration when injected into the eye.
Although aptamers have several practical advantages over antibodies, such as simpler production and logistics, they have not had as much impact on healthcare. Over 100 monoclonal antibodies have been approved by the FDA, including some of the best-selling therapies in recent times.2 By contrast, it took 19 years for the only other aptamer to be approved after pegaptanib, also a treatment for macular degeneration.
Nucleotide modifications in pegaptanib
Pegaptanib was only the second ever nucleic acid therapeutic approved, and it was the first to introduce several important innovations that have continued to be used in later therapeutics.
Aptamers are highly susceptible to nucleases and clearance via the kidneys and so modifications can help to improve the longevity and effectiveness of the oligo therapeutic in vivo.3 Pegaptanib bears hallmarks of these adaptations, with almost all of its 28 nucleotides carrying modifications and a polyethylene glycol (PEG) ligand attached to the 5ʹ end.
All of the pyrimidine bases in pegaptanib have a 2ʹ-fluoro modification and almost all of the purine bases carry a 2ʹ-O-methyl group. The 2ʹ modifications favour a puckered conformation of the ribose ring, which increases the stability of the RNA-like structure, improves binding to the target and increases resistance to nucleases.4,5
These modifications are widely used in small interfering RNAs (siRNAs), as they increase stability and specificity while reducing immunogenicity.6 Their small size means they are well-tolerated in several positions without compromising the ability to hybridise with complementary RNA targets.
Figure 1. The 2ʹ-fluoro and 2ʹ-O-methyl modifications are heavily used in pegaptanib and many other nucleic acid therapeutics that followed.
Protecting the 5ʹ and 3ʹ ends
The PEG group conjugated to the 5ʹ end of the aptamer is included to improve the pharmacokinetics by increasing the time that the drug stays in the body. This large blocking group can reduce the ability of metabolic enzymes to degrade the oligonucleotide.4 The additional molecular weight of the PEG group also reduces the filtration rate through the kidneys.3
Despite the successful application of PEG conjugation in approved therapeutics, it can cause immune reactions and so alternative bulky moieties have also been investigated for drug development, such as cholesterol.1
Capping the 3ʹ end of aptamer can also protect the molecule’s stability by blocking 3′ to 5′ exonuclease attack in the body. 4,7 Pegaptanib has an inverted deoxy-thymidine as a cap, which has a 3’-3’ linkage to the following nucleotide.
Synthesising an oligo like this requires a modified control pore glass (CPG) attached to the 5′-hydroxyl of the first nucleoside.4 The rest of the chain can then be synthesised with standard phosphoramidites.
Advances since pegaptanib
The only other therapeutic aptamer approved to date, avacincaptad pegol (Izervay), carries very similar
modifications to pegaptanib. It makes use of 2ʹ-F, 2ʹ-OMe, an inverted dT 3′ cap and a similar PEG 5′ cap for all of the same reasons listed above.
However, this similarity is not representative of the innovation happening in the field. A wide range of aptamers with a variety of modifications are being developed for uses in diagnostics and therapeutics. With these advances, more aptamers may follow pegaptanib to become important discoveries in healthcare.
For the full range of modifications available, see our toolbox for oligonucleotide therapeutic development.
Related Posts
- 5-Nitroindole – a universal base oligo modification
- BHQ® (Black Hole Quencher®) non-fluorescent quenchers
- Thio C6 linker
- 3' Spacer C3
- Phosphorothioate bonds
- 2'-O-methoxyethyl (2'-MOE)
- Locked nucleic acids
References
- Ni S et al. (2020) Recent Progress in Aptamer Discoveries and Modifications for Therapeutic Applications. ACS Appl. Mater. Interfaces 13(8):9500–9519 doi: 10.1021/acsami.0c05750
- Mullard A (2021) FDA approves 100th monoclonal antibody product. Nature Reviews Drug Discovery 20:491-495 doi: 10.1038/d41573-021-00079-7
- Adachi T and Nakamura Y (2019) Aptamers: A Review of Their Chemical Properties and Modifications for Therapeutic Application. Molecules 24(23):4229 doi: 10.3390/molecules24234229
- Odeh F et al. (2019) Aptamers Chemistry: Chemical Modifications and Conjugation Strategies. Molecules 25(1):3. doi: 10.3390/molecules25010003
- 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
- Shaw JP et al. (1991) Modified deoxyoligonucleotides stable to exonuclease degradation in serum. Nucleic Acids Res. 19:747–750. doi: 10.1093/nar/19.4.747
