Aptamers consist of sequences of nucleotides or peptides that can bind a specific site on a target molecule. Their specificity rivals that of antibodies, and they have many different diagnostic and therapeutic uses.
Oligonucleotide aptamers are made of short sequences of nucleotides. Peptide aptamers are short peptide sequences displayed on a larger protein matrix. The main benefit of both types over antibodies is that they can be made in a test tube, i.e. without breeding cell lines.
Manufacture and uses of aptamers
Oligonucleotide aptamers can be produced in vitro through a process known as the systematic evolution of ligands by exponential enrichment (SELEX) which first emerged in 1990.
A large library of oligonucleotides is exposed to the target ligand, and the sequences which bind the target are eluted and amplified by PCR. This is repeated which increasingly severe elution conditions, so selecting for the tightest binding aptamer.
This relatively simple and cheap production method provides a significant benefit over antibodies. As it relies on chemical synthesis rather than generating cell lines it is highly reproducible as well, ensuring the consistency of the product.
Another benefit of chemical synthesis is that aptamers can easily be designed to evade degradation by enzymes and to maintain integrity within the body. They may also be useful in targeted therapies and controlling drug release at a specific site. Unlike antibodies they have no to little interaction with the immune system, so are far less likely to elicit a complex immune response.
However, despite this and many years of research very few aptamer-based pharmaceuticals, at least anything close to the volume of antibody products. Currently, aptamer therapeutics generally target niche areas due to the entrenchment of antibodies within pharmaceutical companies.
As of 2015, the only aptamer approved for use by the FDA is Macugen, an RNA aptamer that targets vascular endothelial growth factor, an important regulator that promotes angiogenesis and is overexpressed in many cancers.
However, many more are in development, for example, one aptamer with therapeutic applications is NOX-A12, an RNA aptamer in phase II clinical trials that binds CCL2. This target is an important cytokine in regulating angiogenesis and immune response in certain cancers, including non-Hodgkin’s lymphoma and chronic lymphocytic leukemia.
Aptamer-based biosensors have shown wider use in diagnostics compared to therapeutic aptamers. Examples include DNA aptamers based on Salmonella typhimurium and kanamycin for use in food safety monitoring. Other diagnostic uses include monitoring water samples and detecting toxins.
As aptamer technology develops, hopefully, more therapeutic and diagnostic uses will be identified. Particularly as many therapeutic aptamers have completed phase I and II clinical trials we may see more aptamer-based pharmaceuticals in the coming years.
Issues and recent advancements
Understanding the optimal configuration of aptamers to bind the target in a biological context is an important area where aptamers can be improved. Increasing the bioavailability is essential in ensuring they can act as effective therapies in the future.
This low bioavailability is due to aptamers produced by SELEX having different binding mechanics in vitro during production compared to their characteristics in vivo. Another problem with SELEX is that it can be time-consuming and it has a lower success rate, making it a relatively inefficient process.
One method to increase bioavailability is to limit renal filtration. This is done by conjugating aptamers with large nanomaterials, for example, adding polyethylene glycol (PEG) increases the molecular weight of an aptamer over the threshold for filtration (40 kDa).
Macugen uses this technique to increase bioavailability and time in the blood. PEGylation also did not produce any unwanted immune responses. The increased mass can also reduce their susceptibility to nucleases and reduce toxic build-up.
Improving the design of the initial library can reduce waste in the production phase. Adding modified, artificial nucleotides can improve resistance to nucleases and increase binding affinity. They will also increase the diversity of the library, increasing the success rate, as the additional nucleotides allow more binding configurations and oligonucleotide combinations.
Another limiting aspect is to obtain high-affinity aptamers often 10+ rounds of SELEX are required. An innovative technique known as non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) aims to reduce the number of rounds involved.
The target is incubated with the aptamer library until it reaches an equilibrium. Different migration rates in electrophoresis allow bound aptamers to be separated from free aptamers. This can produce high-affinity aptamers in very few rounds of production.
Utilizing innovative techniques such as these and identifying further areas of improvement will help to optimize the production and application of aptamers. This will allow aptamers to be an important area of future therapeutic development.
Increased bioavailability will improve the effectiveness of aptamers in vivo applications, while improving production will increase the rate of research & development, allowing the focus to move onto disease and diagnostic areas to be targeted for aptamer use.
- Sharma, T. K., Bruno, J. G. and Dhiman, A. (2017) ‘ABCs of DNA aptamer and related assay development’, Biotechnology Advances. Elsevier Inc., pp. 275–301. doi: 10.1016/j.biotechadv.2017.01.003.
- Sun, H. et al. (2015) ‘A Highlight of Recent Advances in Aptamer Technology and Its Application’, Molecules, 20, pp. 11959–11980. doi: 10.3390/molecules200711959.
- Zhu, G. and Chen, X. (2018) ‘Aptamer-based targeted therapy’, Advanced Drug Delivery Reviews. Elsevier B.V., pp. 65–78. doi: 10.1016/j.addr.2018.08.005.