G. Thomas Caltagirone, Ph.D.
President & CEO, APTAGEN Labs, Jacobus
How has the aptamer technology evolved in the last decades in terms of discovery, selection, synthesis, and purification? Are there still unresolved issues?
Over the decades, many different selection strategies and methodologies have been developed. Recently, many innovative partitioning strategies have been applied to separate binders from nonbinders, from magnetic force separation techniques to single-particle sorting techniques. We see a lot of innovation in reducing the number of rounds of screening during the selection process, as well as accommodating different library and target presentations to satisfy requirements for specific use cases. Synthesis and purification has been relatively stable with regard to phosphoramidite chemistry and purification by chromatography or polyacrylamide gel electrophoresis.
Are current aptamer discovery platforms efficient? What improvements are needed?
The aptamer discovery process is still time-consuming and has not been fully automated. The average execution of aptamer discovery is still a highly-manual process over many rounds of selection. Improvements could be made in several respects. Greater automation would result in a higher degree of reproducibility and faster round turnover, while improvements to library amplification between rounds with the use of emulsion PCR minimize the effects of factors external to aptamer-target binding on library enrichment. In addition to these aspects, there are reports that aptamers have been identified after a single round of selection. While all of these constitute improvements to aptamer discovery, the key is in wider adoption of these procedures, especially for commercial applications.
Can you describe the application of your aptamer research?
Our customers use aptamers in a variety of applications, from environmental sensors and diagnostics to therapeutic applications. We have also had a small number apply aptamers to manufacturing QC. Our internal research has focused on aptamers in lower-resource applications, as an initial screening step prior to potentially more costly interventions.
Why is aptamer technology superior to conventional strategies in your field (diagnostic imaging, biosensing, therapeutics, delivery, antibiotics, gene therapy, drug monitoring)?
Aptamers have a few benefits compared to materials such as antibodies. Because aptamers are specific DNA or RNA sequences, they can be chemically synthesized, ensuring batch-to-batch consistency. Additionally, because aptamers are relatively short nucleic acids, they facilitate the democratization of biotechnology as any reasonably competent oligo synthesis lab is capable of their manufacture. From a functional standpoint, aptamers display remarkable stability; while they are reliant on their environmental conditions for functionality, they can recover from denaturation. Under appropriate storage conditions, aptamers can be stable for years. In contrast, antibodies (the typical equivalent molecule) are resilient under a set of conditions but have shorter shelf lives. Another benefit of using aptamers over antibodies is based on the smaller size of aptamers. Since they are often one-third the size of antibodies (and can be smaller than that), they can interact with their targets without as much concern for steric interference.
Which application do you believe is currently most appealing for companies? Will this remain the same in the next year? Do you anticipate the development of new applications, or will existing ones become more prevalent?
Diagnostic applications more so than therapeutics targeting small molecules in a variety of sample matrices.
–
DAVID BUNKA
Chief Scientific Officer, Aptamer Group
Aptamers targeted for precision gene therapies
Over the past few years, we have seen a change in the application of aptamers from their typical use as assay reagents, where antibodies have previously failed, to a specific demand for aptamer technology in targeted therapies, with aptamers increasingly being the first choice. Multiple factors underlie this step change, including (1) the technology coming of age with increasing awareness of aptamers, (2) the body of evidence demonstrating aptamer performance has built to a crux point brings credibility, and (3) the revolution in gene therapies, particularly for molecules like antisense oligonucleotides, small interfering RNA (siRNA) and small activating RNA (saRNA).
Since the discovery of aptamers in 1990, development processes have continually improved, allowing faster, better selection of aptamers using high-throughput automation. Rather than the early days of systematic evolution of ligands by exponential enrichment (SELEX)-based development (which, as it sounds, was more of an aptamer soup approach where we worked with what fell out of the process), we now have strategic development approaches tuned for target type, cross-reactivity, and final application, which incorporate the latest technologies like next-generation sequencing and AI modelling for a deeper and broader understanding of the development process. This focus on aptamer development by specific companies in the space has meant we now understand how to deliver quality binders and support their translation into functional end applications. Equally, advances in oligonucleotide manufacture mean that aptamers’ scalability, quality, and low costs have enabled their wider use and easier adoption.
The rise of gene therapies has highlighted the excellent potential for these molecules. In 2019, Alnylam launched Givosiran, a siRNA using GalNAc to target this treatment specifically to hepatocytes in the liver. The success of this targeting method has led to multiple new treatments targeting hepatocytes with improved efficacy. However, beyond GalNAc, limited options are available for precisely delivering gene therapies to their site-of-action and ensuring they can access the intracellular environment. This is currently one of the major challenges in the gene therapy space, and an area where aptamers can provide the much-needed solution.
Viral vectors are the major delivery mechanism for most gene therapies both in the pipeline and those approved by regulatory agencies. However, viral vectors are not highly tissue specific, resulting in off-target effects, have challenges in manufacture, meaning supply is limited, and are immunogenic, preventing the redosing of patients with these treatments.
Antibodies have been trialled for oligo-therapeutic delivery and are showing positive results in phase 1/2 trials for Muscular Dystrophy. Still, as with antibody-drug conjugates for precision chemotherapy, the large size of antibodies hinders tissue penetration. Using smaller affinity ligands as drug delivery vehicles has been demonstrated to allow deeper tissue penetration to reach, for example, the centre of a solid tumour, or deep into fibrotic liver tissue, like our hepatic stellate cell targeting aptamers, where the inflammation and scarring hinder the penetration of large molecules. With the typical aptamer being 10 times smaller than an antibody, they allow improved access to these diseased tissues for better drug delivery.
As aptamers are oligonucleotide-based ligands, there is potential to synthesise the final therapeutic as a contiguous molecule, overcoming bioconjugation issues and ensuring product consistency in manufacturing. Also, being oligonucleotide-based rather than protein-based, aptamers offer the potential for reduced immunogenic responses, and so improved patient tolerability and the ability to redose where needed.
All these aptamer characteristics of target specificity, small size, and being oligonucleotide-based, offer an ideal profile for gene therapy delivery vehicles. The field has now recognised this and traction is building in this space for the use of aptamers as delivery vehicles to enable improved gene therapy efficacy.
Combined with this need in the gene therapy market for targeted delivery vehicles is the demonstration over recent years of the power of aptamers as targeted therapies. Since their initial explosion onto the market, a growing pipeline has demonstrated the promise of aptamer therapeutics across a range of indications, with 13 molecules in clinical trial and 2 approved for use.
Despite initial thoughts that only modified RNA aptamers would have sufficient in vivo half-life for therapeutic effect, multiple unmodified DNA aptamers are now showing good results in the clinic. This increases the confidence in aptamers as therapeutic modalities and offers flexibility in approaches with aptamers. Indeed, these molecules’ short half-life and renal clearance can be used as an advantage, allowing rapid delivery and excretion to prevent side effects from potentially toxic payloads. An example of this is Bicycle Therapeutics’ BT5528, where the small size and renal excretion of Bicycles enabled improved safety profiles with good functionality compared to a larger antibody-based delivery system of the same payload, MEDI-457. Yet, the same strategies apply just as well to aptamers.
Improved development processes for aptamers have given us higher-performing ligands, while advances in their manufacture are reducing cost, enabling scaling and enhancing access to aptamers across the industry. This has opened the door for multiple aptamer applications, from biosensors and affinity purification to targeted therapies. While the conversion of aptamer-based drug delivery vehicles to approved products will likely see further refinements in aptamer development and our understanding of their specific drug delivery niche, the rise in numbers of aptamers in pre-clinical development indicates that we are already well on our way to success in this field.
–
MARÍA ELENA MARTÍN, PH.D.
Senior Research (Aptamer group co-Leader), IRYCIS-Hospital Ramón y Cajal
Aptamers, a growing technology for diagnostic and therapeutic purposes
When aptamers were discovered in 1990, few would have thought of the great impact they would have both academically and industrially. However, more than 3 decades later, they have become a reality, mainly due to their high specificity and affinity for their target. Aptamers, single-stranded nucleic acids (ssDNA and RNA), are relatively easily selected through an in vitro selection method by exponential enrichment in the presence of the ligand (SELEX). Aptamers adopt three-dimensional structures that allow them to bind stably and very specifically to their targets, which can range from small molecules to complex multimeric structures. On the other hand, the growing industrial interest is determined because, in addition to their exceptionally high specificities and affinities, they have interesting advantages over their natural competitors, the antibodies, not in vain aptamers are known as chemical antibodies. Among these advantages are their increased stability, easy regeneration, simple modifications with different reporters during their synthesis and low cost.
Aptamers are important diagnostic and therapeutic tools, and although they are taking time to reach clinical practice – only two aptamers have been approved by the FDA to date – their use in different diagnostic systems, in detection platforms or as technological tools is becoming increasingly widespread. If, at this moment, a search will be made on the clinical trials page with the word aptamer (https://clinicaltrials.gov/search?term=aptamer) we would obtain 58 results. Of these, more than 30% correspond to trials related to macular degeneration, but, If we focus on the remaining trials, we find that a 37% of these trials use, or search to establish and validate, aptamer-based detection systems. It is interesting to note that these clinical trials have been initiated during the last 10 years, showing that aptamers are increasingly becoming an alternative to conventional systems (biosensing, image, discovery), especially for their use in biosensors to measure analytes in liquid biopsy, as well as their inclusion in proteomics platforms, replacing antibodies.
However, we cannot forget the high therapeutic potential of aptamers, despite the fact that there are still few clinical trials investigating their use as drugs in different pathologies. Specifically in cancer, my main research line, although numerous articles have been published on the use of aptamers in several cancer types, to date only three aptamers have entered clinical trials for cancer therapy: AS1411 and NOX-A12 and, more recently AM033. However, I am convinced that aptamers will become antitumor drugs for clinical use, among many reasons because of their high effectiveness and low toxicity and immunogenicity.
It has been more than 20 years since our laboratory established aptamer technology as one of the main lines of research. Our main objective has been to select aptamers both as bio-targeting molecules and as potential therapeutic tools. Since then, we have advanced along with this technology, have successfully obtained and characterized aptamers that are being tested in aptamer-based detection systems, but in addition, and linking the two research lines, selection and characterization of aptamers and cancer, we have obtained aptamers against proteins with an important role in cancer. The study of the use of aptamers as drugs in cancer is currently the main line of my research.
–
VÍCTOR M. GONZALEZ
Senior Researcher (Aptamer group co-Leader), IRYCIS-Hospital Ramón y Cajal
Aptamers: from basic research to clinics. How is the road map?
One of the questions we ask ourselves, almost 35 years after the first articles on aptamers were published by Larry Gold and Andrew Ellington, is why there are only two aptamers (Macugen and Izervay) approved by the FDA for clinical use. It is true that aptamers have characteristics that give them important advantages over other therapeutic molecules such as small molecules, peptides or antibodies. However, it is also true that, since they are novel molecules, it has been difficult to make progress on regulatory issues. Are aptamers chemical or biological substances? This, a priori, simple question is very relevant from a regulatory point of view.
But before we get there, the development of an aptamer starts in a laboratory trying to find a therapeutic solution for a given pathology.
First, we must identify the therapeutic target of interest and the characteristics of the pathology to be treated, which will allow us to establish the precise conditions under which we will select the aptamers. This selection is carried out by means of an in vitro evolution process called Systematic Ligand Evolution by Exponential Enrichment (SELEX). In this technique, aptamers are selected from a set of random oligonucleotides by iterative rounds of selection and amplification. These aptamers can fold into diverse three-dimensional structures, allowing them to bind to a wide variety of targets with high affinity and specificity.
Once aptamers capable of binding with high affinity and specificity to the target molecule have been identified, they are optimized by reducing their size to favor their arrival at the tumor site and, if necessary, by modifying their sequence or including modified nucleotides, which stabilize their structure and, consequently, increase their half-life and improve their biodistribution. These optimized aptamers are characterized in terms of activity in cellular models of the disease (2D cultures, spheroids or organoids) in order to confirm their potential therapeutic capacity.
This beginning of the adventure can last between 2 and 3 years. Subsequently, the preclinical development of the potential drug begins, in which the candidate aptamer is studied in animal models of the disease. During this phase, the potential drug must show its efficacy, as well as the absence of adverse effects, and the most effective dose must be determined. If the results are positive, it is necessary to perform these same tests under regulatory conditions, i.e. using the aptamer synthesized under GMP conditions and analyzing its status of Chemistry, Manufacturing, and Controls (CMC).
Once this preclinical regulatory phase, which lasts between 4-5 years, clinical trials can begin, once approved by the regulatory agencies. These trials are divided into different stages, called phases. Let’s explore each phase: In Phase 0, a very small number of people (usually fewer than 15) participate. Investigators use a very low dose of the medication to ensure it isn’t harmful to humans before proceeding to higher doses in later phases. If the medication behaves differently than expected, additional preclinical research is conducted before deciding whether to continue the trial; in Phase I participate approximately 20 to 80 healthy individuals without underlying health conditions. The goal is to determine the highest safe dose that humans can tolerate without serious side effects. Investigators closely monitor participants to observe how their bodies react to the medication. Besides safety and dosage, they also evaluate the best administration method (oral, intravenous, or topical). About 70% of medications move on to phase II after phase I; In Phase II, several hundred participants with the specific condition the medication aims to treat are involved. The same safe dose from phase I is administered. Investigators assess effectiveness and gather more information about side effects over several months or years. While larger than earlier phases, phase II still doesn’t demonstrate overall safety; in Phase III, randomized and blind testing occurs in large groups of people. The focus is on confirming effectiveness, monitoring side effects, and comparing the new treatment to existing ones. Data collected during this phase supports the safe use of the drug. If successful, the medication moves toward approval; Finally, the Phase IV, also known as Post Marketing Surveillance Trials, occurs after a treatment is approved. Researchers monitor the drug’s long-term effectiveness and its impact on patients’ quality of life.
Following this roadmap, in our laboratory we developed an aptamer against TLR4, since the relevance of this receptor involved in innate immune response in the inflammation that occurs around the core after ischemic stroke had been described. We demonstrated that this aptamer had an antagonistic effect on the receptor in cell cultures and, subsequently, that it produced a decrease in infarct size in experimental animals subjected to experimental stroke, without producing any relevant toxic effect. Finally, the clinical trial was addressed, where the potential drug was shown to have excellent safety and a significant clinical effect, reducing mortality and disability compared to placebo.
As I mentioned at the beginning, although only two therapeutic aptamers have been approved for clinical use, there are currently more than a dozen in different phases of clinical trials, which gives us hope for the future.
In summary, aptamers, with their unique properties, continue to evolve from basic science to clinical impact, promising breakthroughs in medicine.
–
GREGORY PENNER
President & CEO, NeoVentures Biotechnology Inc.
Constraints and solution for successful commercialization of aptamers
How has the aptamer technology evolved in the last decades in terms of discovery, selection, synthesis, and purification? Are there still unresolved issues?
Aptamer applications have expanded in the last decade in regard to electrochemistry, nanofluidics, and aptasensors, but the basis for the development of the aptamers themselves, SELEX has not changed substantially. We have found difficulties with certain aptamers that we have developed in terms of a loss of performance in biological fluids such as blood or saliva.
I think that the primary constraint to the commercial development of aptamers has been the lack of a system analogous to the immune tolerance system that exists for antibody development. Immune tolerance ensures that any antibody that cross reacts even weakly with an existing host epitope is eliminated. There have been efforts to achieve the same effect with SELEX based development through the use of counter selection. Counter selection is very effective at removing sequences from the enriched library that cross react strongly with the counter selection target. It is progressively less effective as the cross reactivity decreases in strength. This is important because an abundant protein like serum albumin is present on average in blood at a concentration of 600 uM. If the target for the aptamer is present at a concentration of 600 pM, there is a need for one-million-fold specificity, or the aptamer will be saturated by binding to serum albumin. In my opinion this is the key constraint to aptamer success.
To overcome this constraint, we have redesigned the selection process.
We have reduced the number of random nucleotides such that it is now possible to apply the same set of initial naïve sequences to different targets. This means that we can apply the same library of sequences to a desired target and to serum albumin. One question that we are often asked about this approach is whether we are concerned about the reduction in structural diversity created by reducing the number of random nucleotides. We have actually increased the structural diversity by removing the constraint imposed by contiguous random nucleotides on their ability to interact with each other by introducing fixed sequences between random sequences. We design thousands of possible templates and screen them for structural diversity in order to choose those templates with the highest level.
Our Neomer reinvention of aptamer development represents the next generation of aptamers by enabling an in-silico analogue to immune tolerance. This concept is protected by patent filings, but we do license neomer libraries out for use by others.
–
JEAN-JACQUES TOULMÉ, PhD
Chief Scientic Officer, Novaptech
Aptasensors for the detection of contaminants in water and beverages.
Nucleic acid aptamers provide a validated alternative to antibodies for therapeutic, diagnostic and analytical applications. They offer a number of advantages over their protein rivals with respect to production and use. In particular, no live material -cell or animal- is required for their production.
Aptamers are chemically synthesized and can be modified at will. They can be conjugated to dyes or grafted onto a support. Once identified their sourcing is guaranteed forever at an affordable cost. Large amounts can be synthesized under GMP conditions if necessary. Since their discovery in the early 90’s multiple aptamers were described against a bunch of targets ranging from small organic molecules to proteins, bacteria and live cells.
Targeting small molecules (molecular weight less than 1,000 Da) with aptamers remains a challenge as they generally offer limited potential for generating interactions (stacking, electrostatic, hydrogen bonding) with oligonucleotides. Consequently, the equilibrium binding constant for aptamer-small molecule complexes lies generally in the micromolar range which might constitute a limitation when the relevant concentration for analysis is low. Thanks to our NOVAswitch technology, which is based on the identification of structure-switching aptamers we perform the selection against free small molecules in solution. We do not require them to be grafted on beads or resin. Such aptamers are then easily converted into sensing elements, the structural change being associated to a signal. Due to the versatility of the oligonucleotide synthesis the conjugation of aptamers to fluorophores, nanoparticles or redox markers allows quantitative optical or electrochemical detection of the target.
Extensive usage of pesticides in agriculture is known to cause severe problems in food and environment. Antibiotics are applied to veterinary medicine due to their broad antibacterial spectrum. Residues of these antibiotics can be accumulated in dairy cattle, leading to contamination of the environment and milk. Pesticides and antibiotics are causing major public health problems.
At Novaptech we selected DNA aptamers specific to benzimidazole fungicides. Carbendazim (CBZ) and thiabendazole (TBZ) are broad spectrum fungicides used to protect a wide variety of crops and fruits against fungal diseases. From a ssDNA library we identified 82 nucleotide long aptamers to TBZ and CBZ. The aptamers with the highest binding affinity were subjected to post-selection optimization including truncation and mutagenesis in order to identify the key nucleotides. The optimized, approximately 40 nt long aptamers have a Kd in the low micromolar range. The unique stem-loop structures were subsequently integrated into optical biosensors for the quantitative detection of fungicide, by fluorescence and colorimetry using gold nanoparticles. In spite of the high homology between CBZ and TBZ, the anti-CBZ aptamer poorly recognized TBZ and vice-versa CBZ was hardly recognized by the anti-TBZ aptamer.
We also selected aptamers to a series of antibiotics used in animal medicine with a focus on aminoglycosides (AG) and cyclins. We identified highly specific aptamers on the one hand and aptamers that recognize the entire class of molecules. For instance, we characterized and optimized an aptamer that discriminates streptomycin from dihydrostreptomycine, highlighting the exquisite specificity of this class of ligands. Another aptamer was able to detect seven different AGs (streptomycine, gentamicin, paromomycin, dihydrostreptomycine, apramycin, neomycin and amikacin).
Taking advantage of the structural switch of these aptamers between target-free and -bound states we engineered fluorescence “turn-on” biosensors based on molecular beacons in which the binding of the target to the aptamer results in an increased distance between a fluorophore and a quencher.
Several analytical methodologies are routinely used to detect pesticides and antibiotics in water, beverages and food, based on physical-chemical techniques and microbiological tests. These are highly selective and sensitive methods, which, however, require expensive instruments and trained professionals to operate the equipment. In addition, the analysis is generally performed in remote laboratories and the results are known after several hours or even days. The development of portable, rapid, multiplexed and cheap tests is of great social and economic interest. This would enable on-site measurements and rapid decision making. This would be beneficial for both manufacturers and consumers.
Aptamer-based sensors could fulfill these requirements. They are cheap reagents and are easily associated to diverse signal transduction modalities (colorimetry, fluorescence, electrochemistry). Indeed, in recent years numerous publications came out on the design of assays and devices for the detection of pesticides, endocrine disruptors and drug residues.
Several hurdles must be overcome for the development of such aptasensors. Beyond the difficulty of selecting aptamers to low molecular weight targets with limited potential for interaction with oligonucleotides, the sensitivity required for relevant applications in the field remains a challenge.
Indeed, the limit of detection of interest is frequently in the low ppb range. The introduction of chemically-modified aptamers with an extended chemical diversity together with their combination with highly sensitive signaling methods (ultra-bright nanoparticles, FETS) could make it possible to achieve the very low levels of detection required. Another constraint comes from the detection of these contaminants in complex matrices.
Carrying out the selection in a medium mimicking the matrix in which the apta-sensor will ultimately be used could help reduce the background. Of note the same difficulty of detection in complex matrices applies to sensors based on other biomolecules. For instance, from this point of view aptamers rival with antibody-based sensors for the detection of antibiotics. Aptamers were also demonstrated to detect biomarkers in body fluids. Diagnostic constitutes another field for developing apta-sensors for point of care tests. No doubt that aptamer-based kits and devices for the detection of molecules of interest for health safety and medicine will be made available in the near future.
