Aptamer stability can be tuned for clearance mechanism and to prevent nuclease activity.

As the number of aptamers in the clinical pipeline grows, it demonstrates that aptamer stability can be optimised to match the therapeutic strategy. Binders can be highly stable when you need them to be or rapidly removed from circulation, with renal clearance to prevent any toxicity.

There are times when either one of these strategies might be the better route for therapeutic development, and our team is on hand to help you decide which way you might need to go to prevent any hurdles in your downstream development.

Multiple different factors contribute to aptamer stability in vivo. Here we’ll talk a bit about each of these and show you a simple modification we made to our library that gave us great aptamer stability in whole blood.

Resisting nasty nucleases for aptamer stability

As short, single-stranded DNA or RNA molecules, aptamers are open to nuclease attack. Nucleases can be endonucleases, which degrade the aptamer backbone to chew it up from the middle of the molecule, or exonucleases that degrade the aptamer from the 5’ and 3’ ends. There are plenty of both types of nuclease in the blood and we can protect against both to ensure aptamer stability in vivo.

There are a host of ways we can protect against nucleases.

Aptamer stability can be tuned to allow degradation once it has reached its site and achieved therapeutic effect or for longer in vivo half-life, by carefully balancing the backbone modifications with the capping of the aptamer.

To stop endonuclease activity, backbone modifications can be made to the nucleotides to include 2’fluoro or 2’-O-methyl groups that prevent the endonuclease enzymes accessing the aptamer backbone for degradation. To prevent exonuclease activity on the ends of the aptamer, we can add caps that turn the end of the aptamer back on itself, add phosphothioate groups to the ends or circularise the aptamer. All these modifications prevent exonucleases from accessing the aptamer 5’ or 3’ ends to increase aptamer stability in vivo.

Not all aptamers are modified for use in vivo. There are examples of clinical aptamers with no nuclease-resistant modifications. Both BC007 for heart failure treatment and ApToll for thrombosis treatment are unmodified DNA aptamers that appear to be stable enough as they are with no modifications. ApToll has shown a half-life of 9.5 hours, which, while not long, is long enough to exert its therapeutic effect in vivo, with promising clinical trial results seen.

Our modified library has built-in nuclease resistance

For RNA aptamers to be used in vivo, our library contains 2’fluoro-modified bases that protect the backbone from endonucleases. Once discovery has identified a specific aptamer sequence that binds to the required target with the right binding profile, then more backbone modifications can be added or some removed to tune aptamer stability.

Optimising the exit strategy

To tune aptamer stability for renal or hepatic clearance, different half-life extension modules can be added to the aptamer to extend half-life and overcome the threshold for renal clearance. Aptamers range in molecular weight from 4 to 30 kDa. Even at their largest, this molecular weight is still below the threshold for renal clearance, meaning the aptamers will be cleared through the kidneys and rapidly leave the body. Adding big bulky groups with extra molecular weight to the aptamer by conjugating PEG groups, or binding to blood proteins to increase circulatory half-life, can help extend the retention time in vivo and promote hepatic clearance over renal clearance.

The small molecular weight of aptamers allows rapid renal filtration for hit-and-run strategies or in vivo half-life modification strategies can extend the half-life for longer residency where required.

Reaping the benefits of renal clearance

First, let’s state that a speedy exit is not always bad. For example, if an aptamer is conjugated to a toxic payload, it can quickly hit the target, deliver the payload, and leave the body. This might prevent any potential problems from long half-lives that can cause uncontrolled payload release and toxicity in non-target tissues. Bicycle Therapeutics uses this strategy for shorter retention times and renal clearance to its benefit when targeting auristatin-derivative payloads, showing improved safety profiles compared to larger antibodies with longer half-lives carrying the same payload.

Similar fast renal excretion of aptamers can be helpful for imaging agents. In this case, long half-lives offer no benefit, and rapid release from the body can mean that patients’ stays in the hospital are shorter.

Playing the aptamer stability long game

Both approved aptamer therapies, Macugen and Izervay, use PEG groups to increase the size of the drug and help reduce renal clearance, along with 2’fluoro and 2’O-methyl backbone modifications for better aptamer stability with a longer half-life. PEG groups are bulky and inert, so they aren’t typically linked to immunogenicity issues. In terms of Macugen and Izervay, their half-lives are 10 and 12 days, respectively, which is comparable to larger antibody therapeutics.

Other methods to increase circulatory half-life include adding cholesterol to the end of the aptamer, adding an albumin binder as a bi-functional aptamer, or adding small-molecule drugs like NSAIDs that bind albumin in the blood to increase the circulatory time.

A quick case study to tune our aptamer stability in blood

We started with a stability study to test the library in human blood at dilutions ranging from 90% all the way down to 10% with an entirely unmodified DNA aptamer.

In concentrations over 30% human blood, there was complete degradation of the aptamers after just 5 minutes, showing they were not stable enough.

A simple modification was performed on both ends of the aptamer with no backbone modifications included, and the aptamer stability was improved. Retesting aptamer stability in whole blood showed no degradation even over a 24-hour period.

So, aptamer stability in vivo can be what you need it to be, offering you a flexible approach to various diagnostic and therapeutic strategies.

Get in touch with our team if you’d like to learn more.

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