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Gene therapies offer powerful treatments to patients, but researchers must ensure they can’t be unknowingly transmitted to others.

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Detecting exposure risk from gene therapy viral vectors

Researchers created an improved method for assays that detect viral vectors in a patient’s blood or urine. It’s used to ensure that bystanders will not be exposed.
Allison Whitten
| 5 min read
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For most of human history, a person born with a genetic disorder lived with the condition for life. Now, gene therapies that edit the genetic mutation offer a potential cure to lifelong diseases like Duchenne muscular dystrophy and hemophilia as well as conditions that emerge later in life, like bladder cancer. 

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Johannes Stanta leads a team at Celerion that rigorously analyzes data for non-traditional endpoints in clinical trials using methods like qPCR and flow cytometry.
Credit: Ryan Learoyd from Shutter Go Click Photography

Before any gene therapy is approved, it must pass through clinical trials to rigorously prove its efficacy and safety — including its safety for others not taking the medicine. If the viral vectors that deliver gene therapies to patients escape into the environment, any individuals in close proximity could be exposed. To avoid this scenario, regulators have called for the addition of shedding assays in gene therapy clinical trials (1). These assays measure the amount of viral vector DNA that ends up in a patient’s blood, urine, tissue, and beyond to confirm that no transmission of the gene therapy could take place.

Recently, Johannes Stanta, Global Director of Molecular and Cell Biology at Celerion, and his team developed a new approach for shedding assays after discovering concerns with the reliability of the data generated by most labs (2). Stanta’s group developed a streamlined method for adeno-associated virus (AAV) vectors, which are currently the most popular vectors in gene therapies (3). 

Although shedding assays are measured as secondary endpoints, Stanta emphasized that getting the data right is just as important as for primary endpoints. “We owe this to the patients, to the volunteers, that we don't just waste their sample on data that might be unreliable. So, we really want to do the best job we can on samples that we get. They are absolutely precious,” he said.

What are the risks of shedding from gene therapies?

Before the field of gene therapy took off, the Food and Drug Administration and European Medicines Agency wrote guidelines suggesting that gene therapy clinical trials should include shedding assays as monitoring tools. Since then, we haven't seen any evidence that any of the vectors that we’re currently using are released into the environment. We especially have good evidence that’s not the case with AAV vectors. The risk of infectivity from AAV vectors is nearly impossible. 

However, there is one aspect where there are still a lot of unknowns. We don't know exactly if vectors are transmitted through semen; and if the partner gets pregnant, it’s possible that the capsid could be transmitted to the offspring. These things are totally untested. From my point of view, that is the only real risk. 

Why did you develop your shedding assay?

Currently, if we give people the task to validate a qPCR-based assay to measure shedding, most people in the industry only focus on validating the qPCR part. They add primers and probes and run cycles and then generate a response to see how much plasmid DNA the machine detects. At the end, they get a beautiful assay because it's easy. They’re just using one machine; they have control of all the materials; and everything's in there. 

I hope that we spark a new bioanalytical revolution in the space of PCR where everyone starts doing it this way
– Johannes Stanta, Celerion

In that scenario, people forget one key thing. Most people do not validate the extraction of DNA from the sample. This is so important because we need to ensure accuracy of the whole workflow, not just the PCR results at the end. If we get a sample from blood, or urine, or tissue, we want to measure how much of the plasmid DNA is in the sample. Our task is not to determine how much DNA is in the sample after it's already been extracted from the machine, which is what many researchers do when they run PCR on the clean sample coming out of the extraction. As soon as we start trying to validate the extraction method as well, we realize that the assay is not as good as we thought.

For our method, we combined the extraction and the qPCR at the stage where we add the standards and the reference material before we start the extraction instead of after. Consequently, we controlled the amount of variability, which allows for more accurate results that answer not just how much DNA is in the extract, but how much DNA from the vector is actually in the original sample that we received from the patient. That is the answer we want.

What did you learn from developing this new approach?

I learned not to underestimate the difficulty of shedding assays. They have very unique challenges, so we can't just say, “I've got a PCR machine in the corner of the lab. I'll do a shedding assay. How difficult can it be?” Actually, it's really difficult.  

Our goal with sharing our work now is to spark discussion around industry harmonization and increase awareness that shedding assays, in particular those used for clinical studies, are their own unique entities. We want to start the best practice so that when it comes time for others to develop a shedding assay for gene therapy clinical trials, they start from a better position. Hopefully, this will accelerate the field and help everyone arrive at a solution much more quickly.

What are your future plans with this approach? 

We hope that we can support as many shedding projects as possible and really put our approach to the test. I want to provide the most streamlined and cheapest way of measuring shedding in a compliant way. 

Hopefully, we will be able to use the same approach when new capsids for gene therapy come on the scene. The next one after AAV vectors appears to be  lentiviruses, so it would be great to test how the type of capsid influences shedding assays. I'm up for that challenge. I look forward to applying the same thought process that we used in this project to come up with the best possible way to analyze lentivirus capsids.

Could this approach be used for other types of assays beyond shedding?

Absolutely. I use the approach of adding the reference material before the extraction for various projects that we have ongoing, including our mRNA projects. It translates well for many other methods as well. 

If we can demonstrate how easy it is to create a solid assay where we don't cut any corners and then apply it to all of our PCR-based assays, we will benefit from much better data without caveats. I hope that we spark a new bioanalytical revolution in the space of PCR where everyone starts doing it this way.

This interview has been condensed and edited for clarity.

References

1. Food and Drug Administration Center for Biologics Evaluation and Research. Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncolytic Products. (2019). at https://www.fda.gov/regulatory-information/search-fda-guidance-documents/design-and-analysis-shedding-studies-virus-or-bacteria-based-gene-therapy-and-oncolytic-products

2. Stanta, J. AAV8 Shedding Assay to Support Gene Therapy Clinical Trials. AAPS National Biotechnology Conference. San Francisco, CA. May 16, 2024.

3. Pupo, A. et al. AAV vectors: The Rubik’s cube of human gene therapy. Molecular Therapy  30, 3515–3541 (2022).

About the Author

  • Allison Whitten
    Allison Whitten joined Drug Discovery News as an assistant editor in 2023. She earned her PhD from Vanderbilt University in 2018, and has written for WIRED, Discover Magazine, Quanta Magazine, and more.

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