Recently, Matthew Pennington, PhD (one of KCAS’s Senior Scientists) was the senior author on a publication entitled “A Validated Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno Associated Viral Vectors in Bio Shedding Studies of Tears”. Below, Matt explains the significance in what they accomplished and how they were able to reach the findings they achieved using the technology and experience at KCAS.

The publication specifically focused on quantifying adeno-associated viral vectors (or, AAV Vectors). Adeno-associated viruses (AAVs) are a type of very simple DNA virus that scientists have been studying for nearly 60 years, and have developed it as a tool for gene therapy. Gene therapies broadly have the goal of deleting a dysfunctional gene, supplementing a poorly performing gene, or otherwise correcting a genetic deficiency, preventing a normal function in a cell.

Gene therapies are being researched for use in addressing any type of genetic disease you can imagine. One of the areas that this kind of treatment has found a lot of success is in ocular diseases – specifically in correcting genetic mutations that lead to early onset blindness. The success of Luxturna showed that AAVs can be powerful tools to correct diseases in the eye and has driven their use in other parts of the body. As part of the development process of any gene therapy product, regulators require different  studies to understand where that vector is going in the body, and how it might be released or shed into the environment, among others.

This is why we turned our focus to developing a method to support bioshedding studies of tears.

It is possible that when you treat these conditions, somebody with this vector could release it in their tears, and into the environment (“shedding”). That vector could then reach other people and either accidentally transfer the viral vector to them, or (and this is the bigger concern) there an exposure to the vector that leads to an unintended immune response in the third party person. Therefore, it is important to understand how long shedding happens and to what extent it occurs to better understand and manage potential off target exposures.

There are many methods that can be used to detect and quantifyshedding. We chose to use dropet digital polerymase chain reaction (ddPCR). This method is a polymerase chain reaction-based method that fractionates the sample into approximately 20,000 oil-in-water droplets, which are then individually amplified to endpoint, and read on a droplet reader. It then uses Poisson statistics to calculate the actual copy number of the target in the sample.

We used ddPCR because it allows for highly accurate quantification of the target without the use of a standard curve. As it is less susceptible to PCR inhibitors, it some cases it can simplify conventional workflows by eliminating the need for an extraction step. Its greater sensitivity allows for lower limits of detection. Collectively, this can allow for more rapid method development and validation timelines.

We work in a regulated atmosphere. These kinds of studies are ultimately reviewed by the FDA, EMA, or other agencies. Therefore, the assays are typically  either qualified or validatied to demonstrate that they are as accurate, precise, and robust as possible. This publication does this type of assay within the context of a GLP validation to show how we would suggest validating the assay, considering the relative lack of regulatory guidance on the matter.

Who will be most interested in this publication?

There are likely two main audiences who will most appreciate the publication we have put together. The first would be developers of viral vector and other gene therapy products. They can see our recommendations for how they approach these kinds of studies from a bioanalytical point of view.

The second audience would be other contract research organizations and other research-based companies looking to support this kind of assay. They will be able to review our approach and determine if it is one that they would like to adopt and align on. There is a lack of clear guidance from the FDA and other regulators on the validation of these types of assays. This study represents our suggested approach to validation.

More about the publication, available on…

This was a project developed entirely internally at KCAS and supported by our Cell & Gene Therapy department staff – of which I am a part. We had two analysts on the paper, Bry and Amanda who were critical in driving this project in the lab. Carrie Vyhlidall, PhD is our associate director and was instrumental in assisting with developing a validation approach in line with current industry standards.

To read the publication, click here:

For any questions about this piece, or any of KCAS’s other publications or services, please use the form below.