A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears

Our protocol quantifies adeno-associated viral vectors in complex matrices. Accurate detection and quantification are essential to assessing their performance in clinical trials. This protocol specifically seeks to fill the current regulatory gaps in the design of good laboratory practices'compliant molecular studies.

As a part of viral vector development, regulatory agencies require the assessment of vector bio-shedding. ddPCR is a promising technology for this assessment as it allows for accurate quantification without the use of a standard curve and improved sensitivity compared to qPCR. Accurate quantification in the complex matrices encountered in clinical settings, such as tears, can be challenging to validate due to PCR interference and inhibition.

Limited clinical sample amounts can also limit the ability to detect rare events if the assay is not sufficiently sensitive. Our approach develops a repeatable and adaptable ddPCR-based framework to validate assays for use in regulated studies. This method avoids a specific DNA extraction step, allowing for more streamlined validation and provides clear criteria against which to evaluate assay performance to ensure robustness and repeatability.

This protocol has the potential to be applied to any viral vector shedding study, not just the detection of AAV in tears, as presented here. It creates a robust and repeatable framework upon which multiple fit-for-purpose, regulated programs can be developed.