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High-Sensitivity ddPCR Assays for Detecting AAV Vector Shedding in Ocular Gene Therapies

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High-Sensitivity ddPCR Assays for Detecting AAV Vector Shedding in Ocular Gene Therapies

High-Sensitivity ddPCR for Ocular AAV Vector Shedding Analysis

CELL & GENE | RNA | BIOLOGICS

Quantifying Vector Biodistribution with Absolute Precision.

For ocular AAV gene therapies, regulatory agencies require robust viral shedding data to assess safety and biodistribution. Detecting minute quantities of vector DNA in low-volume matrices like aqueous humor and tears demands assays with exceptional sensitivity and precision. Franklin Biolabs employs validated Droplet Digital PCR (ddPCR) to provide absolute quantification of vector shedding, generating the high-fidelity data required for IND submissions and clinical trial support.

    What is the LLOQ for your ocular ddPCR shedding assays?

    A: Our assays are validated to achieve the high sensitivity required for detecting vector DNA in distal fluids and low-volume ocular matrices. The specific lower limit of quantification (LLOQ) is established and confirmed during method validation for each unique vector construct and sample matrix combination.

    Which sample matrices do you support for these studies?

    A: We routinely process a range of ocular and systemic matrices, including aqueous humor, vitreous humor, tears, conjunctival swabs, whole blood, plasma, and serum, under a GxP-compliant framework.

    How does ddPCR compare to qPCR for this application?

    A: ddPCR provides absolute quantification without reliance on a standard curve, offering superior precision and day-to-day reproducibility. It is also less susceptible to PCR inhibitors often present in complex biological samples like tears.

    What is the typical turnaround time for a viral shedding study?

    A: Turnaround time is project-dependent. Standard timelines are established at study initiation to meet program goals, and expedited options are available to align with key program milestones.

Quantifying Vector Biodistribution in Ocular Gene Therapy

Assessing the biodistribution and shedding profile of an AAV vector is a foundational component of any preclinical safety program. For therapies targeting the eye, an immune-privileged compartment, understanding the potential for vector migration into systemic circulation or shedding via ocular fluids is a primary regulatory expectation. The analytical challenge lies in the extremely low concentrations of vector DNA expected in these samples, combined with limited available sample volumes.

Our bioanalytical strategy utilizes ddPCR to overcome these limitations. This technology partitions each sample into thousands of nano-liter sized droplets, allowing for the direct, absolute count of target DNA molecules. This approach provides the statistical power needed to confidently measure vector presence at the limits of detection.

“We started collaborating with UPenn Vector core in 2023 and the AAV vector which they manufactured laid a foundation for development of a gene therapy candidate which will enter soon preclinical studies. The key people from UPenn Vector Core joined Franklin Biolabs and our partnership transitioned without interruption from UPenn Vecor Core to Franklin Biolabs Research Vector Division. Franklin Biolabs is our trusted partner in our AAV-vector based gene therapy candidate development and we hope to continue the partnership for years to come.”
— Biotech Partner

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Validated Assays for IND-Enabling Studies

A robust analytical method is only as valuable as its validation. Drawing from principles established in studies requiring high-sensitivity quantification of biomarkers in unique biological matrices (PMID: 39282076), we conduct rigorous, GxP-compliant assay validation. This process establishes key performance characteristics:

  • Accuracy and Precision

  • Selectivity and Specificity

  • Lower Limit of Quantification (LLOQ)

  • Matrix Effect Evaluation

This level of characterization ensures that the data generated is reliable and defensible for regulatory review. This bioanalytical rigor is a key component in navigating the typical 18-24 month IND timeline. Since 2019, programs supported by our scientific leadership have achieved a 100% IND success rate, a legacy we continue at Franklin Biolabs, which launched as a new brand in 2024.

Integrated Bioanalytical and Preclinical Facilities

Our bioanalytical laboratories are co-located within our >100,000 sq ft preclinical facility, enabling efficient sample transfer and chain of custody for in vivo studies. This integration streamlines study execution from dose administration through to final data analysis.

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Scientific Process Diagram

This content is for informational purposes. For guidance specific to your therapeutic program, please contact our team for a consultation.

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