(M1230-02-08) Comprehensive AAV-Based Vector Shedding Assessment by PCR:  A Case Study with Whole Blood, Serum, Semen, Urine, and Buccal Swab

Purpose: In this abstract, we describe a case study focused on lessons learned in PCR testing which commonly support Cell & Gene Therapy as well as Vaccine trials. Recombinant AAV-based gene delivery makes possible transformative and curative treatment options for genetic disorders and is also the leading platform in the field. Studying shedding profiles of viral particles is important to understand the potential risk associated with transmission of the virus and associated gene therapy to individuals (for example, friends, family members, and healthcare workers) and the environment. AAV may be transmitted through respiratory, gastrointestinal, sexual, and possibly vertical (mother to fetus, although pregnant women are currently excluded from gene therapy trials) routes. Therefore, levels of AAV in a comprehensive set of challenging matrices such as whole blood, serum, semen, urine, stool, and saliva swabs are routinely monitored to understand the risks holistically. Given the challenges of detection in the outlined matrices, optimizing the sensitivity of detection can provide important safety data to support clinical development. Currently, quantitative polymerase chain reaction (qPCR) assays are considered the gold standard for studying viral shedding. Here, we describe the development and validation of a sensitive, reproducible, and robust client-specific AAV qPCR assay for use in viral shedding studies.

Methods: Our group developed, optimized, and validated a qPCR assay in whole blood, serum, semen, urine, and buccal swab. The cell-rich matrices (whole blood and semen) used the MagMAX DNA Multi-Sample Ultra 2.0 Kit. Semen samples were pre-processed with MagNA Lyser Green Beads and extraction buffer. Each of the matrices was pooled and spiked with client-provided AAV viral stocks independently at pre-determined concentrations spanning the dynamic range of the assay. Nucleic acid extractions for whole blood, swab, and semen specimens were performed using the KingFisher Flex platform. Nucleic acid extractions for urine and serum were performed using the easyMAG platform. The total nucleic concentration in ng/µL in each extracted whole blood and semen sample was determined by spectrophotometry at 260 nm. Nucleic acid amplification was performed using the ABI 7500 SDS qPCR instrument in a single well format. A seven-point standard curve (5 copies/reaction to 5 x 10⁶ copies/reaction) was created for quantitation by dilution of a 700 bp gBlock. Concurrent research by our group on ThermoFisher’s KingFisher Flex instruments revealed lot-to-lot variability issues with MagMAX Viral/Pathogen (MVP) extraction reagents.

Results: For the client-specific AAV qPCR assay, the Limit of Detection (LOD) ranged from 38 copies/mL to 544 copies/mL (whole blood – 245 copies/mL or 3 copies/µg, swab – 71 copies/mL, semen – 544 copies/mL or 1 copy/µg, urine – 44 copies/mL, and serum – 38 copies/mL) based on the matrix type. The Lower Limit of Quantitation (LLOQ) ranged from 100 copies/mL to 1450 copies/mL (whole blood – 400 copies/mL or 6 copies/µg, swab – 125 copies/mL, semen – 1450 copies/mL or 5 copies/µg, urine – 100 copies/mL, and serum – 100 copies/mL). All matrices demonstrated an intra-assay and inter-assay %CV at or below 40% and 50%, respectively. All matrices demonstrated acceptable Accuracy, Selectivity, Linearity and Dynamic Range.Concurrent independent investigations with ThermoFisher identified pH drifts of binding buffers as a probable cause for the observed MVP1 kit lot-to-lot variability issues. A new lot of kit generated ~4 Cts earlier than the current lot in use. In a qPCR assay, a 3.3 Ct difference is associated with a 10-fold copy number difference. Based on the direction of the shift, such a difference can increase the risk of false positivity or false negativity in a clinical trial.

Conclusion: This assay contributes to enhancing our understanding of viral shedding dynamics, thereby facilitating informed decision-making in the advancement of gene therapy treatments towards broader, life-enhancing clinical applications. The observed variability with the MVP1 kit highlights the need for a robust Quality Control process to support multi-year clinical trials.