(M1530-01-05) Comparative Analysis of Replication-Competent Vector Quantification: Whole Blood vs. PBMCs in Gene Therapy Monitoring

Purpose: The quantification of replication-competent lentiviral vectors (RCL) is a critical component in evaluating the safety and efficacy of gene therapy vectors. While lentiviral vectors are well-suited for gene delivery and stable integration into the host cell genome, they carry potential health risks, including integration related transformation and the possibility of generating replication competent lentivirus (RCL) that could infect unintended cells. Due to this concern, all lentiviral vector modified cell-based products intended for patient treatment must undergo RCL testing. Traditionally, RCL quantification is performed using peripheral blood mononuclear cells (PBMCs) as the target cell population. However, whole blood, which contains a broader range of immune cells, offers more comprehensive sampling. Other advantages of using whole blood include reduced processing time, simpler and lower volumes of sample collection.  Advantages of using PBMCs include less background interference, and possibly higher sensitivity. This study aimed to compare the sensitivity, precision, accuracy, and stability of RCL quantification using whole blood versus PBMCs.

Methods: PBMCs were processed at a cell concentration of 4 million cells/mL. Whole blood was collected in 8.5 mL PAXgene tubes. All PBMC and whole blood samples were pooled and spiked with linearized RCL plasmids at pre-determined concentrations spanning the dynamic range of the assay. Nucleic acids extractions for PBMC specimens were performed using the QIAcube Connect and nucleic acid extractions for whole blood specimens were performed using the KingFisher Flex systems. The total dsDNA concentration in ng/µL in each extracted sample was determined by spectrophotometry at 260 nm. Quantitative PCR was performed using the ABI 7500 Fast (PBMCs) or QuantStudio 7 Pro (Whole blood) instruments in a triple well format. A seven-point standard curve (5 copies/reaction to 5 x 10⁶ copies/reaction) was created using a dilution of a linearized plasmid used for quantitation. Both sample types were validated independently with more than 200 independent full process (extraction and qPCR) samples each.  The performance characteristics for this comparative analysis are Sensitivity (Limit of Detection), Intra- and Inter-Assay Precision, Accuracy and Stability.

Results: The Limit of Detection was 9 copies/µg for PBMCs and 1 copy/µg for whole blood.  Inter-Assay Precision coefficient of variation (CV) was as high as 17% for PBMCs and 40% for whole blood. For both matrices, accuracy was met for the sample set (≤ 0.7 log10 copies/µg from expected). 100% and 99% of PBMC and whole blood samples met accuracy acceptance criteria, respectively.  And finally, stability was comparable for both matrices.

Conclusion: These results indicated that use of whole blood samples permitted higher assay sensitivity for detecting RCL, with advantages in sample handling and reduced processing time. This came at a cost of poorer precision for whole blood. These findings suggest that whole blood may serve as an efficient alternative to PBMCs for RCL quantification in gene therapy trials, offering a less invasive and more practical approach for monitoring viral replication in clinical settings.