(M0930-10-59) Validation of a Quantitative PCR (qPCR) Assay for the Detection and Quantification of Replication Competent Lentivirus for Patient Monitoring in Lentiviral Vector-Based Cell Therapy Studies

Purpose: Lentiviral vectors (gene delivery vehicles) are playing a critical role in the emerging field of cell therapies. These vectors are a common delivery system for the introduction of modified or engineered genetic cargo due to their relative safe and stable integration into the host cell genome. Despite these advantages, there is a potential risk of replication competent lentivirus (RCL) being introduced in patients receiving lentiviral vector-based cell therapies, such as chimeric antigen receptor (CAR)- and T cell receptor (TCR)-T cell therapies. Therefore, it is critical for patients to be regularly monitored for presence of RCL. Current Food and Drug Administration (FDA) guidelines recommend use of PCR-based assays for RCL detection¹. Here, we describe the validation of a qPCR assay based on the detection and quantification of the vesicular stomatitis virus glycoprotein (VSV-G) envelope protein gene sequence for RCL.

Methods: The primer/probe set was designed in-house targeting the VSV-G sequence. Linearized and purified pCMV-VSV-G Envelope Vector was used as positive control in the qPCR assay. Copy concentration was determined using Quant-iT® PicoGreen and diluted in nuclease-free water to a working stock of 1x10⁷ copies/5uL. For each qPCR, 10-fold serial dilutions were prepared in water to achieve a dilution series of 1x10⁶ to 10 copies/5uL reaction and assessed in triplicate. Data generated from the dilution series was used to construct a standard curve and generate quantitative data. To monitor for assay contamination, no template controls (NTCs) and sentinel controls were assessed in duplicate on each qPCR. Quality control (QC) samples were generated by performing independent dilutions of the positive control at 100 (low), 1x10⁴ (medium), and 1x10⁶ (high) copies/reaction in 1ug of human blood genomic DNA (gDNA) and assessed in triplicate on each qPCR. qPCR was performed on the QuantStudio® 6 Flex. Raw data was processed using the QuantStudio® software. Data was exported to excel for further calculation and analysis. gDNA from whole blood and peripheral blood mononuclear cell (PBMC) samples was extracted using the QIAmp DNA Blood Midi Kit and QIAamp Blood Mini Kit, respectively. DNA concentration and purity of samples was determined spectrophotometrically by taking the optical density readings in triplicate at 260nm and 280nm. Up to 1ug of gDNA was assessed in each qPCR reaction, where applicable.

Results: The limit of quantitation (LOQ) ranged from 10 copies/reaction (lower limit of quantitation, LLOQ) and 1x10⁶ copies/reaction (upper limit of quantitation, ULOQ). The limit of detection (LOD) was equivalent to the LLOQ. Robustness of the qPCR assay was assessed using the standard curve with differing reagents, operators, and instruments, displaying consistent amplification efficiencies (90-110%) and Ct signals (%CV ranged 0.27% to 1.79%) within the determined LOQ range. Specificity of the VSV-G primer and probe set was evaluated in human gDNA extracted from whole blood and no detectable copies of VSV-G were observed. Accuracy was assessed by spiking the VSV-G positive control into human gDNA at the determined LLOQ and 1 log above. Respective recoveries were 113% and 102% when compared to the related spike controls. Detection of RCL using linearized VSV-G plasmid as positive control was compared to VSV-G pseudotyped lentivirus extracted from HEK293T cells. Both materials were assessed across all levels of the standard curve, resulting in similar linearity (0.99 and 1.00) and amplification efficiency (96.12% and 96.37%). To assess reproducibility and efficiency of the extraction methodology in human whole blood and PBMCs, 4000 copies of VSV-G DNA was spiked into each matrix, extracted, and assessed by qPCR over 3 separate occasions. Recoveries ranged from 35% to 76% in gDNA extracted from blood, and 28% to 31% from PBMCs. Unspiked matrix displayed no detectable VSV-G copies. Further matrix effects were evaluated by spiking 100 copies of the VSV-G positive control into the respective unspiked replicates with no observed amplification inhibition. Lastly, assessment of inter- and intra-assay precision demonstrated respective %CVs ranging from 0.10% to 2.05% and 0.55% to 2.18%. Application of this RCL detection assay is currently being used for patient monitoring in phase 1 clinical trials evaluating the safety and tolerability of potential CAR-T therapies. Results have indicated no detectable RCL.

Conclusion: Due to the potential pathogenicity of RCL, it is critical to monitor patients post-infusion of lentiviral vector-based cell therapy products, such as CAR/TCR-T therapies. qPCR assays offer rapid assessment of RCL. We demonstrate an RCL detection qPCR assay that is linear from 1x10⁶ to 10 copies/reaction, specific to the VSV-G target sequence, robust with respect to reagents, operators, and instruments, and is sensitive down to 10 copies/reaction. Our data also shows that the assay is accurate in quantifying VSV-G target sequence in the presence of gDNA extracted from human whole blood and PBMCs, indicating no matrix effect. This assay is being used to monitor RCL in patients undergoing CAR-T therapies in phase 1 clinical trials and is validated for patient monitoring of RCL in future clinical studies.

References: 1U.S. Food and Drug Administration. (2020). Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up.

Acknowledgements: The authors have no relevant conflicts of interest.