Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G

Figures

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  Figure 1. Standard Curve Comparison (A and B) Representative standard curves for VSV-G using nuclease-free water as the diluent (A) or control human DNA as the diluent (B). (C) Average slopes for VSV-G standard curves using nuclease-free water or control human DNA as the diluent (n = 7). (D) Average R2 values for VSV-G standard curves using nuclease-free water or control human DNA as the diluent (n = 7). Error bars represent SD.

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  Figure 2. Limit of Detection for VSV-G in Nuclease-free Water and Control Human DNA (A) Quantification cycle (Cq) values for 10, 8, 6, 5, 4, and 3 copies/reaction and NTC. UND, undetectable; sample detection failed to reach the threshold of detection in 40 cycles (n = 4; 3 replicates per assay). (B) Number out of 3 replicates detectable within 40 reaction cycles for 10, 8, 6, 5, 4, and 3 copies/reaction and NTCs (n = 4). Error bars represent SD.

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  Figure 3. Standard Curve Comparison with DMSO Standard Curves (A) Average slopes for VSV-G standard curves using nuclease-free water (n = 7; repeated from Figure 1 for comparison), control human DNA (n = 7; repeated from Figure 1 for comparison), or control human DNA with a final 3% (v/v) DMSO reaction concentration (n = 16) as the diluent. (B) Average R2 values for VSV-G standard curves using nuclease-free water (n = 7; repeated from Figure 1 for comparison), control human DNA (n = 7; repeated from Figure 1 for comparison), or control human DNA with a final 3% (v/v) DMSO reaction concentration (n = 16) as the diluent. Error bars represent SD.

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  Figure 4. Limit of Detection for VSV-G in Control Human DNA with a 3% v/v Final Concentration of DMSO in the Reaction (A) Cq values for 10, 8, 6, 5, 4, and 3 copies/reaction and NTC. UND, undetectable; sample detection failed to reach the threshold of detection in 40 cycles (n = 16; 3 replicates per assay). (B) Number of 3 replicates detectable within 40 reaction cycles for 10, 8, 6, 5, 4, and 3 copies/reaction and NTCs (n = 16). Data from Figure 2B are included for comparison. Error bars represent SD.

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  Figure 5. Detection of VSV-G DNA in Clinical Product Test Samples (A) Cq values for non-transduced control samples and transduced CAR T cell samples compared with the Cq values of the 10 copy positive control in the standard curve for each respective assay. (B) Cq values for sample DNA alone, sample DNA with 10 copies of VSV-G plasmid DNA spiked in, and the 10 copy positive control on the same assay (n = 7). Error bars represent SD.