Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells

Figure 4

Transfer of VL30 genomes by HIV-1 vector particles. (a) To test the hypothesis that human pathogens, including HIV-1, can potentially transfer VL30 genomes, 293T cell clones 6 and 7 (VL30 GCN of 5.17 and 2.68, respectively) were transiently transfected with the HIV-1 vector packaging and the VSV-G envelope expression cassettes. Vector particles were employed on naïve 293T cells either in the presence or absence of 10 μM AZT. DNA was extracted from treated 293T cells, and the VL30 genome copy number was determined by qPCR. (a) Graph bar showing the VL30 genome copy number in naive 293T cells exposed to lentiviral vector particles generated in cell clones 6 and 7, either in the presence or in the absence of AZT. DNA samples extracted from 293T cells comprising the lentiviral vectors pTK1261 and pTK2229 served as negative and positive controls, respectively. The significant reduction in VL30 genome copy number in AZT-treated 293T cells indicates that the observed transfer of VL30 genomes was reverse transcription dependent. PCR amplification products of the endogenous RNaseP gene served as loading controls. values were determined by the 2-way ANOVA test. The experiment was performed in triplicate. (b) Electrophoresis analysis of PCR products following amplification of DNA samples extracted from 293T cells transduced by HIV-1 particles generated in cell clones 6 and 7. DNA samples extracted from 293T cells transduced with the lentiviral vectors pTK1261 and pTK2229 served as negative and positive controls, respectively.

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