Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells

Figure 1

Inadvertently transferred VL30 genomes are transcriptionally active in HEK 293T cells. Detection of VL30 genomes and mRNA in 293T cells following exposure to PG13-conditioned media. (a) Physical map of the lentiviral vector cassette pTK2229 carrying a DNA sequence from the VL30 retrotransposon. The VL30 sequence is shown in a dotted box. It is located downstream of the internal CMV promoter, which is in opposite orientation to the LTRs and thus cannot initiate transcription of the VL30 sequence. The direction of transcription is indicated by an arrow. A CMV promoter replaces the 5 U3. It is followed by the 5 R and U5 regions. The packaging signal (Y), Rev response element (RRE), central polypurine tract (cPPT), woodchuck hepatitis virus posttranscriptional regulatory element (WP), internal CMV promoter, and Puro-T2A-mCherry reporter genes are shown. The modified self-inactivating (SIN) 3 U3 is shown as SINU3. The parental 3 poly purine tract (PPT) is shown. (b) PCR-based analysis testing the presence of VL30 genomes in 293T cells. DNA was extracted from either naïve or treated (exposed to PG13-conditioned media) 293T cells following 4 passages in culture. The presence of VL30 in the abovementioned DNA samples was determined by PCR. PCRs either in the absence of DNA or with DNA extracted from PG13 cells served as negative and positive controls. PCR products were visualized following gel electrophoresis. (c) Bar graph showing vector copy number analysis of VL30 genomes in 293T cells following exposure to PG13-conditioned media. The qPCR-based assay was done in triplicate. Serial dilutions of DNA from pTK2229 vector-transduced 293T cells served as a standard curve. DNA of naive 293T cells served as a negative control. (d) Employing RT-PCR-based analysis to characterize transcriptional activity of VL30 genomes in 293T cells. Total cellular RNA was extracted from either naïve or treated (exposed to PG13-conditioned media) 293T cells following 4 passages in culture. The presence of VL30 mRNA in the abovementioned RNA samples was determined by RT-PCR. RT-PCR reactions in the absence of RNA or with RNA extracted from PG13 cells served as negative and positive controls. PCR of RNA samples prior to reverse transcription served as controls for DNA contamination. PCR products were visualized following gel electrophoresis. (e) Bar graph showing quantitative (q) RT-PCR analysis of VL30 mRNA in 293T cells following exposure to PG13-conditioned media. The qPCR-based assay was done in triplicate. RNA of naive 293T cells served as a negative control. Levels of the endogenous hACTB mRNA served as an internal reference.

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