Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells

Figure 6

qPCR-based analysis of VL30 GCN and -RVV VCN in primary human T-cells. Naïve primary human T-cells were treated with the chimeric antigen receptor- (CAR-) carrying -RVV SFG.iC9.GD2.CAR.IL15 (samples 1, 2, 5, 6, 7, 11, 12, and 13). The vector was produced in the stable packaging cell line PG13. Samples 3, 4, 8, 9, and 10 were not exposed to the abovementioned CAR-carrying -RVV and served as biologic negative controls. DNA samples extracted from the abovementioned controls and vector-treated cells were used to determine GCN and VCN of the murine endogenous retrotransposon VL30 and the -RVV vector, respectively. DNA from naïve 293T cells served as a technical negative control. DNA from 293T cells transduced with the lentiviral vectors pTK2229 and pTK2151 served as positive controls for VL30 and -RVV genome amplification, respectively. Amplification of the endogenous human gene hRNaseP served as a loading control. The assay was performed in technical triplicate. (a) Physical map depicting the structure of the CAR-carrying -RVV, SFG.iC9.GD2.CAR.IL15. The vector’s non-self-inactivating (non-SIN) long-terminal repeats (LTRs) and the packaging signal y are shown. The inducible caspase-9 suicide gene, Ic9, is shown. The self-cleavable peptide from the thosea asigna virus (T2A) and the equine rhinitis virus (E2A) is shown. Sequences encoding the CAR including the single-chain variable fragment (scFv1) directed to the NB-antigen GD2 of the disialoganglioside GD2 (14g2a), the CD8a stalk and transmembrane domain, the CD28 intracellular domain, and the CD3z chain are shown. The human IL15 cDNA containing sequence is shown. (b) Graph bar showing GCN of VL30 in the abovementioned primary human T-cells. The experiment was performed in technical triplicate. (c) Graph bar showing VCN of the -RVV SFG.iC9.GD2.CAR.IL15 in the abovementioned primary human T-cells. The experiment was performed in technical triplicate. (d) Gel electrophoresis analysis of DNA amplification products of VL30, -RVV, and hRNaseP sequences generated in the course of the abovementtioned gPCR assay. DNA of naïve 293T cells (N) served as a technical negative control for amplification of VL30 and -RVV sequences. DNA from 293T cells transduced with the lentiviral vectors pTK2229 and pTK2151 served as positive controls (P) for VL30 and -RVV genome amplification, respectively. (e) Significant correlation between VL30 and -RVV genome copy number in human T-cells transduced with CAR-expressing -RVV. Linear regression graph demonstrating the relation between VL30 and -RVV genome copy number. The R value is indicated.

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