Quantifying the mutational landscape of retroviral and lentiviral vectors in gene therapy patients.

Adenosine deaminase severe combined immunodeficiency (ADA-SCID) is a monogenic disorder caused by mutations in the ADA gene. Gene therapy using γ-retroviral and lentiviral vector gene addition approaches have shown curative results. We sequenced the ADA transgene in transduced CD3+ T cells, and in peripheral blood cells from patients treated with autologous CD34+ cells transduced with either a γ-retroviral or lentiviral ADA gene vector to assess transgene mutational profiles. In both CD3+ T cells and ADA-SCID patients' cells treated with the lentiviral vector, we observed significantly higher occurrences of guanine (G)-to-adenosine (A) base substitutions than with the γ-retroviral vector. We hypothesized that this G-to-A mutational signature was due to the APOBEC3 cytosine deaminase protein family. By knocking out APOBEC3 genes in HEK239T packaging cells, APOBEC3-mediated mutagenesis decreased by 91.2% along the transgene in CD34+ transduced cells in comparison to CD34+ cells transduced with lentiviral supernatant packaged in parental HEK293T cells.