Figure 6

Analyses of IDLV genomic integrations in SmyleDCpp65. Cells from different production batches (RG-ICLV, RG-IDLV, GMP2 and GMP3) were thawed and cultured for 7 days without exogenous addition of cytokines. Total DNA (tDNA) was extracted from the cells for q-PCR or for LAM-PCR followed by high throughput integration analyses of LV sequences. a Reference analyses of pp65⁺ cells detected by FACS for each cell product. b Analyses of number of vector copies/cell performed by q-PCR (amplification of WPRE sequences). c Total matched sequences and unique integration sites (IS) analyses performed after LAM-PCR (LV sequences amplified with LTR primers) of the tDNA followed by NGS. Fold reduction from the total matched sequences to IS is indicated. d Frequency of IDLV integrations per chromosome, showing correlation with chromosome size. e Distribution of IDLV integrations upstream of transcription start sites (TSS arrow) or within genes. f Ten most pre-dominant clones detected in SmyleDCpp65 (RG-ICLV, RG-IDLV and GMP batches 2 and 3). Colored columns represent retrieval frequency as percentage of total sequences. Larger colored bars represent higher frequencies of integration sites clustering in the proximity of that gene. Lower panel Ranking of the 10 most pre-dominant clones with their corresponding color code and gene ID. Red arrows indicate frequent insertion sites observed in DENND1B and WDR74 for GMP2. g Common Integration Site (CIS) statistical analyses performed for RG-IDLV and GMP2 and GMP3 batches combined. Red arrows indicate recurrent insertion sites observed in the analyses (LIF2, MDM4, WDR74, DENND1B).