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Figure 1: Genotypic characterization of HEKExpress® cells . (A) TLA sequence coverage across HEKExpress® genome using the primers targeting the T-antigen coding sequence (set 1). The plot show that the integration site of the plasmid is located on the chromosome 3 equivalent (chr3). (B) TLA sequence coverage across the integration locus in human chr3 using the primers targeting the T-antigen coding sequence (set 1) or chr3 (set 3 and 4). The coverage of set 1 shows a deletion of 550 kb in HEKExpress® genome (green arrows) compared to the human hg38 genome. The coverage of sets 3 and 4 confirm the junctions of the integrated plamid pRTAK. (C) Map of pRTAK plasmid originally integrated in HEKExpress® cell line. The large and small T-antigen sequences are indicated in dark purple and gRNAs (gRNA_beginning and gRNA_end) targeting the gene in orange. (D) Map of chr3 equivalent (in red) with the 550 kb deletion and the integration of the pRTAK plasmid containing the T-antigen sequence. Plasmid-chromosome junctions confirmed by TLA are indicated in blue.
Figure 2: CMC compliant cloning of HEKEpress® cells after CRISPR/Cas9. (A) Images from the single-cell dispenser showing a single cell to dispense. The first four images show the cell coming in the ROI (blue circle). The last image is taken after the dispensing and show that the cell was successfully dispensed. Scale bar: 50 µm. (B) Scan of a 96-well plate at day 13 post-seeding. Wells surrounded in green indicate monoclonally-derived cell populations ready to be transferred for expansion and represent approximatively 21% of the total printed cells. (C) Images from the clone imager device taken at D0 (day 0), D1, D2 and D13 after single-cell cloning. The image at D0 confirms the dispensing of a single cell, that divides into two cells at D1 until forming a single-cell derived colony at D13. Scale bar: 200 µm.
Figure 3: Analysis of T-antigen removal on clonally derived cell populations . (A) A duplex-qPCR was performed on genomic DNA extracts, using two sets of primers targeting the T-antigen and the ß-ACTIN coding sequences. PCR products (5 or 10 µL) were then electrophoretically separated on agarose gel. The PCR products sizes from the T-antigen and ß-ACTIN targeting primers pairs are 107 bp and 141 bp, respectively. (B) Summary of single-cell cloning and KO colonies.
Figure 4: AAV productivity of T-antigen-negative clonally derived cell populations . Triple transfection was performed on cells and AAVs were harvested 72h later. Viral capsid (A) and genome (B) titers were determined by ELISA and dPCR, respectively. Data were normalized with titers from HEKExpress® as reference. Error bars represent means ± standard deviation of two technical replicates.
Figure 5: T-antigen negative cell performance and stability . Cells were maintained in culture for two months, with regular banking every other week. “PXX” represents the passage number of the cell culture after a bank was generated. Viable Cell Densities (VCD) and viabilities were measured (A) and doubling times calculated (B). The data corresponding to clone #8 in (B) are representative of other top clones. (C-D) One vial of each bank was thawed and AAV8 virions were produced as described in Figure 4. Viral capsid (C) and genome (D) titers were determined by ELISA and dPCR, respectively. Error bars represent means ± standard deviation of two technical replicates.
Figure 6: T-antigen negative cell performance in different scale bioreactors . Clone #8 was seeded in different working volumes from 10 mL to 1 L and transfected for AAV9 production as described in Figure 4. (A) Viable Cell Density (VCD) and viability were assessed every day. (B) Viral capsid and genome titers were determined by ELISA and dPCR, respectively.