Extra-viral DNA in adeno-associated viral vector preparations induces TLR9-dependent innate immune responses in human plasmacytoid dendritic cells

Journal article

We sought to engineer mammalian cells to secrete nuclease activity as a step toward removing the need to purchase commercial nucleases as process additions in bioprocessing of AAV5 and AAV9 as gene therapy vectors. Engineering HeLa cells with a serratial nuclease transgene did not bring about nuclease activity in surrounding media whereas engineering serum-free, suspension-adapted HEK293-F cells with a staphylococcal nuclease transgene did result in detectable nuclease activity in surrounding media of the resultant stable transfectant cell line, 'NuPro-1S'. When cultivated in serum-free media, NuPro-1S cells yielded 3.06x1010 AAV5 viral genomes (vg) / mL via transient transfection, compared to 3.85x109 vg /mL from the parental HEK293-F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8x1013 vg /mL from NuPro-1S cells compared to 7.35x1012 vg /mL from HEK293-F cells. AAV9 from both HEK293-F and NuPro-1S showed almost identical ability to transduce cells embedded in a scaffold tissue mimic or cells of mouse neonate brain tissue in-vivo. Comparison of agarose gel data indicated that the DNA content of AAV5 and AAV9 process streams from NuPro-1S cells was reduced by approximately 60% compared to HEK293-F cells. A similar reduction in HEK293-F cells was only achievable with a 50 U / mL Benzonase® treatment

Authors: Bucher, K; Rodríguez-Bocanegra, E; Wissinger, B; Strasser, T; Clark, SJ

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: Scientific Reports
• Volume: 13
• Issue: 1
• Pages: 1890-1890
• Article number: 1890
• Publication date: 2023-02-02
• DOI: 10.1038/s41598-023-28830-7
• EISSN: 2045-2322
• ISSN: 2045-2322
• Language: English
• Keywords: Plasmacytoid dendritic cell; Innate immune system; Gene expression; Genetics; Dendritic cell; DNA methylation; Recombinant DNA; Vector (molecular biology); Immune system; DNA; Biology; Virology; TLR9; Gene; Immunology