Optimizing Lentiviral Vector Production: Insights Into PiggyBac Transposase and Concatemeric Array Strategies.

Lentiviral vectors (LVVs) are essential tools in gene and cell therapy due to their ability to transduce both dividing and non-dividing cells. Conventional production by transient plasmid co-transfection is variable, costly, and difficult to scale, prompting development of stable producer cell lines. Historically, the GPRTG cell line has been generated using concatemeric-array integration, which requires high DNA input, complex workflows, and can cause genetic instability. To address these limitations, we evaluated a transposase-mediated integration strategy. Compared with the concatemeric-array method, transposase-based integration enabled faster recovery after selection with only a mild viability crisis and required substantially less DNA. This approach generated highly diverse and heterogeneous producer pools, providing a strong basis for subsequent clonal selection. During LVV production, both methods maintained comparable cell growth stability. However, concatemeric-derived pools exhibited greater variability in recovery kinetics, viable cell density, and LVV titers, despite achieving the highest maximum titers overall. In contrast, transposase-mediated pools showed more consistent performance, supporting their reliability for large-scale applications. In summary, transposase-based integration offers a robust and scalable alternative to concatemeric-array methods for generating stable LVV producer cell lines, with significant potential to streamline LVV manufacturing for gene therapy.