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.