The growing demand for high-quality plasmid DNA (pDNA) in cell and gene therapy, including chimeric antigen receptor T-cell therapy (CAR-T) manufacturing, is constrained by the limited availability of transfection-grade plasmids. This study describes a practical, scalable, and cost-effective acidic phenol extraction method based on a modified alkaline lysis protocol. This approach efficiently removes endotoxins and residual genomic DNA without requiring chromatography or ultracentrifugation. When benchmarked against CsCl density gradient ultracentrifugation and a commercial midiprep kit, the acidic phenol method delivered comparable plasmid purity and functional performance while providing superior scalability, yielding 5–10 mg of pDNA per 500 mL culture. Notably, residual endotoxin levels were negligible for downstream applications such as transfection and lentiviral vector packaging. Lentiviral vectors produced with these plasmids reached titers exceeding 5 × 10⁶ TU/mL, with transduction efficiencies statistically indistinguishable from those obtained with CsCl-purified DNA. The acidic phenol extraction method is a robust, scalable, and cost-effective alternative to industry-standard methods, matching them in yield and purity while being readily scalable, making it a practical tool for both academic research and preparative laboratory production.

Lentiviral vectors (LVs) are indispensable tools in cell and gene therapy. Rising demand has created a global shortage of LVs, driving the development of novel packaging approaches. We report a novel vector packaging approach using autonomously replicating cytoplasmic RNAs (replicons) to express packaging proteins. Yellow fever virus (YFV) was used as a source of replicons encoding the HIV-1 Gag–Pol polyprotein together with reporter or selectable markers. YFV replicons were able to establish chronic infection in HEK293FT cells. Replicons expressing HIV-1 Gag–Pol containing the wild-type HIV-1 protease caused strong cytotoxicity, which prevented the selection of polyclonal cell pools harboring the replicon. In contrast, a replicon carrying the T26S mutation in the HIV-1 protease gene showed no measurable cytotoxic effects, enabling the generation of stable replicon-containing cell pools. The replicon cell pools were established using antibiotic selection and maintained Gag-Pol expression for at least ten passages under selection pressure. Using these first-generation replicon cell pools as packaging cells, LV production required only transient transfection of a transfer vector, a Tat/Rev plasmid, and an envelope plasmid. Yields reached ~10⁶ TU/mL prior to concentration and ~10⁹ TU from multilayer cell stacks, which fall within the range typically reported for conventional transient transfection systems under similar culture conditions. The resulting vectors efficiently transduced target cells, and no replication-competent lentivirus (RCL) was detected using a two-phase RCL assay with p24 ELISA detection. This demonstrator platform utilizing replicon cell pools represents a novel approach for LV packaging.

Lentiviral vector (LV) production for CAR-T therapy remains challenging due to the limited scalability of adherent HEK293 cell cultures. To address this, we adapted HEK293FT cells directly to serum-free FreeStyle 293 Expression Medium, generating a novel suspension cell line, HEK293FT-DS. We characterized growth kinetics, stability over 35 passages, and transient transfection parameters (PEI:DNA ratio, cell density). The suspension-adapted cells grew with >93% viability and a specific growth rate of 0.54 day⁻¹, and maintained stable viable cell density (1.51 ± 0.03 × 10⁶ cells/mL) over 35 passages. The optimal PEI:DNA ratio was 2.5:1, and increasing the transfection cell density to 16 × 10⁶ cells/mL boosted functional titers to approximately 9 × 10⁶ TU/mL in clarified supernatant. Scaled production (1 L culture supernatant per batch) yielded up to 5 × 10⁹ TU. The resulting vectors efficiently transduced primary human T cells (57% CAR-positive cells) in a CliniMACS Prodigy-based process. The platform based on suspension-adapted HEK293FT-DS cells enables high-density transfection and provides a cost-effective, scalable alternative to commercial LV production systems, particularly suited for academic CAR-T cell manufacturing.

The rapid advancing of cell and gene therapy has increased the demand for plasmid DNA used in viral vector manufacturing. The production of transfection-grade plasmid DNA remains a critical bottleneck, particularly for academic and emerging manufacturing facilities, due to the high cost and limited scalability of existing purification technologies. The aim of this study was to develop a practical and cost-effective approach for producing transfection-grade plasmid DNA suitable for lentiviral vector manufacturing. The described acidic phenol extraction method was evaluated using plasmids commonly employed in lentiviral packaging systems and compared with a reference method, CsCl density gradient ultracentrifugation. The resulting plasmid preparations showed efficient removal of genomic DNA and other impurities, including endotoxin – the acidic phenol method consistently reduced endotoxin levels to 1.4–3.8 EU/µg DNA. Moreover, the purified plasmid DNA was predominantly in the supercoiled (covalently closed circular) form. Functional validation in transfection assays confirmed that plasmids purified by the proposed method supported lentiviral vector production with efficiency comparable to the reference method, yielding titers exceeding 5×106 TU/mL, with no statistically significant differences in transduction efficiency between the groups. These findings confirm that the acidic phenol extraction method is a simple, scalable, and economical approach for isolating transfection-grade plasmid DNA for lentiviral vector production. The method represents a practical alternative to ultracentrifugation-based purification and may facilitate broader use of cell and gene therapy by reducing dependence on specialized equipment and costly commercial kits.