Enhancing Gene Delivery in NB-4 Cells: Overcoming Transduction and Selection Challenges

• Published in: Cells (Basel, Switzerland) Switzerland), 2024-11, Vol.13 (22), p.1849
• Main Authors: Leto, Stefano, Gehlot, Sonakshi, Sheth, Bhavwanti, Ratti, Stefano, Manzoli, Lucia, Divecha, Nullin, Fiume, Roberta
• Format: Article
• Language: English
• Subjects: Acute promyeloid leukemia; Arsenic; Cell culture; Cell death; Cell Line, Tumor; Cell Survival; Cell viability; Cells; Efficiency; Experiments; Flow cytometry; Gene therapy; gene transduction; Gene transfer; Gene Transfer Techniques; Genes; Genetic engineering; Genetic vectors; Genetic Vectors - genetics; Health aspects; HEK293 Cells; Humans; Lentivirus - genetics; Leukemia; Leukemia, Promyelocytic, Acute - genetics; Leukemia, Promyelocytic, Acute - pathology; Leukemia, Promyelocytic, Acute - therapy; NB-4 leukemia cells; Phosphoglycerate kinase; Phosphoglycerate Kinase - genetics; Phosphoglycerate Kinase - metabolism; Polyethylene glycol; Polyols; Promoter Regions, Genetic - genetics; Promyeloid leukemia; Puromycin; Secondary metabolites; Therapeutic applications; Toxicity; Transduction; Transduction, Genetic; Tumor cell lines; Ultracentrifugation; Vectors (Biology); viral vector engineering
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells13221849

Efficient gene transduction and cell viability are critical factors in genetic manipulation for research and therapeutic purposes. In this study, we explored the challenges associated with transducing the NB-4 cell line, a well-established model for acute promyelocytic leukemia (APL), using lentiviral vectors. While the initial transduction efficiency in NB-4 cells reached approximately 30%, we observed a significant decrease in cell viability, a phenomenon not observed in other acute leukemia cell lines such as THP-1 cells. We identified that this toxicity could be mitigated by purifying viral particles through ultracentrifugation or polyethylene glycol (PEG) precipitation, indicating that toxic substances, potentially secondary metabolites released by HEK293, could be responsible for the cell death. Nevertheless, cell selection by puromycin was still ineffective; crucially, we discovered that the human phosphoglycerate kinase (hPGK) promoter, commonly used in the PLKO1 vector, may become silenced in NB-4 cells, preventing effective selection with puromycin. By replacing the hPGK promoter with the elongation factor-1 alpha (EF1α) promoter, we successfully achieved high transduction efficiency and robust selection, demonstrating the potential for this modified vector system to facilitate genetic studies in APL models. These findings provide important insights into optimizing gene transduction protocols not only for NB-4 cells but also for other challenging cell lines, offering a refined approach for gene delivery and selection in cell models.