pCMV-dR8.2 dvpr

Gene therapy revolves around inserting exogenous nucleic acids into target cells through gene delivery methods to change the genetic makeup to treat specific diseases. Because primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system needs to be robust, and virus-based vectors (such as lentiviral vectors) are best suited to deliver transgenes into cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CC) as a model system, this study aimed to optimize lentiviral production by studying various experimental factors such as lentiviral system generation, concentration methods, and selection marker types. The results of this study showed that the lentivirus yield of the second-generation system using pCMV-dR8.2 dvpr as the packaging plasmid was 7.3 times higher than that of psPAX2.

In this study, quantification using ImageJ showed that the lentiviral yield of the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid (2A) was 7.3-fold higher than that of the 2nd generation system with psPAX2 packaging plasmid (2B). At the same time, compared with the 3rd generation lentiviral plasmids 3A and 3B, the virus yield of 2A was increased by 1.7 times and 2.6 times respectively. The expression of GFP in cells 48 hours after transduction with 2A and 3B lentiviral vectors is shown in Figure 1.

Figure 1. GFP expression in cells 48 h after transduction with the second and third lentiviral vectors. (A) Lentiviral vector 2A, which uses pCMV-dR8.2 dvpr as the packaging plasmid, produces the most lentiviral particles compared to 2B. (B) Lentiviral vector 3B produces a smaller number of lentiviral particles than 3A. (Kalidasan V, et al., 2021)