A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

Abstract

Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 10⁵ infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

Figure 1

GFP expression in cells at 48-h post-transduction with the 2nd and 3rd lentiviral vectors. (A) Lentiviral vector 2A with pCMV-dR8.2 dvpr as the packaging plasmid produced the most lentivirus particle compared to 2B, and (B) Lentiviral vector 3B produced a smaller number of lentivirus particle compared to 3A.

Figure 2

Lenti-X™ GoStix™ viral titration. Two bands on the stick indicate the presence of virus at greater than 5 × 10⁵ IFU/mL. (A) Negative control, (B) Lentiviral vector 2A, (C) Lentiviral vector 3A, and (D) Lentiviral vector 3B.

Figure 3

Lentiviral transduction CCs. (A) Successful transfection was observed in HEK293T cells using Lipofectamine 3000 after 24 h. (B) GFP-transduced CCs using lentivirus concentrated by Lenti-X™ Concentrator, (C) GFP-transduced CCs using lentivirus concentrated by ultracentrifugation, and (D) GFP-transduced CCs after passaging for one day.

Figure 4

The minimum inhibitory concentration (MIC) of puromycin for the cells at day 5. (A) HEK293T with 0 µg/mL puromycin showed confluent adherent cells with a characteristic morphology, (B) HEK293T with 10 µg/mL puromycin showed 10 µg/ml dead cells, (C) CCs with 0 µg/mL puromycin showed confluent adherent cells with cell wall spikes, and (D) CCs with 7 µg/mL puromycin showed dead cells without spikes and floating.

Figure 5

Diagrammatic workflow of the experiment. Preparation of complete growth media for CCs, optimizing different lipofection reagents, lentivirus production and the relative ratio of transfer, packaging and envelope plasmids, viral titering and purification, concentration, as well as selection method of cells carrying the transgene.

Figure 6

Cardiac-derived c-kit expressing cells (CCs) are self-renewal, clonogenic, and multipotent give rise to cardiomyocytes, smooth muscle, and endothelial cells. c-Kit is a type III receptor tyrosine kinase (RTK) transmembrane receptor involved in multiple intracellular signalling and considered a ligand for stem cell factor (SCF).

Figure 7

Comparison between non-viral and viral vectors gene delivery systems. (A) Lentiviral vector has up to 10 kb packaging capacity with high transduction efficiency. It is a stable expression system that integrates with host DNA and allows for long-term expression. (B) Non-viral vectors such as lipofection complexes DNA with liposomes and protects DNA from enzymatic degradation once released into the cell cytoplasm. It is a simple method with high packaging capacity and low immunogenicity. It is a transient expression system. (C) Adenoviral vector has up to 36 kb packaging capacity with high transduction efficiency. It is a transient expression system and immunogenic, which elicits a strong immune response.