This study demonstrates a simplified lentiviral vector optimization that is cost and time effective and produces highly efficient transduction in CCs in transfection method, the relative ratio of transfer, packaging and envelope plasmids, viral titering and purification, concentration method, as well as selection method of cells carrying the transgene, as illustrated in Fig. 5. HEK293T cells act as host cells to assemble and generate viral particles before transduction was optimized.
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.
In this study, lipofection was preferred for transportation of exogenous DNA by liposomes due to the properties and chemical composition of the cationic and neutral lipids. The transfection efficiency between Lipofectamine 2000 and 3000 was compared, and Lipofectamine 3000 worked for transfecting cells, in agreement with many past studies. Although transfection efficacy and toxicity of transfection reagents are highly cell-dependent10, Lipofectamine 2000, which has been widely used, displayed high toxicity in most cell types tested. In contrast, Lipofectamine 3000 demonstrated a high transfection efficacy in most cells, including in HEK293 and human umbilical vein endothelial cells (HUVECs) and showed stable co-transfection with multiple linear DNA11. Overall, in agreement with past studies, we found Lipofectamine 3000 to be an efficient and reproducible transfection reagent for biologically relevant cell models compared to other lipid-based reagents (i.e., Lipofectamine 2000 and FuGENE reagents).
After optimizing the transfection reagent, the transfection efficiency, the titer yield of 2nd and 3rd generation lentiviral vectors and their transduction efficiency on CCs were investigated. The packaging vector of the 2nd generation of lentivirus contained Gag, Pol, Tat and Rev in a single plasmid, while in the 3rd generation, the packaging vector was divided into two different plasmids (i.e., one encoding Gag and Pol and another encoding Rev) to improve the safety of the system12,13. This study showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid (2A) had an increased efficiency and higher reproducibility compared to the 3rd generation lentivirus vector (3B). Similarly, a study reported that the 2nd generation lentivirus vectors produced approximately fifty times more total yield than 3rd generation vectors. The latter also did not have any beneficial effect on the ability of the viral vector to transduce primary human CD45RA+ T cells14. Although deemed to be safe, the 3rd generation of lentiviral necessitates the successful co-transfection of four different plasmids (including the transfer vector and envelope) to produce functional particles15,16. Due to the high number of plasmid requirements, lower viral titers and reduced transduction efficiency were observed in this study. Overall, the plasmid ratios and amounts were optimized based on previously published papers to ensure successful transfection. Despite the differences in promoters in the transfer vectors, different plasmids were used based on the recommended combinations from groups that had previously worked with these vectors. This approach also enabled the efficacy of publicly available lentiviral plasmids to be gauged, reducing the need to only purchase specific commercialized plasmids, which are typically less affordable.
The next step after selecting the most suitable lentiviral vector is to ensure an adequate number of viral particles for transduction. Viral titering is performed to analyze the quality of lentiviral vector production, gene transfer efficiency, and the level of therapeutic gene expression17. There are various methods to titer the virus, such as p24 ELISA, limiting dilution, quantitative PCR, and reporter genes expression18,19,20. Although these methods are efficient, they can be tedious and time-consuming. Thus, a commercially available kit, Lenti-X GoStix, was used in this study, which was quick and produced results within ten minutes. The virus particles from 2A, 3A and 3B expressed two bands when titered with Lenti-X GoStix, implying that the virus was successfully produced, in agreement with the data seen with fluorescent microscopy. Previous studies have demonstrated the usage of semi-quantitative assessment of viral titer using Lenti-X GoStix for transducing cardiomyocytes. Furthermore, the GoStix Value (GV; equivalent to ng/ml p24) has been proven to be correlated to infectious titer when using traditional p24 ELISAs, or qRT-PCR assay as a suitable substitute for other methods of functional titration. This technique requires minimal technical skills, it can easily be adopted even by beginners in virus-related work.
To improve the viral transduction efficiency in subsequent testing, virus can be purified and concentrated using several methods, such as ultracentrifugation or ultrafiltration14. In this study, we compared ultracentrifugation and a commercially available kit, Lenti-X Concentrator. Lenti-X Concentrator is simple, scalable to any volume and concentrates virus in less than an hour. On the other hand, ultracentrifugation, a more established method, requires a longer time to concentrate the virus (90 min). Our study showed higher transduction efficiency in CCs using virus concentrated from ultracentrifugation compared to Lenti-X Concentrator. Numerous studies have shown that the concentration of the culture supernatants achieved an increase in the viral titers by ultracentrifugation and was the preferred method since there is no evidence of toxicity by lentivirus vectors prepared using this technique19,21. Newer methods related to ultracentrifugation have recently been established, such as sucrose gradient centrifugation with a relatively low speed (≤ 10,000 g), which robustly produces a high-titer virus (up to 2 × 108 TU/ml)22. This method is an efficient and easy-to-handle protocol for high-titer lentivirus purification and surpasses the instrumental barrier for routine laboratory operations.
Following transduction, not all target cells might receive the transgene. Cell enrichment can be conducted to select successfully transduced cells containing the transgene from a mixture of cells. Different methods can be used to determine efficient transgene delivery23, such as drug-resistant genes, intracellular enzymes, and fluorescent proteins (GFP). As the viability of cells can be affected by cell sorting, this study used transgene with puromycin as a selection marker that enabled an effective positive selection of cells expressing the puromycin-N-acetyltransferase (pac) gene. Similar to many of the methods used in this study, substituting cell sorting during cell enrichment with this method would eliminate the need for high-end equipment. Our study showed at day 0, the CCs showed adherent confluent cells with wall spikes, which later degenerated without spikes on day 5 (at 8 µg/mL of puromycin), proving that this simple method can be completed over a short time span, in less than a week.
Overall, we provided a comprehensive overview of lentiviral vector production for hard-to-transfect cells, using CCs as a model system. Cells that are c-kit positive have properties of cardiac stem cells, including the capability to self-renew, are clonogenic, multipotent, and give rise to myocytes, smooth muscle, and endothelial cells24,25, as shown in Fig. 6. c-Kit is a type III receptor tyrosine kinase (RTK) transmembrane receptor involved in multiple intracellular signalling and considered as a ligand for stem cell factor (SCF), which participates in vital functions of the mammalian body and holds immense potential for therapeutic purposes26,27,28.
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).
Current transgene delivery utilizing adenoviral or adeno-associated vectors that directly target adult cardiomyocytes are still under investigation for cardiac gene therapy29,30,31,32,33. However, this approach is limited by the ability of cardiomyocyte proliferation34,35. Thus, genetic materials cannot be passed down to sustain therapeutic benefits. Adenoviral vector has up to 36 kb packaging capacity with high transduction efficiency36,37,38,39,40. While it has a large capacity, it is a transient expression system and immunogenic, which elicits a strong immune response, making it an unsuitable delivery method for gene therapy. On the other hand, the adeno-associated viral system is affected by neutralizing antibodies, which will significantly reduce the transduction efficiency41,42,43. In contrast, lentiviral systems allow stable genome integration into the host cells and have the added advantage of high transduction efficiency in hard-to-transfect cells and cardiovascular gene therapy6,44. The balance of important features in lentiviral systems makes it a suitable system for long-term gene delivery. The comparison between lipofection, lentiviral and adenoviral vectors gene delivery systems is illustrated in Fig. 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.
Cardiac c-kit cells remain interesting regenerative cells for repairing damaged hearts. Despite the ongoing debates over its cardiomyocyte commitment45, nonetheless, evidence has supported the function of cardiac c-kit cells in salvaging damaged hearts46,47. Labelling purified rat cardiac cells with reporter genes was previously achieved by employing a closed commercial lentivirus system46 or by direct isolation from GFP-expressing transgenic rat hearts48. Whilst the underlying mechanism and the function of the cells would need to be further explored, this manuscript offers the most effective and precise molecular tracking method to unveil the cell fate and functions of such cells in a more complex setting such as coculture, three-dimensional tissue engineering and the heart in vivo.
We acknowledge that the limitation of this study is that there are several other options for specific stages of lentivirus production that was not investigated in this study. However, the primary purpose of this study was to identify steps that can be easily replicated in most laboratories when working with hard-to-transfect cells without having to purchase any additional equipment or incurring extra costs. Using CCs as a model system for lentiviral vector production, this encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells. In the future, we plan to compare the efficacy of fourth-generation lentiviral plasmids to take into account safety when establishing delivery systems for gene therapy.
