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.

Orthogonalized human protease control of secreted signals

“…Clinically, ex vivo gene delivery to engineer therapeutic cells is bottlenecked by viral packaging limits of ∼5 kb (adeno-associated virus) to ∼10 kb (lentivirus) 43,44 . Compact genetic constructs are therefore a critical requirement for translational applications.…”

Multi-scale in vivo imaging of tumour development using a germline conditional triple-reporter system

“…For example, while somatic gene transfer of both Cre and reporter gene sequences on the background of a conditional GEMM is resource-e cient, it may be limited due to lack vector tropism [40]. There are also payload considerations, with a ceiling at approximately 10 kbp for lentiviral vector, limiting the total number of reporter genes that are deliverable somatically [41]. Combining different transgenic alleles in a single composite animal through cross-breeding although straightforward in principle, requires prolonged and costly breeding.…”

CD9 and folate receptor overexpression are not sufficient for VSV-G-independent lentiviral transduction

“…First of all, we used a different packaging plasmid for LVs production: psPAX2 and pCMV-dR8.91 are both second generation plasmids and share similar architecture and gene compositions [ 38 ], but there could be differences in efficiency of virus production. For example, a recent study showed that pCMV-dR8.2 dvpr was able to stimulate a lentivirus production at least 7 times stronger than psPAX2 [ 39 ].However, since we always used the same titre of virus for transduction, this should not affect transduction efficiency per se. Further, the cell line used to produce the LVs might determine several characteristics of the EVs.…”