Abstract
BackgroundEffective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudovirus-based systems and electro-transfection are the most popular strategies for genetic material transduction. Compared with viral-particle-mediated approaches, electro-transfection is theoretically safer, because it does not promote transgene integration into the host genome. Additionally, the simplicity and speed of the procedure increases the attractiveness of electroporation. Here, we developed and optimized an electro-transfection method for the production of engineered chimeric antigen receptor (CAR)-T cells.ResultsStimulation of T cells had the greatest effect on their transfection, with stimulation of cells for up to 3 days substantially improving transfection efficiency. Additionally, the strength of the external electric field, input cell number, and the initial amount of DNA significantly affected transfection performance. The voltage applied during electroporation affected plasmid permeation and was negatively correlated with the number of viable cells after electroporation. Moreover, higher plasmid concentration increased the proportion of positively transfected cells, but decreased cell viability, and for single-activated cells, higher cell density enhanced their viability. We evaluated the effects of two clinically relevant factors, serum supplementation in the culture medium and cryopreservation immediately after the isolation of peripheral blood lymphocytes. Our findings showed that our protocol performed well using xeno-free cultured, fresh T cells, with application resulting in a lower but acceptable transfection efficiency of cells cultured with fetal bovine serum or thawed cells. Furthermore, we described an optimized procedure to generate CAR-T cells within 6 days and that exhibited cytotoxicity toward targeted cells.ConclusionsOur investigation of DNA electro-transfection for the use in human primary T cell engineering established and validated an optimized method for the construction of functional CAR-T cells.
Highlights
Effective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications
Isolated lymphocytes were incubated with magnetic beads coated with anti-CD3/CD28 antibodies for stimulation
green fluorescent protein (GFP) expression remained stable for 3 days after electroporation, after which the percentage of positive cells gradually decreased, but remained detectable (6–7%; Fig. 1c, green line)
Summary
Effective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudo gamma-retroviral and lentiviral systems have been widely applied for gene transfer into T lymphocytes and result in an average of 30% to 80% transduction efficiency [8,9,10,11,12] These viral vectors can integrate into the host genome and lead to permanent transgene expression, with integration-related safety concerns largely resolved during long-term follow-up clinical studies [1, 4]. The preparation and transduction of high-quality pseudoviral particles are labor-intensive and time-consuming processes, with large technical requirements During this procedure, the viral vector is transfected into a package cell line along with two or more package plasmids to generate
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