In this study, we used an RNA electroporation system to improve transfection efficiency and reduce or eliminate transfection-related toxicity. A transfection efficiency of over 90% could be easily achieved for human PBLs. Similar results were observed in murine T cells that were stimulated with ConA or antigen-specific peptide. The results from optimization experiments altering electroporation parameters for stimulated human PBLs demonstrated that, except at high voltage and long pulse lengths (500 V/2 ms and 500 V/5 ms) the viability of transfected T cells was 63–86% at 24 h postelectroporation () and transgene expression of 99% was achieved at 3 days postelectroporation (). The length of time poststimulation does not significantly influence RNA electroporation as similar efficiencies can be achieved with cells stimulated from 2 to 18 days. Recently, Smits et al.
electroporated PHA-stimulated human PBLs with GFP RNA and 50% of transgene expression was detected using flow cytometry [17
]. Differences in PBL stimulation, model of electroporator, and/or the parameters for electroporation may account for the higher transfection efficiency achieved herein.
RNA electroporation may have distinct advantages over plasmid DNA-based electroporation. Following electroporation of plasmid DNA, the plasmid must enter the nucleus before the DNA can be transcribed into mRNA. Transcription efficiency is largely dependent on the promoter used in the plasmid vector and can be greatly influenced by target cell type-specific transcription factors, which ultimately determine transgene expression efficiency. By introducing mRNA directly into the cytoplasm of the cells, transcription is bypassed and the target gene can be immediately translated into protein. Indeed, we observed easily detectable GFP expression within 30 min postelectroporation (). RNA electroporation also introduces a molecule (although completely synthetic) that is almost identical to native mRNAs (in vitro
-transcribed RNAs have both poly(A) tails and 5′-methlylated cap analogues). This may be a critical distinction between RNA and plasmid DNA, as bacterial-based plasmids are known to contain “danger signals” and signal through Toll-like receptors leading to alterations in T cell biology [18
]. These difference in how the T cells recognize RNA versus plasmid DNA may in part account for the difference in cell viability following electroporation of these two different nucleic acid species.
Any transfection method, including electroporation, may induce unforeseen adverse affects on transfected T cells in terms of viability, proliferation, or functionality. The optimized electroporation conditions used in this study did not significantly alter T cell viability or cell proliferation and did not induce apoptosis ( and and ). Functionality of electroporated T cells was verified by testing electroporation of antigen-specific T cell recognition of limiting dilutions of gp100 peptide (). The most important potential application of this technology would be to engineer T cells with new biological functions. When T cells without tumor recognition were electroporated with in vitro-transcribed α and β TCR mRNAs, electroporated T cells were redirected to recognize not only tumor antigen peptide-pulsed APC (T2 cell line and dendritic cells), but also tumor antigen-expressing melanoma lines (). This simplified method of redirecting tumor antigen recognition of T lymphocytes by RNA electroporation makes it possible to screen TCR α and β cDNA libraries rapidly for functional TCR α/β pairs.
Promising results have recently been reported in adoptive immunotherapy trials for the treatment of patients with metastatic cancer [19
]. Engineering T cells with new functions using RNA electroporation may provide a powerful tool for altering T cell biology in circumstances in which long-term transgene expression is not necessary or may be undesirable. Molecules such as some cytokine genes and their receptors (IL-2, IL-15, and IL-7) [22
] or costimulatory molecules (CD28, CD27, 4-1BB, and OX40) [24
] can influence T cell functionality and homeostasis but do not need to be constitutively expressed. Furthermore, some molecules, such as CD62L and CCR7, are important for T cell homing and migration [15
], but long-term expression of such molecules on transferred T cells may be undesirable. CD62L expression is associated with T cell migration to lymph nodes, but continuous expression of this molecule on the T cell surface could prevent T cells from migration out of the lymph node to the effector site [27
]. The molecules Hlx, which promotes CD4 T lymphocyte polarization from Th2 to Th1 [28
], and telomerase reverse transcriptase, which extends the replicative potential of T cells [29
], are also possible candidates to be transferred into T cells for the adoptive immunotherapy of cancer or infectious diseases. Finally, the down-regulation of genes that may have a negative effect on T cell function (e.g., CTLA-4 or Foxp3) could be achieved by electroporation of siRNAs.
This highly reproducible and cost-effective technology is an attractive methodology for clinical development because it permits the use of naked RNA and does not involve additional reagents that must pass rigorous production standards and quality control testing. mRNA electroporation also provides a valuable alternative to viral transduction of T lymphocytes, which implies a more complex and laborious manipulation. Moreover, there may be lower safety concerns compared to viral vector transduction or plasmid DNA transfection that can result in the inactivation or activation of genes as a result of random integration of the introduced DNA.
In summary, mRNA electroporation provides a powerful tool to introduce genes easily into both human and murine primary T lymphocytes with high efficiency and low toxicity. The availability of 96-well electroporation cuvettes and the MaxCyte system [30
] make electroporation amenable to high-throughput screening protocols and potentially useful for large-scale genetic modification of T lymphocytes required for clinical applications.