Introduction
Chimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy in which a patient’s T cells are harvested, genetically modified to express a chimeric antigen receptor, and then infused back into the patient to seek out and eliminate any cells that express a target antigen that is bound by the CAR. For example, anti-CD19 CAR-T cell therapy is approved by the FDA for the treatment of B cell leukemia (e.g., non-Hodgkin’s lymphoma,1 chronic lymphocytic leukemia,2 and acute lymphoblastic leukemia3–5). In these treatments, the CAR is delivered to the T cells using a gammaretrovirus or lentivirus.6,7 These viruses are generally regarded for their high transduction efficiency, but it is worth noting that their genomic integration patterns are semi-random.11For example, gammaretroviral vectors have been shown to have a preference for integrating near transcriptional start sites. This type of semi-random integration could potentially lead to mutagenesis, but years of clinical scrutiny have demonstrated these vectors to be safe thus far.8–10,12,13 Nonetheless, these vectors are still considered by the FDA to be potentially oncogenic and thus must be tested for replication competence during manufacturing and patients must be monitored for up to 15 years after receiving treatment.13–15 It is also important to note that the transduction efficiency achieved with retroviral delivery systems can vary significantly between patients (2.3-80%),1–6although this variation may be due to differences between their genotypes and treatment regimens.
These challenges have motivated researchers to investigate non-viral transfection methods for delivery of the CAR gene, which has been a formidable task. Indeed, T cells have proven to be notoriously hard to transfect, perhaps because they are uniquely adapted to clear the body of viral infections and restrict viral replication.20For example, lymphocytes have been shown to expel mitochondrial DNA in inflammatory webs after recognizing CpG oligodeoxynucleotides (a pathogenic signature unique to bacteria).7Nonetheless, several groups have shown that transfection efficiencies as high as 60-70% can be achieved in primary T cells with electroporation.21,22 This technique applies an electric field to a sample of cells that exceeds the capacitance of the cell membrane to create pores in the cell membrane that allow delivery of DNA.15,23 This physical method of introducing DNA to T cells is generally quick and inexpensive, but it can be difficult to scale up and is relatively harsh, leading to significant decreases in T cell viability.24–27 Alternatively, a few groups have investigated the use of cationic polymers (e.g., PEI and pDMAEMA) for gene delivery to T cells.82-85 These studies have demonstrated highly efficient delivery of siRNA and mRNA to T cells, but the maximum transfection efficiencies for pDNA with these vehicles (1.5-25%) tend to be relatively low compared to electroporation and lentiviral transduction.86
The goal of this work was to investigate the use of Lipofectamine LTX as a lipid-based alternative to cationic polymers, electroporation, and lentiviral transduction for T cell gene delivery. Transfection with the cationic lipid Lipofectamine (i.e., Lipofection) involves the formation of a lipoplex consisting of negatively charged plasmid DNA and positively charged liposomes. The lipoplex can then enter the cell via endocytosis and escape into the cytoplasm by lysing the endosome through the proton-sponge effect.28,29 The effects of additional transfection variables (e.g., media type, promoter, et al.) on Lipofectamine transfection efficiency were also investigated to determine the most efficient means of non-viral gene delivery to both Jurkat (a T cell leukemia line) and primary T cells. Finally, the transcriptome of the T cells was also analyzed to detect potential mechanisms of resistance to transfection or transduction.