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.