Introduction
Immunotherapy, which uses the patient’s immune system to target and kill
cancer cells, has become a promising tool for cancer treatment (Koury et
al., 2018). Adoptive T cell therapy is a type of immunotherapy involving
the isolation and in vitro expansion of patient-derived T cells
and re-infusion into the cancer patients. In this context, peripheral
blood T cells are used to produce genetically modified-T cells
expressing transgenic T cell receptor, known as TCR and CAR-T cells
(Met, Jensen, Chamberlain, Donia, & Svane, 2019; Morgan, Dudley, &
Rosenberg, 2010). CAR T-cell, a living drug, has been investigated for
more than two decades. Cumulative research data have demonstrated the
remarkable success of CAR-T cells in some hematologic malignancies and
solid tumors. In the beginning, CAR T-based therapy has been intensively
used against hematologic malignancies, especially for patients with
B-cell Acute Lymphoblastic Leukemia (B-ALL) (Sadeqi Nezhad et al.,
2020). Consequently, FDA approved three CAR products, YESCARTA
(axicabtagene ciloleucel), KYMRIAH (tisagenlecleucel), and TECARTUS
(brexucabtagene autoleucel) to treat adult patients with certain types
of large B-cell lymphoma, patients up to 25 years of age with B-cell
precursor ALL, and patients with relapsed or refractory mantle-cell
lymphoma. (S. L. Maude et al., 2018; S. S. Neelapu et al., 2017; M. Wang
et al., 2020). Three other potential CAR-T cells with an International
Nonproprietary Name (INN), bb2121 (idecabtagene vicleucel),
vadacabtagene leraleucel, and JCAR017 (lisocabtagene maraleucel) are
expected to be approved for clinical use (Chow, Shadman, & Gopal, 2018;
D. W. Kim & Cho, 2020; Rodríguez-Lobato et al., 2020).
CAR-T cells have several limitations that stop them from performing
successfully and efficiently. Despite the tremendous clinical efficacy
of CAR-T cell therapy in hematologic malignancies, there are multiple
hurdles and barriers that restrict successful therapeutic outcomes.
CAR-T cells found to have a limited persistence, proliferation, and
expansion in some individuals, especially patients with CLL (Fraietta et
al., 2018; Porter et al., 2015). Studies demonstrated that defects in
intrinsic autologous T cells may prevent the success of CAR-T cells in
patients (Fraietta et al., 2018). In some cancer types (e.g., ALL),
patients diagnosed with rapid progressive disease whom requires an
immediate treatment with CAR-T cells, the therapy may fail due to the
long-time autologous CAR-T manufacturing process. Additionally, adequate
number of T cell collections from hematologic malignancy patients is
sometimes laborious and impracticable due to lymphopenia from recent of
prior chemotherapy or underlying disease (Singh, Perazzelli, Grupp, &
Barrett, 2016). Furthermore, CAR-T therapy has also been used against
solid tumors and showed promising therapeutic approaches; however, up to
now FDA approved no CAR-T products for solid tumors. This signifies that
the challenges in solid tumors are much more serious and require a
thorough investigations. A major hurdle to the success of CAR-T cell
therapy against solid tumors is tumor microenvironment and lack of
tumor-specific antigen (K. B. Long et al., 2018).
By the advent of genome editing technology, such as CRISPR/Cas9,
transcription activator-like effector nucleases (TALEN), and zinc finger
nucleases (ZFNs), there is an opportunity to address many of these
hindrances posed on CAR-T cell therapy (Berdien, Mock, Atanackovic, &
Fehse, 2014; Liu et al., 2017; J. Ren et al., 2017; Hiroki Torikai et
al., 2010). Genome editing refers to the delivery of an editing
machinery system in cells of interest to modify their genome either
through the replacement of faulty genes or inserting new genes to treat
diseases or boost the therapeutic outcomes (Gaj, Sirk, Shui, & Liu,
2016). CRISPR/Cas9 has surpassed the two other genome editing systems in
the following ways: (a) CRISPR/Cas9 recognizes the DNA site through
RNA-DNA interaction; (b) is easy designing; (c) results in higher
specificity and efficiency; (d) provides an easy way to manipulate
multiple target DNA, simultaneously (high-yield multiplexing); and (e)
is a budget-friendly technology (H. Li et al., 2020).
In the following sections, we will provide an overview of the
CRISPR/Cas9 technology, challenges and barriers posed on CAR-T cell
therapy, and finally methods thereby CRISPR/Cas9 system can potentially
improve the success of CAR T-cell therapy.
Overview of CRISPR/Cas9 Technology: Mechanism of Action as a
Genome Editing Platform
The immune system of many bacteria and almost all archaea harbor
RNA-guided adaptive immune systems encoded by CRISPRs and
CRISPR-associated (Cas) proteins to fight the invading bacteriophage or
block the foreign plasmid transfer (Rath, Amlinger, Rath, & Lundgren,
2015; Sorek, Lawrence, & Wiedenheft, 2013). The short sequence of
invading bacteriophage or plasmid DNA fragments stored in the CRISPR
region is known as a protospacer sequence. The CRISPR RNA (crRNA)
biogenesis process occurs upon the same entrance of pathogenic virus or
plasmid in the future. The protospacer selected for transcription is
based on protospacer adjacent motifs (PAMs) within the invading phage
genomes and plasmids. These protospacer sequences, which serve as a
genetic record of previously invaded viruses, transcribe into a long
precursor (pre-crRNA) and subsequently form into mature crRNAs by
endonucleolytic cleavage. Finally, the mature crRNA combines with Cas
protein to generate a ribonucleoprotein complex structure that detects
the target DNA by crRNA spacer and degrades the viruses and plasmids
(Garcia-Robledo, Barrera, & Tobón, 2020; Ishino, Krupovic, & Forterre,
2018; Makarova et al., 2019).
There are different types of
CRISPR systems. Among them, type II CRISPR locus recruits
CRISPR-associated protein, Cas9, to produce double-stranded breaks
(DSBs) in DNA of interest. Cas9 has a multitude of functions and is
considered as a multi-functional protein. It possesses two distinct
domains named HNH-nuclease and RuvC-like nuclease, which break the
target DNA strand and the non-target DNA strand, respectively (Makarova
& Koonin, 2015; Wu, Kriz, & Sharp, 2014). In genome engineering, the
trans-activating CRISPR RNA (tracrRNA) and crRNA are engineered as a
single-guided RNA (sgRNA), which is a 17-20 nucleotide sequence
corresponding to the target DNA. To further simplify, CRISPR/Cas9 system
is defined as sgRNA and Cas9 protein combination. sgRNA has a PAM
sequence after the 3′ end of its sequence (5′-NGG-3′ for streptococcus
pyogenes (Sp)-Cas9), which is essential for guiding the Cas9 protein to
the target DNA wherein the complementary PAM sequence is present. Upon
the interaction between the sgRNA and the target DNA, the Cas9 protein
generates a DSB from three nucleotides upstream of the PAM sequence
(Cao, Xiao, & Yan, 2018; Fonfara et al., 2014; Xiao-Jie, Li-Juan, Koo,
& Kim, 2017). Afterward, DSBs undergo two different mechanisms of
repairs, homologous directed repair (HDR) and non-homologous end joining
(NHEJ). The former approach is used to knock-in a specific DNA that
creates either an aberrant gene to develop a specific disease or repair
a particular defective gene with homologous DNA. The NHEJ system is
error-prone and a quick fix mechanism throughout the cell cycle (Allen
et al., 2019; Jasin & Rothstein, 2013). This pathway requires no
homologous sequence for ligation of DNA end and generates frameshift
mutations through insertion and/or deletion (indel) mutations at the
repair junctions (Figure 1 ). Once the DSB is induced, certain
proteins, named Ku70 and Ku80, quickly bind to the DSB end and form the
Ku heterodimer. The Ku complex forms a ring-shape to serve as a scaffold
to recruit the NHEJ pathway molecules (Bischoff, Wimberger, Maresca, &
Brakebusch, 2020).
Overview of CAR-T Cells
In recent years, CAR-T cell emergence has revolutionized the cancer
immunotherapy, particularly against refractory hematological
malignancies (Sadelain, 2017) In this approach, patient’s own T cells
are genetically modified to express a synthetic receptor (CAR) linking
antibody binding domains (scFv) with T cell’s activation and
costimulatory molecules. This chimeric molecule is able to bind a
specific tumor antigen and transmit signals through intracellular
domains leading to activation and cytotoxic function of T cells (D. Li
et al., 2019).
Currently the most commonly used strategy for generating CAR-T cells is
harvesting patient’s derived autologous T cells, transducing them with
CAR gene normally embedded in viral vectors, and then infusing back into
the patient after lymphodepleting therapy (Poorebrahim et al., 2021).
This process involves high level of variability and potential
manufacturing failure due to low quality of cancer patient’s autologous
T cells. Also, there is the risk of insertional mutagenesis caused by
randomly integrating viral vectors. Moreover, in case of leukemias,
blood- derived T cells might be contaminated with leukemic cells (Han,
Xu, Zhuang, Ye, & Qian, 2021). Therefore, there are several
manufacturing challenges that can affect the quality of CAR-T cell
products (Marofi et al., 2021). Generating CAR-T cells through genome
editing with CRISPR/Cas9 might address these challenges, and
simultaneous TCR-knock out in T cells provides the opportunity to
develop safe allogeneic T cell therapy (Roth et al., 2018).
Principle of CRISPR/Cas9 system gene delivery into T cells
The delivery of CRISPR/Cas9 to edit the genome of interest is defined
into three distinct strategies. The first approach is using plasmid DNA
encoding the Cas9 protein and sgRNA from the same vector. The second
format is to deliver the mixture of the Cas9 mRNA and the sgRNA. The
last strategy is a ribonucleoprotein (RNP), the complex of Cas9 protein
and sgRNA, which is considered advantageous compared to the two other
systems (Luther, Lee, Nagaraj, Scaletti, & Rotello, 2018). The RNP
method does not require the delivery of foreign DNA and the complex of
Cas9-gRNA degrades over time, which may minimize the off-target effects.
RNP-based delivery displayed a fast, efficient and cost-effective method
to modify the genome of the target. Another advantage of using RNP is
the variety of methods that can be used to deliver the Cas9-gRNA
complex, including electroporation (Gundry et al., 2016).
Although the first strategy of delivery, plasmid-based CRISPR-Cas9
system, is a simple and straightforward approach, it tends to cause
off-target mutation in primary T cells (Kornete, Marone, & Jeker,
2018). The plasmid-based system encounters several challenges. Upon the
entering of plasmid into the desired nucleus, it undergoes the
transcription and translation processes to express the encoded proteins.
These processes require more time to effectively target the gene of
interest (Fujihara & Ikawa, 2014). More importantly, this format of
delivery was found to result in an irreversible off-target cleavage site
(Cradick, Fine, Antico, & Bao, 2013; Fu et al., 2013). The other
negative aspect of the plasmid-based approach is its size limitation, as
many current vectors have restrictions for large-sized genes. Moreover,
transfection of plasmid DNA may activate the cyclic GMP-AMP synthase and
as a consequence leads to host immunogenicity (Xu et al., 2019).
The second strategy is direct delivery of Cas9 mRNA and sgRNA into the
target cells to form a Cas9/sgRNA complex inside the cells. One
advantage of this approach is the use of mRNAs that can be translated in
the cytoplasm, therefore requiring intracellular delivery which is much
more convenient rather than delivery to the nucleus. Furthermore, mRNA
translation process reduces required time for genome editing. Besides,
mRNA-based delivery demonstrated a low rate of off-target effects
compared to the plasmid DNA strategy. However, this approach is limited
because mRNA is fragile and may get degraded during the delivery or
preparation process (Givens, Naguib, Geary, Devor, & Salem, 2018; Shen
et al., 2014).
The last form of CRISPR/Cas9 delivery is RNP. This approach avoids the
processes of transcription and translation, and provides the fastest
means of gene editing compared to the other two methods (Schumann et
al., 2015; Seki & Rutz, 2018). The delivery of RNP gives a myriad of
advantages including the less off-target effect due to the fast
degradation of Cas9 nuclease and no need for codon optimization and
promoter selection (Hendel et al., 2015; Liang et al., 2015). RNP
editing is very rapid, and indels can be measured after 3-24 hours. Cas9
protein is rapidly degraded from cells within 24h, compared to the
plasmid electroporation method that persists nearly 73h (Sojung Kim,
Kim, Cho, Kim, & Kim, 2014).
Currently, several non-viral nanovectors are used for RNP delivery into
the cells in vitro, including DNA nanoclews (the yarn-like DNA
nanoparticles synthesized by rolling circle amplification), cationic
lipid nanoparticles and lipoplexes (cationic liposomes, composed of
nonviral (synthetic) lipid carriers of DNA), gold-based nanoparticles,
and zeolitic imidazole frameworks (Wei, Cheng, Min, Olson, & Siegwart,
2020; Xu et al., 2019).
CRISPR/Cas9 system can be applied either before the generation of CAR-T
cells or after the production of CAR-T cells, as it is illustrated inFigure 2 . Currently, RNP delivery of CRISPR/Cas9 technology
into the T cells represented as a promising approach compared to the
other methods of delivery. T cells have been targeted by lentiviral and
adenoviral for delivery of CRISPR components. These deliveries seem to
be ineffective due to low gene disruption efficiency, feeble
site-specificity insert, and randomly disruption of unwanted genes (C.
Li et al., 2015; W. Wang et al., 2014). The in vivo transfection of
CRISPR/Cas9 model encounters different problems, including low
disruption efficiency, insertional mutagenesis, off-target effects,
toxicity and immunogenicity (Lino, Harper, Carney, & Timlin, 2018;
Mout, Ray, Lee, Scaletti, & Rotello, 2017). These adverse effects will
be explored later in the next section.
CRISPR/Cas9 system generates off-the-shelf CAR-T cells
Lymphocytes used in genetically modified-T cell therapies are dominantly
derived from patient’s autologous T cells. This source of T cells has
limitations including having insufficient number of T cells,
time-consuming and laborious isolation process (Sharpe & Mount, 2015).
These hurdles have brought the concept of universal or off-the-shelf T
cells, wherein the allogeneic T cells derived from third party donors
are genetically manipulated and can be used for different patients.
Using allogeneic T cells as the main source of T cells in CAR-based
therapy is not simple. The infused allogeneic T cells expressing αβ TCR
can recognize the recipient’s cells as foreign and destroy them, leading
to a phenomenon known as graft versus host disease (GVHD). (Ju et al.,
2005; Townsend, Bennion, Robison, & O’Neill, 2020).
Several factors are found to promote the development of GVHD, including
human leukocyte antigen class I (HLA-I) mismatched related donor or HLA
matched unrelated donor. The most important factor is
beta-2-microglobulin (β2M), a pivotal subunit of HLA-I protein that
plays a key role in the removal of allogeneic cells expressing non-self
HLA-I molecules in the recipient (Salas-Mckee et al., 2019; H. Torikai
et al., 2013). Therefore, knocking out endogenous TCR and HLA (or β2M)
as two crucial receptors of T cells may realize the development of
off-the-shelf CAR-T cells by eliminating the risk of GVHD.
Eyquem et al. disrupted T-cell receptor α constant (TRAC) locus
through sgRNA targeting the 5’ end of the first exon of TCR α, and using
adeno-associated virus (AAV) vector encoding the promoter-CAR-polyA
cassette flanked by homology arms to knock in the CD19 CAR gene. Nearly
95% of transfused CAR+ T cells were negative for TCR
expression. NALM-6 mouse with pre-B acute lymphoblastic leukemia was
introduced with 1 × 105 doses of CD19 TRAC-CAR
T-cells, which successfully achieved tumor control, and just 2% of
these cells expressed exhaustion or co-inhibitory receptors such as PD1,
LAG3, and TIM3, and maintained more effector memory phenotypes. The low
expression of inhibitory receptors is mainly associated with greater in
vivo anti-tumor activity and resulted in superior tumor eradication.
These results underscore that TRAC has a pivotal role in the regulation
of CAR expression in two different ways. One is by enhancing the optimal
baseline expression, which participates in CAR internalization upon
either the interaction with antigens or receiving signals. The other is
the recovery of baseline CAR expression upon exposure to the antigen by
controlling the transcriptional response. More importantly, targeting of
TCR locus may mitigate the probability of insertional oncogenesis and
TCR-induced autoimmunity and alloreactivity, leading to safer modified-T
cells and resulting in perpetual CAR expression. Hence, this study
depicted that how by genome editing technology, a T cell-based therapy
can be improved and yield a robust treatment (Eyquem et al., 2017).
Likewise, Ren et al. have knocked out three different genomic
loci, including TCR, β2M, and PD-1 simultaneously in human T cells via
CRISPR/Cas9 system electroporation. They introduced CAR transgene
through lentiviral transduction and generated allogeneic CAR-T cells
deprived of TCR, HLA-I, and PD-1, are known as universal CAR T-cells.
The targeting efficiency of sgRNA yielded over 90%, and the disruption
of HLA-I and TCR resulted in a low rapid rejection of CAR-T cells in
allogeneic recipients. Also, it did not lead to GVHD in in vivo model.
The anti-tumor activity of CAR-T cells increased substantially by
knocking PD-1expression out. One significant concern of this study is
that triple loci-knocked out CAR-T cells may trigger the NK cell
activation due to the absence of HLA-I in CAR T-cells. NK cell specific
antibody or NK cell depletion via chemotherapy may potentially avoid or
mitigate NK mediated rejection of transferred HLA-I negative CAR-T cells
(Jiangtao Ren, Xiaojun Liu, et al., 2017).
Inconsistent with these data, Georgiadis et al. introduced human
T cells with lentiviral vector encoded CD19 CAR and sgRNA targeting the
TRAC region, and Cas9 mRNA was delivered by electroporation. 5 ×
105 of CD19-CAR TCR- T-cells infused
into the humanized murine model of Daudi B cell leukemia resulted in
significant clearance of tumors, with no GVHD and no evidence of the
overexpression of engineered T cell’s exhaustion markers such as PD-1.
(Georgiadis et al., 2018).
Similarly, CRISPR/Cas9 was applied to CD19 CAR-T cells to ablate the
constant TCR β-chain. CD19 CAR-T cells lacking TCR were highly
functional and showed no alloreactivity in patient-derived xenografts of
CD19+ childhood ALL in a murine model (Stenger, Stief,
Käuferle, et al., 2020). These studies suggest that CRISPR/Cas9
technology can be used as a promising tool for the development of
off-the-shelf CAR-T cells by knocking out the TCR and β2M loci in
allogeneic T cells, and further it can lead to remarkable GVHD reduction
and alloreactivity by TCR disruption.
Development of CAR-T cells that are resistant to suppressive
molecules
The expression of inhibitory receptors such as cytotoxic T
lymphocyte-associated antigen 4 (CTLA-4), T-cell membrane protein 3
(TIM-3), lymphocyte activation gene 3 (LAG-3), and programmed cell death
protein-1 (PD-1) on T cells control and restrict T cell activities and
responses (L. Long et al., 2018). These inhibitory molecules mitigate
the immune responses and cause exhaustion of T cells. The exhausted T
cells have altered transcriptional program which distinguishes them from
memory and prototypic effector T cell populations (Wherry & Kurachi,
2015).
Tumors can suppress the immune responses and escape from immune cells by
expressing negative regulatory pathways, known as immune checkpoints.
One important key player of the immune checkpoints is PD-1, a type I
transmembrane receptor inhibiting T cell proliferation and performance
(Seliger, 2019). PD-1 is normally expressed on the surface of activated
T cells, and its interaction with cognate ligands, PDL1 and PDL2, limits
T cell activity and inhibits excessive stimulation, which leads to an
immune escape for tumor cells (Zak et al., 2015). PD-1 expression by
CAR-T cells has the same deteriorating impact; therefore, disrupting
PD-1 can boost T cell anti-tumor responses (Table 1).
In addition, Fas receptor (CD95), a cell surface protein that belongs to
the tumor necrosis factor α family of death receptors, contributes to
the regulation of T cell activity. Interaction of Fas molecule with its
ligand (FasL) induces the T cell apoptosis cascades which may reduce the
engineered T-cell response via inducing the activation-induced cell
death (AICD) (Jiangtao Ren, Xuhua Zhang, et al., 2017).
Rupp et al . have transfected human T cells with Cas9 and sgRNA
targeting PD-1 exon 1 through electroporation and subsequent
introduction of lentiviral vector containing the CD19 CAR transgene.
CD19+ PD-L1+ tumor xenograft models
were injected with 4×106 PD-1-deficient CD19 CAR-T
cells and resulted in clearance of tumors in all treated mice. This
finding highlights the suppressive role of the PD-1/PD-L1 axis on CAR-T
cells upon the engagement with the antigen of interest on tumor cells.
Further, the study revealed the applicability of CRISPR-Cas9 genome
editing as a viable tool for the enhancement of CAR T-cell performance
(Rupp et al., 2017).
In contrast to the previous study wherein T cells were transfected at
least twice using a combination of electroporation and lentiviral
transduction, a new study transfected T cells using plasmids encoding
Cas9, PD-1 targeting sgRNA, and the piggyBac transposon vector encoding
CD133-CAR in one reaction via nucleofection process. This method led to
89.5–95% insertions and deletions in PD-1 gene site.
2×106 doses of PD-1-deficient CD133 CAR-T cells were
infused to the orthotopic glioma xenografts in immunodeficient mice and
led to outstanding outcomes. The modified T-cells demonstrated
persistence and the survival of mice was enhanced. Besides, no sign of
GVHD and CAR T related side effects and aberrant proliferation of
PD-1-deficient modified T-cells were detected due to the rapid
elimination of these modified T-cells within 28 days, highlighting the
role of PD-1 in the survival of CAR T-cells. Importantly, this study
highlighted the use of plasmid DNA as a more efficient approach due to
the low cost and easier preparation compared to the RNA, protein and
virus delivery methods (B. Hu et al., 2019). Other studies are also
consistent with these findings (W. Hu et al., 2019; Nakazawa et al.,
2020).
More encouragingly, Choi et al. exerted the CRISPR-Cas9 system
application to EGFRVIII CAR-T cells in which three different loci
including PD-1, β2M and TRAC regions were targeted to generate universal
CAR modified-T cells resistant to PD-1 suppression. T cells were
electroporated with CRISPR/Cas9 complex targeting TRAC and β2M and Pdcd1
loci and were subsequently transduced with adeno-associated virus
encoding the EGFRVIII CAR. More than 80% of the T cell population was
double knocked out for surface expression of TCR and HLA-I.
5×103 triple gene-deficient EGFRvIII CAR-T cells were
administered through intravenous delivery or intraventricular infusion
in murine models of human GBM. The former route of delivery did not
significantly increase the survival rate of mice, while the latter means
of infusion showed to be more efficacious against the GBM mice model.
The triple gene-deficient EGFRvIII CAR-T cells also depicted highly
anti-tumor response in preclinical glioma models (Choi et al., 2019).
CRISPR/Cas9 technology enhances CAR T-cell performance in
solid tumors
The efficacy of CAR T-cell therapy towards solid tumors has been
severely restricted by some physical and physiological barriers, such as
tumor microenvironment (TME) hypoxia, acidic environment, and
nutritional deficiency and presence of immunosuppressive cells
(regulatory T cells, myeloid-derived suppressor cells, and
tumor-associated macrophages and neutrophils) (Yazdanifar, Zhou, &
Mukherjee, 2016). Solid tumors create a complex zone containing many
cell types, tumor’s vasculature, extracellular matrix components,
connective tissues, and inflammatory mediators which can impair T cells
infiltration and function (Joyce & Fearon, 2015; Turley, Cremasco, &
Astarita, 2015).
Other factors which hamper the efficacy of CAR-T cells include
suppressive molecules (Indoleamine 2,3-dioxygenase (IDO) (Yazdanifar et
al., 2019), transforming growth factor-beta (TGF-b), PDL1, IL-10 and
arginase-1), immunosuppressive inhibitor receptors, soluble factors
(prostaglandin E2 and indoleamine-2,3-dioxygenase) (Anderson, Stromnes,
& Greenberg, 2017; J. H. Chen et al., 2015; Koyama et al., 2016).
To overcome the hostile TME, recently, a group of researchers
successfully disrupted the endogenous TGF-ß receptor II (TGFBR2) gene in
modified T-cells expressing mesothelin CAR, using CRISPR/Cas9
technology. The modified T cells diminished the induced Treg conversion
and restrained the exhaustion. TGF-ß knockout mesothelin CAR-T cells
eradicated the tumor cells completely by day 28 in pancreatic carcinoma
patient-derived xenograft models expressing mesothelin and
TGF-ß1receptors. This study further shed light on the negative
regulatory role of TGF-ß receptors in CAR T-cell cytotoxicity responses.
It also highlighted that disruption of the TGFBR2 gene would enable
modified T-cells to survive and proliferate effectively and exert higher
anti-tumor activity. Encouragingly, it was shown that knocking out of
other immune checkpoints such as PD-1 simultaneously with TGF-ß1 may
lead to a better therapeutic outcome in CAR T-cell therapy (Tang et al.,
2020).
Diacylglycerol kinases (DGKs) is an enzyme that phosphorylates
diacylglycerol (DAG) signaling to encourage phosphatidic acid (PA)
production. Both DAG and PA are bioactive molecules that regulate a
multitude of intracellular signaling proteins involved in innate and
adaptive immunity (S. S. Chen, Hu, & Zhong, 2016). Upon the interaction
between TCR on T cells and antigen presenting cells, a cascade of
signaling initiates by the activation of phospholipase C γ1 (PLCγ1),
which cleaves phosphatidylinositol-4,5-biphosphate
(PIP2) to form the second messengers DAG and inositol
triphosphate (IP3) (Baldanzi, Bettio, Malacarne, &
Graziani, 2016). DAG plays an important role in activation of different
downstream signaling pathways, such as AKT, NF-kB, and Ras pathways. The
vital role of DGKs is to control DAG metabolism in T cells. Two DGK
isoforms, DGKα and DGKζ, control DAG signaling in T cells. DAG engages
with pivotal proteins present in CD3 signaling such as Ras activating
protein (RasGRP1) and protein kinase C (PKC); thus, activation of DGK
leads downregulation of TCR distal molecules via metabolizing DAG at
immune synapse (Riese, Moon, Johnson, & Albelda, 2016). Accordingly,
DGKs control T cell polarization and function during migration and
activation, anergy, and response to tumor cells.
The production of CARs with appropriate signaling mechanism is an
essential issue to boost the cellular activation, persistence, cytokine
secretion, and cytotoxicity of CAR-T cells. Since DGKs participate in T
cell signaling, a group of researchers used CRISPR/Cas9 genome editing
technology to disrupt DGK in CAR-T cells. Findings revealed that
DGK-deficient CAR-T cells were significantly more resistance to soluble
immunosuppressive components such as prostaglandin E2 and TGF-β inin vivo model. DGK-deficient CAR-T cells had increased effector
functions in vitro and robust TCR signaling. This finding clearly
suggested that DGKs can be considered as a potential therapeutic
component to tackle suppressive factors present in solid tumors that
prevent CAR T-cell activities (Jung et al., 2018).
6. CRISPR/Cas9 technology can reduce CAR-T cells associated
toxicities
Although CAR T-cell therapy is demonstrated as a promising therapeutic
option for different cancers, this novel treatment is not exempt from
adverse events, and needs thorough consideration to tackle its
limitations. One potential adverse event is on-target off-tumor effects,
where CAR-T cells target the healthy tissues sharing the same epitope of
antigen (Jung & Lee, 2018). Second adverse event is cytokine release
syndrome (CRS), which mostly occurs by proinflammatory cytokines such as
granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte
chemoattractant protein-1(MCP-1), TNF-α , IL-6, IFN-γ , and
IL-2 (Shannon L Maude et al., 2018; Sattva S Neelapu et al., 2017; Park
et al., 2018). One eminent strategy to tackle these obstacles is the use
of CRISPR/Cas9 gene-editing technology.
Sterner et al. applied the CRISPR/Cas9 system armed with sgRNA
targeting GM-CSF in CD19 CAR-T cells to control the CAR-T associated
toxicities. These GM-CSF-deficient CD19 CAR-T cells reduced the GM-CSF
secretion and enhanced the anti-tumor effect with low side effects of
CRS and neurotoxicities in leukemia NALM6 xenograft model (Sterner et
al., 2019). This study clearly proposed that GM-CSF is associated with
CRS; thus, future studies need to target this gene to reduce the side
effects.
CRISPR/Cas9 can be utilized to generate safer and more controllable
CAR-T cells by adding inducible safety switches or suicide genes which
provide a tool for eliminating CAR-T cells in case of potential
toxicities. An inducible Cas9-based suicide gene was incorporated in
IL-15-expressing CD19 CAR-T cells by Hoyos et al. and their
results confirmed that >95% of CAR-T cells could be
efficiently ablated within 24 hours upon pharmacologic activation of the
suicide gene (Hoyos et al., 2010). Currently, there are three clinical
trials (NCT02107963, NCT01822652, and NCT02439788) incorporating the
Cas9-based suicide gene into CAR-T cell products to provide a means for
eliminating the autologous CAR-T cells in case of unexpected off-target
toxicity. Insertion of safety switches in CAR construct is another
approach to terminate the adverse effects without jeopardizing clinical
responses. Inducible Cas9-based safety switch was tested in a CD19 CAR-T
cells and results confirmed its feasibility for eliminating CAR-T cells
in a dose dependent manner in a humanized mouse model (Diaconu et al.,
2017). This approach allows for both selective suppression of CAR-T cell
activation in a case of CRS, and also complete depletion on demand.
As discussed here, many limitations of conventional CAR-T cells can be
addressed using CRISPR/Cas9. However, there are concerns surrounding the
safety of using these gene-edited cells in clinic and careful
investigation must be applied. Several factors such as off-target
effects, unintended mutations and unwanted Cas9 activity can affect the
safety of CRISPR/Cas9 system (S.
Kim et al., 2018). Besides, CRISPR/Cas9 might alter the function of
gene-edited CAR-T cells which could lead to the activation of unintended
innate/adaptive immune responses
(S. Kim et al., 2018). Although
these events are rare, they can cause adverse effects in patients and
need to be addressed in gene-edited T cells before clinical use.