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.