Elucidation of CRISPR-Cas9 application in novel cellular immunotherapy

Novel cellular immunotherapy with engineered T cells has improved cancer treatment and established therapeutic promises to prevent tumor formation in clinical studies. Due to certain restrictions and difficulties, CAR and TCR T-cells therapies were inadequate at points. CRISPR Cas9 genome-editing tool has significant potential for these two cell-based therapies. As a specialized gene-editing technique, CRISPR Cas9 is used to repair genetic alternations with minimal damage. It is used as an adjunct to immunotherapy to stimulate a more robust immune response. CRISPR has long outpaced other target-specific genome editing methods such as ZFNs and TALEN because of its high efficiency, competence in targeting, and stable operating conditions. CRISPR can overcome the two major drawbacks of universal CAR T cells: allorejection and graft-vs-host disease. TCR-based T cell treatment can reduce inappropriate binding between endogenous and transgenic TCR, resulting in a reduction of severe toxicity. The CAR and TCR T based cell therapies uphold an excellent future for tumor malignancies. This article has elucidated the administration of CRISPR Cas9 in novel cellular immunotherapy, CAR, and TCR T cell therapy. However, this article did not fail to observe this technology's ethical concerns, limitations, and challenges. Furthermore, the article compares CRISPR-mediated allogeneic CAR T cell to TCR-T cell therapy.


Introduction
Cancer is one of the most lethal diseases in the world. Following cardiovascular disease, it is the second leading cause of mortality [1]. Carcinogenesis can be triggered by genetic diversity in an individual's DNA, inducing a complicated immunological response. It encompasses both tumor cells' immune evasion and the tumor microenvironment's immunosuppressive nature [2]. Recently, scientists have used various genetic manipulation techniques to repair the DNA in the genome of a cell via site-specific modification. These techniques can edit tumor cell's genome to meet apoptosis, decrease the drug resistance and replace mutant genes [3][4][5][6]. Aside from traditional cancer treatments like chemotherapy, radiation, and surgery, newer approaches like oncolytic virotherapy are being developed to use the immune system against cancer cells [7,8].
Immunotherapy, including checkpoint inhibitors and adoptive T cell therapy, has been very successful in treating different malignancies. It functions by reversing the tumorinitiated immunosuppression and boosting immunity against cancers [9]. Anti-programmed death 1 (PD-1/PDL-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA-1) are immune checkpoint inhibitors that play a significant role in blocking immunosuppressive signals promoting T cell renewal. In comparison, ACT or adoptive cell transfer could be accomplished through distinct immunogenic processes such TILs, engineered CAR T, and TCR T cells [10,11]. Recent development in molecular biology has led to the advancement and refinement of gene editing technologies. A gene-engineering tool is required to achieve the desired outcome in novel cellular Immunotherapy, particularly in CARs and TCR T cells.
'Clustered Regularly Interspaced Short Palindromic Repeats associated with Cas protein nine is defined as CRISPR Cas9. It outperforms alternative gene engineering tools, Zinc Finger Nucleases and Transcription Activator-Like Effector Nucleases (TALENs), over its straightforwardness, high accuracy, and multiple gene targeting capabilities 1 3 [12]. It is a sophisticated technique for gene editing that has been using in cancer research.
Researchers have used CRISPR technology to design cancer models, identify novel immunotherapy targets, potentially accredit genes for drug trials, examine individuals' drug resistance mechanisms, and gain a brief understanding of the mode of action of non-coding gene regions, among other applications [13][14][15][16][17][18][19][20]. CRISPR maybe used to treat tumors using cancer immunotherapy by enhancing efficiency and safety. The application of CRISPR Cas9 with cancer immunotherapy has a great potential to revolutionize cancer treatment by alleviating patients' suffering from untargeted therapies like chemotherapy and radiation.

CARs-T cell immunotherapy through CRISPR Cas9 system
CARs (chimeric antigen receptors) were initially developed in 1989 [21]. Over the last few decades, engineered T-cells with CARs have shown significant effectiveness in the treatment of various types of leukemia [22]. Chimeric antigen receptors are composed of three components: a ScFv derived from a monoclonal antibody, an extracellular antigen-recognition site, a hydrophobic domain, and intracellular signaling transduction, which contains a CD3 chain with costimulatory molecules such as CD137, CD28, OX40 4-1-BB, CD3ζ, (Fig. 1). According to their endo-domain structure, CAR T cells can divide into four generations.
The discovery of genetically modified CAR T-cells has unfolded a new revolutionary era in cancer treatment [21,23,24]. Kymriah and Yescarta were the initial CAR T-cell therapy authorized by the US Food and Drug Administration after their successful impact on B-cell malignancies [25]. The two CD19-directed CAR T-cell therapies were authorized to treat B lymphoblastic leukemia in adolescents and large B-cell lymphoma in adults. The FDA authorized two more CAR T-cell treatments, Tecartus and Breyanzi, in 2020 and 2021, respectively, to treat Mantle Cell Lymphoma (MCL) and highly resistant large B-cell lymphoma. While the success rate of this treatment was rising, many patients were unable to get it for various reasons. As laborious and costly, patients with advanced level diseases could not make the grade to Immunotherapy. The second instance was the production of the finest quality of T-cells from individuals with lymphopenia [26]. Compared to individuals' CAR T-cells, allorejection and GVHD (graft versus host disease) were the primary concerns with allogeneic CAR T-cells, caused by pre-existent endogenous TCR and HLA I on donor's T-cells. CRISPR technology has outperformed the previous genome modifying technologies in terms of efficiency and versatility in the production of CAR T-cells [27].
Researches have demonstrated that using multiple guided RNAs inserted in a CAR lentiviral vector allows CRISPR to knock off gene loci such as TCR/TCR class 1, PD-1, HLA class1, Fas, in one shot [28,29]. Ren et al. published another study in 2017 demonstrating that utilizing CRISPR to initiate CAR T-cells production can decrease endogenous TCR, MHC I corresponded HLA I, and PD1, thus increasing anticancer immunity in clinical investigations [30].
Finally, the CRISPR-mediated knockout system for these checkpoint inhibitors has demonstrated no evidence of alloreactivity. Consequently, it enhanced CAR T cell functional activity and opened the path for universal CAR T cell development from healthy donors [31][32][33].
Several researchers have employed CRISPR Cas9 to disrupt the PD-1 gene from CAR T-cells in primary brain cancer, hepatocellular carcinoma, and K562 tumor cell lines. The Programmed death one removal from CAR T cells increased the tumor-killing activity. It also reduced exhaustion and improved the cancer-killing ability of T cells [34][35][36]. The application of CRISPR technology to remove additional genes from CAR T cells, including LAG 3, GM CSF, CD7, TRAC, DGK, and CTLA-4, showed remarkable improvement in CAR T cell performance.

TCR based T cell immunotherapy by CRISPR Cas9
Engineered TCR T cell immunotherapy upholds a higher affinity in targeting a wide range of antigens in numerous cancers. It has shown significant curative promises in multiple cancer, along with melanoma [43,44], sarcoma [43], and multiple myeloma [45]. Antigen-recognition receptors present on the surface of T cells are known as T cell receptors. T cell receptors are heterodimers that contain two chains: α chain and β chain. The α chains and β chains include a variable antigen-binding region, an extracellular area, and a hydrophobic domain. The complex formed between TCR and CD3 is called TCR/CD3 complex. It is composed of TCR and CD3 chains with γ, δ, ζ, ε (Fig. 2). Through an MHC recognition pattern, this TCR/CD3 complex may identify tumor-specific antigens.
Genetically modified TCRs' are inserted in the recipient T cells through retroviral gene transfer [46]. Due to preexistent endogenous TCR, the recombinant TCR engages in a competition to form CD3 and cell surface expression. Therefore, the mispairing between the recombinant and endogenous TCRs forms a mixed dimer, resulting in dilution of the surface expression and detrimental autoreactivity [47,48]. Endogenous TCRs' have low efficiency in targeting self-antigens on normal cell-like TAAs (tumor-associated antigens) than foreign invaders like bacteria and viruses [49]. Thus, treating cancer with natural TCRs has delayed the treatment progression because cancer-specific antigens are also like TAAs on normal cells. In 2006, a study carried out by Morgan et al. performed a clinical experiment to treat patients with melanoma. He targeted melanocyte differentiation antigen (Mart1) using natural TCRs with different levels of targeting efficiency. The study uncovered that DMF4 (low-affinity of T cells) was well tolerated but lacked anti-tumor efficacy. He then used DMF5 (higher affinity of T cells) in the research. The result indicated an increased degree of anti-tumor efficacy and significant toxicity [50].
To overcome this limitation barrier, CRISPR Cas9 was employed with a manufactured tumor-specific TCR sequence to disrupt the α and β genes present on the endogenous TCRs [45,51,52]. Legut et al. reported that replacing endogenous TCRs from T cells upgrades surface expression and transgenic TCR T cells [52]. Researchers adopted another technique to reduce the inappropriate pairing between the endogenous and engineered TCRs. CRISPR Cas9 was used with the transduction of a stabilized single-chain TCRs to disrupt the mispairing, which showed promising results [53].
In 2020, the first clinical study of CRISPR mediated T cell therapy w applied to cancer patients who were not responding to previous treatment [54]. By using the lentiviral transduction process, the autologous T cells from patients were modified. Later on, CRISPR disrupted the endogenous TCR and PD1. Then the modified TCR T cells therapy was applied to three patients where two of them showed stable results; then another showed disease advancement [54].
CRISPR Cas9 mediated transgenic cell receptor T cell therapy may bring unprecedented results in cancer remission in the upcoming future by eradicating the limitations and challenges.

CRISPR-mediated CAR T cell therapy over TCR T cell therapy: present and future
Chimeric antigen receptor-T cells could detect peptide antigens and effectively identify carbohydrates and glycolipid antigens, allowing for a broad spectrum of the target of cancer antigens [55]. When CARs T cells were compared to TILs and TCR-T cell therapy, CARs T cells were more effective on many criteria, including lower cell therapy dosages, increased specificity, a clear target for selecting cancer-specific antigens, and a small number of single transfusion cells used in the treatment [55]. CAR T cell immunotherapy has demonstrated promising outcomes in hematological malignancies. Still, regarding solid tumors, it has a minor impact due to the absence of specific antigens for tumors, antigen heterogeneity, and tumor microenvironment. Additionally, threatening adverse events including cytokine release syndrome (CRS), neuroinflammation, and difficulties associated with alloreactivity, GVHD, and overexpression of immune checkpoint inhibitors during manufacturing of universal CAR T cells made this therapy less effective than TCR-T cell therapy.
T cells based on transgenic cell receptors could detect antigens generated by MHC molecules, regardless of whether they were intracellular, cell surface, or neo-antigens. The intracellular antigens recognition by TCR-T cells produced a broad range in targeting antigens [55]. Therefore, researchers used various methods of TCR-T cell therapy in hematological and solid tumors, which have positive outcomes in cancer treatment. As TCR-T cells treatment is MHC-centered, there is always a risk of mispairing between endogenous and transgenic TCR. Consequently, such mispairing between two TCRs may lead to significant toxicity, inducing GVHD in patients. Adjusting affinity levels was another issue in this cell treatment because a greater affinity level may provide misleading findings, while lower affinity reduces the anti-tumor effectiveness. Though TCR-T cellsbased immunotherapy had demonstrated promising therapeutic benefits on cancer treatment, the constraints made it tough to co-operate in clinical trials.
Until recently, experts have tried a variety of methods to cure cancer fully. Immunotherapy has changed the scenario in the fight against cancer treatment. Lately, the US Food and Drug Administration has approved a variety of immunotherapies against carcinomas. Despite this, new cellular immunotherapies based on CAR and TCR T cells have a more significant effect on therapy. Researchers have used CRISPR Cas9 genome editing to address the challenges of these two T cell-based immunotherapies.
We already know that the pre-existent endogenous HLA and TCR on donor T cells may result in alloreactivity and GVHD during CAR T cell generation. Numerous groups believe that the primary cause of GVHD is the αβ chain on T cells used in developing CAR T cells [56]. In recent years, significant advancements in gene-editing technology have given the tools required to suppress endogenous TCR expression and reduce GVHD risk.
Furthermore, it increases the risk of off-target cleavage, leading to more significant CAR T cell proliferation when tumor suppressor genes are lost [59,60]. One of the unique methods for obtaining functional benefits and avoiding GVHD in a more regulated manner is using CRISPR to directly insert the chimeric antigen receptor transgene into the T Cell Receptor Alpha Constant. Moreover, to GVHD, this modification enables the CAR to be expressed homogeneously and controlled under the TCR promoter, a characteristic that has reduced differentiation and exhaustion [57,[61][62][63]. Additionally, CRISPR was capable of mitigating the adverse effects of this treatment. In clinical trials, Sterner et al. showed that CRISPR could disrupt the granulocyte-macrophage colony-stimulating factor gene, improving T cells anti-tumor activity while also lowering the chances of cytokine release syndrome and neuroinflammation (Table 1) [38]. Tang et al. found that using CRISPR technology to disrupt the endogenous TGF-receptor II (TGFBR2) reduces CART cell fatigue and improves tumor-killing efficacy [41]. Such changes in CAR T cells therapy after CRISPR modification have led researchers to apply it clinically. Several clinical studies on CRISPR Cas9-mediated CAR T cells are currently ongoing.
According to Clinical.gov, eight clinical studies are actively enrolling participants for this modified T cell therapy.
On the other hand, CRISPR Cas9 application in TCR T cell treatment has demonstrated promising outcomes in decreasing undesirable effects associated with prior TCR T cell therapy.
The first clinical trial performed by Stadtmauer et al., using CRISPR-mediated TCR T cell treatment, demonstrated that it has a bright future but requires further investigation [54]. Before applying CRISPR, TCR-T cell treatment was preferable because of its capacity to target a wide variety of tumor antigens. However, the scenario has reversed by the development of the CRISPR Cas9. CRISPR-mediated 1 3 universal CAR T cell immunotherapy can target solid and hematological malignancies by CRISPRs' diverse gene targeting capability. As a result, universal CAR-T cell-based Immunotherapy engineered by CRISPR may reign in a revolution in the coming years (Table 2).

Limitations and advantages of CRISPR Cas9
The initial limitation that concerns researchers in CRISPR mediated gene editing are CRISPR's off-target effects [64]. Bacteria and archaea have been seen to be benefited by these  1 3 off-target's effects of CRISPR [65]. However, in numerous preclinical trials with C. elegans and rabbits has demonstrated that CRISPR can induce unintentional genomic alternation or gene deletions [66][67][68]. It has been reported in studies that CRISPR has also shown these effects in T cells. The random mutation is one of the results of these offtarget effects which affect tumor suppressor genes and also activates oncogenes. The off-target effects of CRISPR were identified due to the insertion of TRAC or TRBC locus of CAR-T cell using it. When whole-genome sequencing was done on a CRISPR-Cas9-edited animal, a contentious study found that CRISPR gene editing could generate hundreds of unexpected changes in the genome [69]. A further study found that CRISPR/Cas9 genome editing in human retinal pigment epithelium cells resulted in a p53-mediated DNA damage response [70]. Activation of the p53 gene may result in chromosomal rearrangements and other tumor-causing alterations in cells. Even though consequence of CRISPRinduced p53 activation is unknown, it appears to reduce the effectiveness of gene editing. As a result, off-target difficulties must be considered in future CRISPR/Cas9-edited CAR T cell development. To manage the potential hazard of clinical CAR T treatments, off-target assays during CRISPR target selection may be used [71]. Another limitation regarding this technology includes unanticipated translocations between double strands breaks in DNA when CRISPR is editing multiple genes in a row. Though it is an uncommon scenario in case of T lymphocytes, but transformation analysis should be always done to ensure the effectiveness of CAR T cell therapy. Besides the potential for translocations to cause harm, the altered functionalities of gene-edited CAR-T cells would almost certainly cause harm to patients. CRISPR gene disruption, for example, can result in unexpected innate immune responses in CAR T cells [72].
Despite of the limitations, the advantages of CRISPR Cas9 made it an extremely reliable technology in gene editing. CRISPR-based gene editing has the potential to significantly improve the safety and efficacy of CAR-T cell treatments. CRISPR is a considerably more precise tool for genome engineering than previously available gene editing methods, which means it has fewer off-target consequences that can be harmful to patients. Additionally, it can generate several gene modifications in T cells, a feat that has traditionally been difficult [72].
CRISPR may be utilized in a variety of ways, one of which is to precisely insert the CAR into the exact location in the T cells' genome to ensure that it is produced at appropriate amounts. Additionally, it can be utilized to fix genetic abnormalities in autologous T cells, which can impair the lymphocytes' ability to successfully target and destroy cancer cells. Furthermore, CRISPR modifications can increase the efficiency of CAR-T cells in destroying cancer cells. A further significant application of CRISPR in CAR-T therapy is its ability to manufacture universal CAR-T cells, or "offthe-shelf" CAR-T cells. Universal allogeneic CAR-T products eliminate the need to extract, modify, proliferate, and transfuse T cells from each unique patient, hence saving time and resources [72].
With expressing the CAR on allogeneic (healthy donor) T cells by knock-in and then knocking down the genes responsible for the immune system's detection of these non-selfcells, the cells can be successfully transfused into patients without risk of immunological rejection. These CRISPRedited designer cells have the potential to be mass manufactured, then cryopreserved and kept in hospitals or other facilities across the country, obviating the requirement for individual patient transportation of living cells between production locations [72].

Ethical challenges and concerns
The ethical debate on the termed "CRISPR" is an on-going topic for many years. There has been a lot of discussion recently regarding CRISPR because of a disagreement among academics over the ethics of genetically editing human germ lines. The discussion boils down to a "go or no-go" confrontation between the two sides. Scientific and clinical benefits can be gained through human germ line editing research; however, some researchers feel that it is too risky or crosses an inviolable ethical line to proceed with the research [73]. Another ethical issue that concerns researchers is GMO or genetically modified organisms. Ethical considerations are more pressing than whether to utilize CRISPR to change human germ cells and embryos. It is already being utilized to alter insects and mammals as well as microbes, as well as develop human treatments, using CRISPR [74].
CRISPR may not appear to generate new ethical difficulties in certain circumstances because similar work has been going on for years-or even decades-before it was developed. However, the affordability and efficiency of CRISPR raises the possibility that technology will trample on genuine concerns about the creation and release of genetically engineered organisms (GMOs). The recent characterization of Francisella novicida's up-to-date type 2 CRISPR system shows that the toolkit of genome editing techniques is constantly growing. As a result, effective global standards for GMO testing and environmental discharge are now required [75].
Despite of such ethical challenges, CRISPR is considered as the most powerful gene editing technology that not only modify genomes but also upholds a variety of significant possibilities such as cure a wide range of diseases, or mend fatal gene abnormalities in a human embryo through genetic engineering.

Conclusion
Humanity has overcome complex challenges in the past and emerged victorious [76]. However, humankind is engulfed in darkness, with no apparent cures for illnesses such as cancer and HIV. Scientists have worked effortlessly to find a natural remedy for cancer. Though chemotherapy and radiation may kill all cancer cells and prevent the illness from returning, individuals with suppressed immunity found it challenging to cope with the disease's retreat and adverse effects. In recent years, Immunotherapy has been well known for its contribution to vital cancer. Immunotherapy works by reinforcing the immune system's ability to identify and destroy tumor antigens. Since the early decades, drugs development and repurposing have had a long and complicated history. The discovery of a new drug remains a lengthy process, requiring approximately ten to twelve years to bring effective medicine or potential drug molecule from the laboratory to the marketplace [77]. It reported that the average cost of getting medication to market is more than 3 billion dollars [78]. Several companies worldwide have been advancing their ideas to the clinical stage. Therefore, CAR-T cell treatments for various cancers are anticipated to be available in the future [55]. The third and fourth generations of CRISPR Cas9 mediated CARs T cell therapy have shown higher efficiency and specificity in clinical trials, opening a new door for the transformation of the treatment of various blood cancers and solid tumors [79][80][81]. On the other side, CRISPRmediated TCR-T treatments can be more beneficial for solid cancers. The goal of these two therapies is to increase their specificity and efficiency and decrease the adverse effects of cancer treatment. This purpose may be achieved by using CRISPR gene editing as a tool for gene therapy, identifying particular tumor antigens, and enhancing cellular immunotherapies; however, any limits and concerns should be considered before proceeding with any clinical trials.
Funding No Funding was received.

Conflict of interest
The author declares that they have no conflict of interest.