A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies



Concerning the engineered or bacterial nucleases, the progress of genome editing machinery has provided the possibility of direct and specific recognition and modifi- cation  of genomic  sequences  in practically all eukaryotic cells   [1,   2].   Genome    editing    has   resulted    in  the advancement of our knowledge  respecting  the finding of innovative  therapeutic options  for  treating  a wide spectrum of human  disorders,  ranging  from  infection  to cancer.  Current development in evolving programmable nucleases, including zinc finger nucleases (ZFNs), tran- scription  activator-like  effector  nucleases  (TALENs),  as well as clustered  regularly interspaced short  palindromic repeat   (CRISPR)-CRISPR-associated   protein   9  (Cas9), has critically accelerated  the development of gene editing from notion  to clinical practice  [3]. As CRISPR-Cas9 has been  suggested  as  an  encouraging   tool  for  generating gene  knockouts,   its  competence  to  offer  capable  gene editing  in primary  T cells presents  a pronounced study tool to support  a paradigm shift in T cell-based im- munotherapies, more  importantly,  next-generation chimeric  antigen  receptor  (CAR)-T cells [4].


CAR-T  cell therapy  includes  the  genetic  modification of patients’  autologous  T  cells or  allograft  cells to  effi- ciently express a CAR involving a fusion protein  of a se- lected   single-chain   fragment   variable   (ScFV)  from   a specific monoclonal antibody  and one or more  T cell re- ceptor  intracellular   signaling  domains.  This  chimer  re- ceptor    can   selectively   and   efficiently   recognize   the related   tumor-associated  antigen   (TAA)  expressed   by tumor  cells [5]. Nonetheless,  severe and life-threatening toxicities,   such   as  cytokine   releases   syndrome   (CRS), graft-versus-host disease (GVHD), on-target/off-tumor toxicity,  neurotoxicity,  and  tumor  lysis syndrome,  com- monly  constrain  its clinical  utility  [6]. Correspondingly, it  seems  that   further   progress   in  the  next-generation CAR-T  cells  with  more   optimized   construction, pro- moted  efficacy, and moderated toxicities is of paramount importance. Meanwhile,  the  production of the  universal “off-the-shelf” CAR-T cells from healthy donors can cir- cumvent   the  restraints   and  possibly  be  a  milestone  in the  future.  For  overcoming  the  GVHD  occurrence and potent  rejection  upon  CAR-T  cell, CRISPR/Cas9-medi- ated   ablation   of  the   endogenous  αβ  T  cell  receptor (TCR) has resulted  in a pronounced success  in preclin- ical studies  [7]. The  endogenous αβ TCR on  adoptively transferred donor  lymphocytes  can  identify  alloantigens in  human  leukocyte  antigen  (HLA)  mismatched recipi- ents, and thereby leads to the GVHD; on the other  hand, detection   of  foreign  HLA  molecules  on  donor  T  cells can cause rejection [7]. Further, ablation of beta-2- microglobulin   (β2M),  a  pivotal  subunit   of  HLA-I  pro- teins, can potently  avert swift eradication  of allogeneic T cells those express foreign HLA-I molecules.

Also, it has been suggested that dual blockade of pro- grammed  cell death  protein  1 (PD1), lymphocyte  activa- tion  gene  3 (LAG-3), or  cytotoxic  T  lymphocyte- associated antigen-4 (CTLA-4) using genome editing technologies  can sustain  the improved  T cell effector ac- tivities, facilitating an abrogation  in tumor  growth [8]. Moreover,   knockout    of  diacylglycerol   kinase   (DGK), which  metabolizes   diacylglycerol  to  phosphatidic  acid, using CRISPR/Cas9 supported CAR-T cell anti-tumor functions  against  U87MGvIII  glioblastoma  cell in  vitro and xenografts [9].


Herein,   we  deliver  a  brief  overview  concerning the CAR-T  cell-based  therapy  to  treat  human  cancer,  ran- ging  from  hematological  malignancies  to  solid  tumors. Also, we discuss  recent  findings  respecting  the  applica- tion  of genome  editing  platforms,  in  particular   CRISP-Cas9, for potentiating the  safety and  efficacy of CAR-T cells in the context  of tumor  immunotherapy.


CRISPR/Cas9 therapeutic  application


Early in 1987, CRISPRs were firstly discovered  in E. coli and after that in a large number of other  bacteria species [10]. Various investigations  in 2005 displayed their like- nesses  to  phage  DNA, and  succeeding  studies  indicated that  these  sequences  contribute to bacterial  and archaea adaptive   immune   responses   toward   offending   foreign DNA  by  stimulating   the   RNA-guided   DNA   cleavage [11]. Today, the CRISPR-Cas systems are largely catego- rized into two main classes according to the structural dissimilarity   of  the  Cas  genes  and  their   construction shape [12]. Meanwhile,  a class 1 CRISPR-Cas system in- volves multiple  effector  complexes,  while a class 2 sys- tem includes only a single effector protein. To date, six CRISPR-Cas types  and  approximately 29 subtypes  have been discovered  [13, 14]. The most  commonly  employed subtype  of CRISPR systems  is the  type II CRISPR/Cas9 system, enabling targeting specific DNA sequences  by a single  Cas  protein  from  Streptococcus  pyogenes (SpCas9) [15]. The  CRISPR/Cas9 system consists  of two main  parts,  including  a single-stranded guide RNA (sgRNA) as a particular  17–23 base-pair (bp) sequence intended  for specific identification  of target  DNA region in a sequence-specific style, and  also a Cas9 endonucle- ase [15]. The  sgRNA sequence  is required  to  be trailed by a short  DNA sequence  upstream to facilitate efficient compatibilization with the Cas9 protein  [16]. Corres- pondingly,  the sgRNA causes a connection with a target sequence  by Watson-Crick base pairing and Cas9 exactly cuts  the  DNA  for  establishing   a  DNA  double-strand break (DSB) [16]. Upon  the DSB, DNA-DSB repair  tools start genome  repair. The DSBs can be repaired  by one of the  two main  appliances  that  largely rein  almost  all cell types and organisms,  including  homology-directed repair (HDR) and nonhomologous end-joining  (NHEJ), leading to the  targeted  integration or gene disruptions, respect- ively [17].


The  further  description concerning detailed  mechan- ism of the CRISPR-Cas9 function  and parameters impli- cated   in   the   determining  its  efficacy  is  beyond   the scopes of this article, and thereby  audiences  are referred to the some excellent review in this context  [18–20].


Compared to  ZFN  or  TALEN  tools,  CRISPR-Cas9  is more  suitable  because  of its flexibility and  the  capacity for  multiple   gene  editing   [21].  Indeed,   endonuclease- based ZFN or TALEN technologies request the reengi- neering  of a unique  enzyme,  which  should  be manufac- tured  distinctly  regarding  each target  sequence  [21], but, as the nuclease  protein  Cas9 is the same in all cases, can be appropriately engineered to detect novel regions by varying   the    guide    RNA   sequences    (sgRNA)   [22].


Moreover,  compared to CRISPR-Cas9, ZFNs and  TALE Ns  request  much  more  labor  and  are  more  expensive. On  the  other  hand,  the  unique  competence of CRISPR/ Cas9 to edit multiple  loci concurrently signifies that  this toll is easier, more efficient, and more scalable in com- parison   to  the  ZFNs  and  TALENs  [23].  Thus,  in  the context  of CAR-T  cell-based  targeted  therapy,  it is cur- rently applicable to concurrently affect several genes and accomplish   loss  of  function   (LOF)  of  potentially   any genetic or epigenetic  target  utilizing CRISPR-Cas9 [24].


CAR construction


Concisely, CAR is an engineered  modified fusion protein structurally  similar to the TCR and involves an extracel- lular antigen detecting domain linked to one or more intracellular  signaling domains  [5]. The  CAR extracellu- lar domain  is structurally  an antibody  single-chain  vari- able fragment  (scFv) and identifies the target antigen virtually overexpressed on the tumor cells in the HLA- independent  manner   [25].  The   CAR  intracellular   do- mains  typically involve CD28,  4-1BB, or  OX40  to  sup- port  effector  cell activation,  and  also  include  CD3ζ for the exertion  of the cytotoxicity against transformed cells. The first generation  of CARs involves only an intracellu- lar signal domain  CD3ζ, while the  second  generation  of CARs includes a costimulatory molecule in addition to CD3ζ, and also the third generation  of CARs contains another   costimulatory  domain.   The  recently  advanced fourth generation of CAR-T cells could potently stimulate  the downstream transcription factor to trigger cytokine release following the detection  of the tumor- associated antigen (TAA) with CAR. Importantly, the fifth generation of CARs which has been constructed respect- ing the  second  generation utilizes gene editing  to inhibit the expression of the TCR (TRAC) gene, facilitating the ablation of TCR alpha and beta chains (Fig. 1) [26]. As de- scribed,  CRISPR system  is widely used during  the  recent years to establish novel generation of CAR-T cells. T cells are engineered to generate transgenic cytokines, such as interleukin  (IL-12) within  the  targeted  tumor  and  there- fore attract  higher  quantities  of anti-tumor immune  cells (e.g., natural  killer (NK) cells and macrophages) to provide next-generation CAR-T cells for better toxicity manage- ment  [27]. Moreover, CAR-T cells are equipped  with che- mokine  receptors   to  circumvent  their  poor  homing   to tumor  sites. These strategies like knocking in cytokines or chemokine  receptors  eventually augment  CAR-T cell cytotoxic   functions   against   tumor   cells.  As  well,  ap- proaches   like  knocking   out  immune   checkpoint   mole- cules, and also ablation  of TRAC or B2M can ameliorate CAR cell persistent in vivo and  also enables  CAR-T  cell generation form allogeneic donors  [28]. As well, knocking out   the   endogenous  TGF-β  receptor   II  (TGFBR2)  in CAR-T  cells using  CRISPR/Cas9  method  largely attenu- ates the elicited Treg conversion and thus hinders the ex- haustion  of CAR-T cells [29].


The   CAR-bearing   modified   T   cells   can   recognize


CAR-targeted  antigen  and thus  elicit T cell proliferation,cytokine manufacture, and  critical and  targeted  cytotox- icity versus tumor  cells [30]. Therefore,  CAR-T cell treatment has supported appreciated attainment to treat hematological   malignancies,   including   lymphoma, chronic  lymphocytic  leukemia  (CLL), and acute lympho- blastic  leukemia  (ALL) [31,  32].  CARs  deliver  a  wider array of functional  impacts  than  transduced TCRs; how- ever, CARs and TCRs have their advantages and disad- vantages   [33].  Although   the   flexibility  and   dynamic range  of CARs are striking,  existing  CARs are restricted to identify cell surface antigens  [33] while TCRs identify both cell surface and intracellular  proteins. Nonetheless, antigen  processing  and presentation by HLA are not  re- quired   for  CARs,  making  them   more   applicable  than TCRs to HLA-diverse patient  populations [34].


The  CAR’s engineering  into  T  cells  demands   that  T cells be cultivated to permit  for transduction and suc- ceeding  expansion.  Although   the  transduction  can  ex- ploit diverse methods,  steady gene transfer  is essential to facilitate  continued CAR expression  in clonally expand- ing and persisting  T cells.


CAR-T cells generation  from autologous and allogeneic T cells


The  genetic  alteration  of autologous   or  allogeneic  per- ipheral  blood T lymphocytes  to create  tumor-targeted T cells  has  become  an  inspiring  therapeutic option.  The great and pronounced competencies of TCR and CAR therapies are best exemplified through the stimulating clinical  results  achieved  with  NY-ESO-1  TCR  [35] and CD19 CAR-T cells [36, 37]. CAR-T cell construction processes   combine   T  cell  activation   and  transduction stages for providing genetically targeted T cell products. Indeed,  engineered   T  cells  to  express  particular   CARs can  be  generated   from  Ficoll-purified  PBMCs  followed by their activation with anti-CD3 monoclonal antibody (mAb)  in  the  existence  of  irradiated   allogeneic  feeder cells, and  finally efficient transduction with a vector  en- coding the CAR [38]. The encouraging  clinical outcomes of CAR-T cell therapy may be more enlarged by estab- lishing  the  potent  and  histocompatible T  cells. Autolo- gous  methods  have a confirmed  track  record,  but personalized  products can be challenging  in some cases, for instance  in patients  with chemotherapy or HIV- mediated  immune   deficiency  [39]. Accordingly,  though T cells can be simply achieved from donors, their appli- cation is potently hindered  by the high alloreactive cap- ability.  Indeed,  TCRs  have  the  natural   competence to respond toward non-autologous tissues, identifying both allogeneic   HLA  molecules   and   other   minor   antigens [40]. This tendency  inspires  the incidence  of graft rejec- tion in transplant recipients  and also the occurrence of GVHD in recipients  of donor-isolated T cells [41]. Given these problems,  inhibition  of the alloreactive potential  of allogeneic T cells to obtain  an acceptable  risk-benefit  ra- tio is of paramount importance. To  date, two main  tac- tics  have  been   designed   to  defeat   the   risk  of  graft- versus-host reaction  (GVHR) concerning the selection  of virus-specific  TCRs  devoid  of GVHR or  the  ablation  of TCR expression  [39]. As described,  three  main technolo- gies, containing  ZFNs, TALEN, and CRISPR/Cas9, facili- tate  gene disruption in the  human  cell. Remarkably, the ablation  of endogenous TCR expression  largely obtained through utilizing  genome-editing technologies   abrogate the  continuous  districts   of  TRAC  genes,  and  thereby offer  the   chance   for  manufacturing  universal   CAR-T cells [7, 42].


To CAR-T cells hold potential as a safe and rapidly evolving therapeutic strategy for treating human  malig- nancies,  the development of methods  to pharmacologic- ally control  them  in vivo is required.  Owing to this fact, some strategies, in particular,  suicide mechanisms are developing [43, 44]. For example, Amatya and her col- leagues  designed  a  construction  including  CD28- containing  anti-signaling  lymphocytic  activation  mol- ecule F7 (SLAMF7) CAR and a suicide gene [45]. SLAM F7 is a capable  target  for CAR-T  cell treatment of mul- tiple myeloma  (MM)  because  of their  robust  expression on the surface of MM but not normal  nonhematopoietic cells. The suicide gene encoded  a dimerization domain bonded  to a caspase-9  domain  [45]. They showed that  T cells expressing  the SLAMF7-specific CAR accompanied with suicide-gene construct specifically identified and eradicated   SLAMF7-positive   cells  in  vitro  and   tumor cell-bearing mice. Interestingly, engineered  T cells were eradicated  on demand  through injection  of the  dimeriz- ing  agent  AP1903  [45].  However,  as  suicide  strategies mainly result  in the complete  elimination  of the CAT-T cells, they will possibly lead to the premature end of the intervention. Consequently, carrying out non-lethal con- trol of CAR-T cells is required  to expand  the CAR-T cell both   efficacy  and   safety   [46].  In   this   regard,   small molecule-based plans as described  by Lim et al. can offer a possibility to turn the CAR-T cells “on” or “off” [47]. Further, synthetic splitting receptor  [46], combinatorial target-antigen  recognition  [48],  synthetic  Notch  recep- tors [49], and bispecific T cell engager [50] along with inhibitory   chimeric   antigen   receptor   (iCAR)  [51]  are other suggested strategies for improving the safety of engineered  T cell.


CAR-T cell in clinical trails


Valuing the hopeful results achieved from a myriad of preclinical studies, numerous clinical trials have been conducted or are ongoing  to address  the  safety, feasibil- ity,  and  efficacy  of  CAR-T  cells  in  patients   suffering from hematological  malignancies  or solid tumors  (Fig. 2) (Table 1).


Hematological malignancies


Anti-CD19 CAR-T cell therapy has presented notable activity in patients with refractory or relapsed acute lymphocytic  leukemia  (ALL). Several anti-CD19  CAR-T cell constructs have been investigated  and responses  dif- fer extensively among  various  studies  [52]. In 2017, the Food and Drug Administration (FDA) granted regular approval to axicabtagene ciloleucel or Yescarta as a therapeutic option  for large B cell lymphoma  (BCL). Yescarta is a CD19-specific  CAR-T cell mainly exploited for the treatment of adult patients  with relapsed or re- fractory  large  BCL following  two  or  more  lines  of sys- temic  treatment. However,  a  trial  in  101  patients  with BCL who received a single injection  of axicabtagene  cilo- leucel followed by lymphodepleting chemotherapy using cyclophosphamide and fludarabine indicated that inter- vention  led to severe unwanted  events in 52% of partici- pants.   Also,  recurrence  of  the   CRS  and   neurologic toxicities in 94% and 87% of participants, respectively, signified   the   importance  of  the   operation  of  a  risk

assessment  and mitigation  strategy [53]. Nonetheless,  in- fusion  of the  axicabtagene  ciloleucel  to 111 participants with diffuse large B cell lymphoma  (DLBCL) at the dos-age of 2 × 106   CD19-CAR-T  cells/kg  displayed  signifi-cant efficacy. While the complete  response  rate was 54%, a  significant   number   of  patients   experienced   neutro- penia,  anemia  accompanied by thrombocytopenia. Also,13% and  28% of  the  patients   experienced   robust   CRS and neurological  effects, respectively [54]. Furthermore, brexucabtagene autoleucel  (KTE-X19), another  CD3ζ/ CD28-based  CD19-specific CAR-T cell, is specified for mantle  cell lymphoma  (MCL) therapy. A phase 2 trial in 74 participants with relapsed or refractory  MCL revealed that brexucabtagene autoleucel could elicit durable re- missions  in a majority  of patients  who received 2 × 106 CD19-CAR-T cells/kg. However, similar to the previous reports,   the  intervention  exerted  severe  and  life- threatening toxic  influences  [55]. As well, KTE-C19  as an  autologous  CD3ζ/CD28-based CD19-specific  CAR-T cell product at a target  dose of 2 × 106  CAR-T  cells/kg showed an acceptable  safety profile along with an overall response  rate  of  about  71%, and  a  complete   response rate   of  about   57%  in   a   participant  with   refractory DLBCL [56]. On  the  other  hand,  anti-B  cell maturation antigen  (BCMA) CAR-T  cell therapy  has  been  revealed to have desired activities in patients with relapsed or re- fractory multiple myeloma (MM) [57]. As well, a small subgroup   of  MM   cells  typically  express   CD19,   and thereby  CD19-CAR-T  cell therapy  has displayed  a posi- tive anti-tumor effect in some of these patients  [57]. Evaluation  of the  safety and  efficacy of combined  treat- ment with anti-CD19 and anti-BCMA CAR-T cells in participants with  relapsed  or  refractory  MM  have  indi- cated  that  administration  of  humanized  CD19-CAR-T cells accompanied by murine  BCMA CAR-T cells at the similar dosage of 1 × 106 cells/kg following lymphocyte depletion   may  result  in  significant  preliminary  activity. But, the intervention led to the higher  unwanted events, containing   neutropenia, anemia,  and  thrombocytopenia in 86%, 62%, and  62% of enrolled  participants, respect- ively, concomitant with one intervention-related death possibly due to the thrombocytopenia [57]. Besides, tisa- genlecleucel, an autologous  T cell with a lentiviral vector encoding a CD19-specific CAR, presented a significant efficacy along with a manageable  safety profile in a sub- group  of Japanese  patients  with relapsed/refractory (r/r) B-ALL [58] and DLBCL [59], making them  a rational treatment strategy in patients  with B-ALL and DLBCL.


In addition  to the  cited  trails, a myriad  of trials based on the  targeting  BCMA in MM  ([60–65], CD19 in ALL [32,  66–74]  and  non-Hodgkin’s lymphoma   (NHL)  [69,75–79], CD20 in BCL [70, 80–82], and CD22 in ALL [83–86] have shown the significant efficacy in the clinic.


Solid tumors


CAR-T  cell therapy  is more  restricted   in  solid  tumors than in hematological  malignancies as CAR-T cells are circulated  to the bloodstream and lymphatic  system, and thereby have more interaction with blood tumor  cells. Nevertheless,  in  solid  tumors,  these  redirected   effector cells may  not  be able  to  penetrate tumor  tissue  by the vascular endothelium [87]. Overall, studies have recog- nized various roadblocks for administered CAR-T cells, comprising  a restricted   spectrum of targetable  antigens and heterogeneous antigen expression, restricted T cell survival before  reaching  tumor  region,  incapability  of T cells  to  proficiently  recruit  to  tumor   region  and  pene- trate  physical barriers,  and finally an immunosuppressive TME  [88]. 


Nonetheless,   various  tumor-associated anti- gens (TAA) have been targeted  by redirected  effector immune   cells  to  elicit  an  anti-tumor response  in  vitro and  in  vivo.  For  instance,   anti-prostate-specific mem- brane  antigen  (PSMA) CAR-T cells could selectively tar- get  PSMA-positive   cells  in  vitro  and  eradicate   tumor cells in vivo [89]. A trial in 6 patients  with prostate  can- cer  revealed  that  infusion  of the  PSMA-specific  autolo- gous CAR-T cell led to no anti-PSMA toxicities and reactivities.  Moreover,  the  use of PSMA-specific  CAR-T cell plus IL-2 resulted  in more  prominent anti-tumor re- sponses than monotherapy and thereby suggested that pharmacodynamics of “drug-drug” interactions could improve the efficacy of their co-application [90]. Further, it has been  found  that  the  potent  activity of anti-PSMA CAR-T  cells  could  be  improved  through the  co- expression of a dominant-negative TGF-βRII (dnTGF- βRII). Meanwhile,  expression  of the dominant-negative TGF-βRII in CAR-T cells could support  improved lymphocyte proliferation,  augmented cytokine secretion, resistance  to  exhaustion,  prolonged  in  vivo persistence, and also the stimulation of tumor  elimination  in vivo. As well, this strategy  could be effective for the treatment of patients   suffering  from  relapsed   and  refractory   meta- static  prostate  cancer  [91]. Interestingly,  combine  treat- ment  with  GD2  specific CAR-T  cell with  CD3ζ, CD28, and  OX40 signaling  domains  and  pembrolizumab (anti- PD-1 mAb)  may augment  the  anti-tumor activity of the effector T cells by improving their persistence  and ex- pansion in patients with GD2-positive tumors, such as melanoma  [92].


On the other  hand, constructing and injecting  anti-EGFRvIII CAR-T cells is feasible and  safe, without  indication  of off-tumor  toxicity or CRS [93, 94]. However, systemic injection of a single dose of EGFRvIII-specific CAR-T cells into 10 patients with glioblastoma  mediated  antigen  loss and stimulated  adap- tive  resistance   in  patients   with  recurrent  glioblastoma [93]. These findings have shown that while systemic in- fusion could support  on-target effect in the brain, defeat- ing    the    adaptive     variations     in    the    local    TME concurrently addressing  the antigen  heterogeneity are required to improve EGFRvIII-directed approaches in glioblastoma  [93].


Moreover,  a phase  I/II  clinical  study in  19  patients  with  recurrent/refractory  human   epider- mal growth factor receptor  2 (HER2)-positive sarcoma showed  that  injections  were well tolerated  in the lack of no  dose-limiting   toxicity  [95]. This  study  was  the  first trial  of the  safety and  efficacy of HER2-CAR-T  cells in patients   with  tumors   showing   that   the   administrated cells  persisted   for  6  weeks  without   obvious  toxicities [95]. Similarly, the safety and feasibility of HER2-CAR-T cell therapy  were  shown  in  patients  with  advanced  bil- iary tract cancers (BTCs) and pancreatic  cancers [96]. Besides, transplantation of the carboxy-anhydrase-IX (CAIX)-specific CAR-T cell into 12 patients  with CAIX- expressing  metastatic  renal  cell carcinoma  (RCC) deliv- ered  in-patient  proof   that   intervention  could   lead  to positive anti-tumor responses  [97].


In  addition  to  the  listed  reports,  CAR-T  cell therapy based on the targeting tumor-associated glycoprotein (TAG)-72 in colorectal cancer [98], carcinoembryonic antigen  (CEA) in lung cancer [99] and liver cancer  [100], mesothelin [101], and  EGFR [102] in pancreatic  cancer, fibroblast   activation   protein    (FAP)   in   mesothelioma [103], IL13Rα2 in glioblastoma  [104], and  mucin-1 (MUC1)  in seminal  vesicle cancer  [105] have been  con- ducted  or are ongoing  to address  the  safety and  efficacy of redirected  effector T cells in patients  with tumors.


CRISPR/Cas9 potential to overcome potent challenges of CAR-T cell-based therapies


Currently,  CRISPR/Cas9-mediated genome  editing offers the potential of more effective immunotherapy, by manufacturing  a  universal  “off-the-shelf”  cellular  prod- uct or modifying immune  cells to defeat resistance in hematological  or solid tumors  (Table 2). Despite  the ex- istence of several challenges concerning the safety, effi- ciency, and scalability of this strategy, the CRISPR/Cas9 approach  will undeniably  reign in the context  of CAR-T cell-based therapies  for tumors  [119].


Disruption of inhibitory  molecules and signaling axis


It has been  suggested  that  merging  lentiviral  delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 B2M, and PD-1 simul- taneously   cause  preparing   the  universal  “off-the-shelf” CAR-T  cells. Meanwhile,  TCR and  HLA class I double- deficient  T cells     potentially show diminished alloreactivity and commonly  cause no GVHD [109, 120]. Moreover,  concurrent triple  genome  editing  could  sup- port   ameliorated  in  vivo  anticancer  functions   of  the gene-disrupted redirected  effector T cells [109, 120]. Similarly, triple gene-disrupted CAR-T cells displayed raised activity in glioma mice models leading to the ex- tended overall survival rate in mice bearing intracranial tumors  following intracerebral, but  not  systemic admin- istration  [24]. Moreover, marked PD-1 gene disruption lonely can  be an  attractive  plan  to  enhance  the  efficacy of CAR-T cell therapy in an immunosuppressive TME [110]. Hu et al. found that PD-1 gene disruption by CRISPR/Cas9 and using piggyBac transposon system for expressing  CD133-specific  CAR in one reaction  resulted in the comparable  rates of cytokine releases, while led to the promoted growth and cytotoxicity in vitro. Also, engineered  CAR-T cells displayed robust resistance to inhibitory  molecules  in the  glioma  murine  model  com- pared  to  conventional CD133-CAR-T  cells [110]. Like- wise, PD-1-disrupted EGFRvIII-specific CAR-T cells exerted  evident suppressive  impacts  in vitro on EGFRvIII positive glioblastoma cells (U-251MG and EGFRvIII- expressing  DKMG)  without  any significant  influence  on the T cell phenotype  and the expression  of other  check- point   receptors  [111].  Thereby,   Nakazawa  et  al.  sug- gested   that   the   sgRNA/Cas9-mediated anti-tumor activities of EGFRvIII-specific CAR-T cells are intensely dependent on PD-1 disruption [111]. Besides, PD-1- deficient   CD19-specific   CAR-T   cells  showed   elevated anti-tumor activity against and improved clearance of CD19+  PD-L1+  K562  myelogenous   leukemia   cells  in NOD-SCID-IL-2Rγ−/− (NSG) mice compared to the conventional CD19-specific  CAR-T  cell [121]. Albeit, it was found that ectopic PD-L1 expression could not sig- nificantly  modify  intrinsic  tumor   proliferation in  K562 cell-bearing  mice since there  was no alteration  in growth kinetics of CD19+ and CD19+ PD-L1+ cells in the ex- perimental  model   [121].  Too,   PD-1   deficient mesothelin-specific CAR-T cell diminished  PD-1+ population  in   triple-negative  breast   cancer   (TNBC) [122].  Although   observed   ttenuation  had  no  signifi- cant  impact  on  CAR-T  cell proliferation,  it stimulated CAR-T    cell   cytokine    generation   and    cytotoxicity against PD-L1-expressing TNBC cells in vitro. More efficiently,  PD-1  deficient   mesothelin-specific  CAR-T cells demonstrated a more prominent effect on tumor control    and    relapse    prevention   in   the    preclinical model than conventional CAR-T cells [122]. Besides, lymphocyte  activation  gene-3     (LAG-3)  knockout CD19-specific   CAR-T   cells  by  CRISPR-Cas9  elicited strong antigen-specific  anti-tumor effects in vitro and lymphoma  Raji cell-bearing  NOD-Prkdcscid Il2rgnull (NPG)   mice.   Nonetheless,  LAG-3   knockout  CAR-T cells showed  no  superiority  in terms of the anti-tumor  response  and the reduction in tumor  burden compared  to  the  conventional  CAR-T  cells [112].


Reducing CRS and GVHD occurrence


As  described,   TCR  and  HLA  class  I  double-deficient CAR-T   cells  robustly   display  attenuated  alloreactivity and universally result in no GVHD occurrence. As well, these cells’ anti-tumor activity can be potently  intensified by simultaneous ablation  of PD-1  and  CTLA-4  [123]. It has been documented that fratricide-resistant “off-the- shelf” CAR-T, known  as UCART7, as a novel anti-CD7


CAR-T cell with a deficiency in TCR could exert  robust cytotoxicity   against   CD7   expressing   malignant    cells in vitro and in vivo without  GVHD development.  Both UCART7 and anti-CD7 CAR-T cells could detect and eliminate   CD7+  leukemic   cell  lines,  MOLT3,   CCRF-CEM, and HSB-2 in vitro with similar efficiencies, repre- sentative  of no impairment in activity upon  double  dele- tion  of CD7  and  TCR  [114].  Thereby,  UCART7  as  an allo-tolerant “off-the-shelf”  CAR-T  cell product signifies an  efficient  and  applicable  option   for  treating   the  re- lapsed and refractory T-ALL and non-Hodgkin’s T cell lymphoma  [114].


Given the importance of the granulocyte-macrophage colony-stimulating factor (GM-CSF) in the simulation  of CRS, some studies have focused on the attenuation of its effect on  the  CRS induction upon  CAR-T  cell therapy. GM-CSF is a colony-stimulating factor that adjusts the proliferation  and  differentiation  of  hematopoietic  cells. This cytokine is abundantly  generated  by CAR-T cells following activation and exists in the TME at high levels [124]. In 2019, Sterner  et al. investigated  the use of CRIS


PR/Cas9 gene editing in CD19-specific CAR-T cells by transduction with a lentiviral construct including  a guide RNA to GM-CSF and Cas9 [115]. They found  that  GM- CSF deficient anti-CD19  CAR-T cells efficiently released less  GM-CSF,  whereas  maintained pivotal  T  cell func- tion. Importantly, these redirected  effector T cells exhib- ited a more prominent anti-tumor effect than wild-type CAR-T cells in vivo [115]. In another  study, they found that GM-CSF neutralization with lenzilumab did not elicit any negative  effect on  anti-CD19  CAR-T  cell activity in vitro and in vivo. Furthermore, anti-CD19  CAR-T cell prolifera- tion was improved  and durable control  of ALL was amelio- rated in patient-derived xenografts following GM-CSF neutralization with  lenzilumab  [116].  Finally, they  found that GM-CSF deficient CAR-T cells upheld  normal  activity and  had a superior  anti-tumor function  in vivo leading to an improved  overall survival rate in comparison to the con- ventional anti-CD19  CAR-T cell [116].


Manufacturing allogeneic universal CAR-T cells


It  is mainly  difficult  in  newborn   and  elder  patients   to achieve sufficient  and  good quality T cells for manufac- turing   the  patient-specific  CAR-T  cells.  For  providing more  accessible CAR-T cells, it is greatly wanted  to pro- gress an allogeneic adoptive  transfer  plan, in which uni- versal CAR-T cells are produced from healthy donor’s T cells to treat  numerous patients  [123, 125].


As cited, allogeneic universal CAR-T cells can potently be established  by impairing  TCR and  B2M gene expres- sion in CAR-T cells by genome  editing strategies. Corres-pondingly,   CAR+TCR_T   cells  seem   to   be   a  rational


approach  to introduce as the new generation CAR-T cell, providing an “off-the-shelf”  therapy for the tentative  treat- ment  of B-lineage malignancies  [114]. Genetically edition of anti-CD19  CAR-T cells to disrupt  expression  of the en- dogenous   TCR  for  inhibition   of  GVHD  progress  could display the anticipated  property  of conventional CD19- specific CAR-T cells without  responding to TCR stimula- tion [126]. Likewise, another  report  has implied that directing  CD19-specific  CAR to the  TCR locus may sus- tain the uniform  CAR expression  in T cells and simultan- eously improve T cell potency [117]. Remarkably, Eyquem et al. found that TCR-deficient  CD19-specific CAR-T cells could  trigger  better  anti-tumor response  compared to conventional CAR-T cells in a mice model  of ALL [117]. In addition,  directing  the CAR to the TCR locus prevents tonic  CAR signaling  and  enables  effective internalization and re-expression of the CAR upon  the single or repeated exposure  to antigen, which in turn leads to the delayed ef- fector T cell differentiation and exhaustion.  Indeed, target- ing CARs to a TCR locus offers a safer therapeutic T cell by reducing  the risk of insertional  oncogenesis  and TCR- stimulated  autoimmunity and alloreactivity in addition  to providing   a  more   potent   T   cell,  as  documented  by minimizing  the constitutive  signaling and abrogation  of T cell depletion  [117].


Resistance to the suppressive effects of TGF-β


Despite  CAR-T  cells’ remarkable  activity against  cancer, this therapeutic option still faces various challenges, in particular,  immunosuppressive tumor  microenvironment (TME) for eradicating  solid tumors  [29]. Although  TGF- β exerts tumor-suppressive influences  through inhibiting cell  cycle  development  and  inducing   apoptosis   in  the early stages of tumors, TGF-β elicits tumor-promoting influences  leading  to  the  boosted  tumor  invasiveness  as well as metastasis  in late stages [127]. Besides, the TGF- β signaling axis creates  interactions with other  signaling axes in a synergistic or antagonistic  mode and controls biological  procedures. Taken  together,  given the  critical role  of TGF-β in tumor  progress,  this  pathway  is a ra- tional target for tumor therapy. Various therapeutic strategies, comprising TGF-β antibodies, antisense oligo- nucleotides,  and small molecules inhibitors  of TGF-β receptor-1 (TGF-βR1),  have  exposed  huge  competence to negatively regulate TGF-β signaling [127].


It  has  been  robustly   evidenced   that   suppression  of TGF-βR signaling improves the anti-tumor activities of receptor  tyrosine kinase-like orphan  receptor  1 (ROR1)- specific  CAR-T  cells toward  TNBC.  Meanwhile,  block- ade  of  the  TGF-βR  axis  using  the  specific  inhibitors could  largely  protect   CD8+  and  CD4+  ROR1-CAR-T cells from  the suppressive  impacts  of TGF-β, facilitating their  tumor-suppressive activity in the  3D tumor  model [29]. Similarly, dominant-negative TGF-βR promotes PSMA-specific CAR-T cell proliferation  and strongly in- creases prostate cancer elimination. These CAR-T cells demonstrate improved  cytokine generation,  resistance  to exhaustion,  and also prolonged  persistence  in vivo [91]. Moreover, the knocking out of the endogenous TGF-β receptor   II  (TGFBR2)  in  anti-mesothelin  CAR-T  cells using the CRISPR/Cas9 technique may decrease the acti- vated  Treg  conversion  and  avoid CAR-T  cells depletion [29]. Importantly, TGFBR2-edited  CAR-T cells exhibited a more obvious capability to eliminate mesothelin- expressing CRL5826 and OVCAR-3 cells in tumor cell- bearing  mice  when  injected  locally or  systemically [29]. As well, TGF-βRII-edited  CAR-T cells are mainly resist- ant  to  TGF-β inhibition,  and  also elicit augmented  cell killing compared to the conventional CAR-T cells in the existence of TGF-β against B cell maturation antigen (BCMA)-positive tumor  cells [128]. Furthermore, CRIS PR/Cas9-mediated knockout  of the DGK, as a possible regulator  of TGF-β, boosts  the anti-tumor activity of the CAR-T versus U87MGvIII glioblastoma  cell in vitro and murine   models  mainly  by  the  triggering   resistance   to TGF-β and also PGE2 [9].


In  addition   to   the   CRISPR-Cas9  technology,   other well-known genome-editing techniques have shown the pronounced capability  to  support   the  broader   applica- tion of CAR-T cells (Table 3).


The off-target effects of CRISPR-Cas9 technology Several classes of CRISPR-Cas systems have yet been ad- vanced,  while their  comprehensive use  can  be hindered via off-target effects. Efforts are being accomplished  to at- tenuate  the off-target  effects of CRISPR-Cas9 through  es- tablishing   the  multiple   CRISPR/Cas  systems  with  high fidelity  and  accuracy  [137].  Thereby,  a  myriad  of  tech- niques  have been utilized to identify off-target  mutations, and  restore  the  on-target effects  and  conversely  reduce


potent  off-target  effects. As the  genomic  frameworks  of the targeted  DNA concurrently the secondary  structure of sgRNAs and  their  GC  content are  mainly  contribute to determining cleavage efficiency, designing of the appropri- ate  sgRNAs  with  high  on-target activities  using  specific tools  is severally suggested  [137]. Recently, the  amelior- ation  of the specificity [138] of genome  editing  tools and the identification  [139] of off-target  effects are swiftly de- veloping  research  areas.  Such  research  incorporates de- signer  nuclease  development  [140],  discovery computational  prediction  programs   and  also  databases [141] and  also finding  high-throughput sequencing  [139] to diminish mutational occurrence. Overall, the amelior- ation   of  the  off-target   specificity  in  the  CRISPR-Cas9


system undoubtedly will deliver solid genotype-phenotype associations, and therefore  empower faithful interpretation of gene-editing  statistics,  facilitating the basic and clinical utility of this CRISPR-Cas9 technology [142].


Conclusion and prospect


The progress  of genomic  editing techniques enlarges  the landscape   of  CAR-T  cell-based  therapies   for  adoptive cell therapy.  Among the several technologies  that  can be exploited,   CRISPR/Cas9  is  comparatively   easy  to  use, simple to design, and cost-effective concurrently remark- able multiplex genome engineering competencies [143]. Now, CRISPR/Cas9-based genome editing provides the capability of further streamlining immune  cell-based therapies,  more  prominently, through the  generation  of a universal “off-the-shelf”  cellular product or engineering these redirected  effector cells to overcome resistance in human malignancies, ranging from hematological malig- nancies  to  solid  tumors   [144]. These  findings  have  re- sulted   in   the   execution   of  several   clinical   trials   to evaluate  the  therapeutic safety and  efficacy of CRISPR/ Cas9-mediated genome editing in CAR-T cell therapy (Table  4). However,  for further  human  trials,  designing and expanding  large-scale  approaches for CRISPR/Cas9- mediated  target  ablation  in mature  T cells is of principal significance. These  protocols  must  simplify the  transfer- ence of sgRNA, and Cas9 concomitant with a gene en- coding   the   CAR,  maintain   cell  survival  and   support strong  in vitro cultivation  of modified  T cells upon  gen- etic manipulation [119]. These means may comprise transduction of CRISPR/Cas9 machinery and CAR transgenes  employing   the   retroviruses   or   lentiviruses [145, 146] or using non-integrating viruses, including  ad- enoviruses    and   adenovirus-associated   viruses   (AAV) [147, 148]. Further,  the development of innovative  strat- egies to  attenuate off-target  CRISPR/Cas9  editing,  such as  varying  the   Cas9  endonuclease  using   novel  PAM ants,  and  also exploiting  truncated sgRNAs can support more  prominent consequences in vivo [119]. In sum, we guess that  conduction of the  more  comprehensive stud- ies  based  on  the  CRISPR-Cas9  application   to  improve CAR-T  cell safety, efficacy, and  accessibility  could  lead to the desired therapeutic outcomes  in the clinic.