# Applying CRISPR Technologies for the Treatment of Human Herpesavirus Infections: A Scoping Review
## 3 Results
### Overview of CRISPR Strategies Used to Target Different Human Herpesviruses
#### 3.2.7 Applying CRISPR Systems for the Enhanced Efficiency or Development of Cellular Immunotherapy  
Patients receiving an allogeneic hematopoietic stem cell transplant (HSCT) are subjected to severe glucocorticoid treatment to avoid various complications related to graft-versus-host disease [75]. However, impairment of the host's immune system by glucocorticoid treatment is frequently followed by reactivation of latent herpesvirusirus infections such as HCMV and EBV. In order to control life-threatening complications associated with virus reactivation, immunosuppressed patients can be infused with virus-specific T cells (VSTs). However, the survival of these cells is unstable as binding of the glucocorticoids to the glucocorticoid receptor (GR), encoded by _NR3C1_ (_nuclear receptor subfamily 3 group C member 1_), of the VSTs leads to apoptosis and/or inhibition of proliferation [75]. Therefore, three studies [75; 76; 77] created HCMV- and/or EBV-specific _NR3C1_-knockout VSTs that are resistant to the lymphocytotoxic effect of glucocorticoids. This was achieved upon electroporating human peripheral blood mononuclear cells (PBMCs) with CRISPR-Cas9 ribonucleoproteins targeting exon 2 of _NR3C1_. The modified VSTs showed high viability and high proliferation capacity in vitro in the presence of dexamethasone without alteration of their maturation phenotype, effector function, cytotoxic activity, or cytokine release [75; 76; 77]. In vivo experiments infusing immunodeficient mouse models with the _NR3C1_-KO VSTs and dexamethasone showed a high frequency of human T cells in the bone marrow [75]. Eventually, good manufacturing practices (GMP)-grade _NR3C1_-KO VSTs were manufactured for clinical implementation [75; 77]. Another study [43] manufactured EBV-specific cytotoxic T cells (CTLs) targeting EBV-associated gastric carcinoma cells (EBVaGC cells) by sensitization of peripheral blood lymphocytes with the EBV-LMP2A antigen. However, upregulation of programmed cell death protein 1 (PD-1) on the EBV-LMP2A-CTLs was observed along with the impairment of their cytotoxic function, leading to the hypothesis that EBVaGC cells could escape the CTL killing effect. Therefore, PD-1 was knocked out in EBV-LMP2A-CTLs using CRISPR-Cas9, leading to enhanced secretion of IFN-\(\gamma\) and in vitro cytotoxic activity against EBVAGC cells. This was confirmed in vivo, as human EBVAGC xenografted mice showed improved survival and higher cytokine release upon infusion of PD-1-disrupted LMP2A-CTLs compared to controls. Interestingly, the combination of low-dose radiotherapy and consequent immunotherapy of the modified CTLs led to significant tumor regression [43]. Additionally, one study [78] applied CRISPR-Cas9 editing in PBMCs to insert a green fluorescent protein (GFP)-Barcode transgene into EBV-specific T cells through HDR. Since the latter occurs primarily during active phases of the cell cycle, reactive T cells with enhanced EBV-specificity and cytotoxicity against EBV-lymphoblastoid cell lines in vitro can be selected [78]. [PERSON] et al. [79] applied the CRISPR-Cas9 system to create CAR-T cells against EBV-infected Burkitt lymphomas by knocking out the _TRAC_ (_T cell receptor-\(\alpha\) constant_) gene and knocking in EBV glycoprotein 350 chimeric antigen receptor (gp350 CAR) or cluster of differentiation 19 (CD19) CAR templates via homology-directed repair. The CD19\({}^{\text{Kl}}\)CAR-T cells showed strong cytotoxic effects against two human [PERSON] lymphoma cell lines. Gp350\({}^{\text{Kl}}\)CAR-T cells could efficiently kill HEK293T cells stably expressing EBV gp350, but exhibited only mild cytotoxic effects against the two human [PERSON] lymphoma cell lines. Similarly, CD19\({}^{\text{Kl}}\)CAR-T cells, but not gp350\({}^{\text{Kl}}\)CAR-T cells, could significantly reduce the EBV DNA load in the bone marrow of mice xenografted with these Burkitt lymphoma cell lines. These observations could be explained by the weaker and more variable expression of gp350 instead of CD19 [79].