# 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
The development of ALICE cells is based on generating transgenic HEK293 cells with SV40 large T antigen (HEK293T cells), stably expressing a destabilized STING (stimulator of IFN genes) protein in the cytoplasm which detects intracellular exogenous dsDNA or RNA and subsequently activates a synthetic immune signaling pathway, resulting in the expression of a given gene of interest under the control of a virus-inducible promoter \(\mathrm{P_{ALICEX}}\). Two interesting ALICE systems for treating HSV-1 infection include \(\mathrm{ALICE_{Cas9}}\) and \(\mathrm{ALICE_{Ab}}\), as they result in Cas9 and HSV-1-specific gRNA expression and the production of HSV-1 monoclonal neutralizing antibodies E317, respectively, upon detection of HSV-1 DNA. Both systems can also be combined (\(\mathrm{ALICE_{Cas9+Ab}}\)). In vitro, all three systems, but mostly the \(\mathrm{ALICE_{Cas9+Ab}}\) system, inhibited HSV-1 replication and transmission among the HEK293T cells. In vivo, prevention, but also treatment mouse models showed reduced HSV-1 mRNA expression and virus titer in different tissues following intraperitoneal implantation of hydrogels containing transgenic \(\mathrm{HEK_{ALICE-SEAP-Cas9}}\) (\(\mathrm{ALICE_{Cas9}}\) with gRNAs targeting the HSV-1 genes _US8_ [L], _UL29_ [E], or _UL52_ [E]), transgenic \(\mathrm{HEK_{ALICE-E317 Ab}}\) (\(\mathrm{ALICE_{Ab}}\)), and/or transgenic \(\mathrm{HEK_{ALICE-Cas9-E317 Ab}}\) (\(\mathrm{ALICE_{Cas9+Ab}}\)) cells [59]. The Application of CRISPR Systems to Edit Quiescent HSV-1 Genomes and Disrupt Viral Reactivation  
Transducing an HSV-1 latency model of human cerebral organoids with an AAV2 vector carrying Cas9 and two gRNAs against the IE _ICP0_ or _ICP27_ gene reduced HSV-1 reactivation rates by 2.5 to 5-fold, respectively, compared to control upon the induction of reactivation [51]. Two studies [45; 56] applied the CRISPR-Cas9 system to quiescently HSV-1-infected MRC5 human lung or foreskin fibroblasts by lentiviral transduction of Cas9 and gRNAs targeting the viral E genes _UL8_, _UL29_, _UL30_, or _UL52_. In both cell types, all single gRNA-Cas9 complexes resulted in a reduction in viral reactivation upon stimulation [45; 56]. This effect was enhanced following the simultaneous delivery of two gRNA-Cas9 complexes targeting _UL29_ and _UL30_[56]. However, in both studies, no loss of HSV-1 genomes nor the induction of large INDELs was detected, suggesting that the CRISPR-Cas9 system had low efficiency in editing quiescent HSV-1 genomes compared to lytic viral genomes [45; 56]. The Application of CRISPR Systems for the Prevention or Treatment of Herpes Simplex Keratitis  
As HSV-1 is the leading infectious cause of blindness, multiple studies [47; 49; 50; 59; 62] have explored the application of CRISPR-Cas systems for herpes simplex keratitis (HSK) therapy. A first approach implied in vivo corneal scarification in HSV-1 latently infected rabbit keratitis models to deliver CRISPR-Cas9-carrying AAVs (type 8 or 9) targeting _ICP0_ and _ICP27_, which resulted in absent viral shedding in the tear swabs of approximately 50-60% of the treated eyes upon virus reactivation [50]. Still, AAV presence in the cornea and the trigeminal ganglia was low. Higher vector concentrations could be achieved following intravenous injection of the CRISPR-Cas9 AAVs. All eyes with local AAV presence showed a complete absence of viral shedding. Additionally, a 50% reduction of HSV-1 viral load and up to 80% reduced transcription of the latently expressed _latency-associated transcript_ (_LAT_) gene were observed in the trigeminal ganglia, indicating partial HSV-1 removal from the latent reservoir [50]. A second approach explored the application of ALICE systems in an established HSK mouse model [59]. Two AAVs encoding ALICE\({}_{\text{Ca89}}\) and gRNAs targeting _ICP4_ or ALICE\({}_{\text{Ab}}\) were administered intravenously prior to HSV-1 infection.