# 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
This led to significantly reduced viral titers in the trigeminal ganglia, eye, and brain samples as well as a complete blockage of HSV-1-induced upregulation of different inflammatory molecules [59]. A third approach involved the development of HSV-1-erasing lentiviral particles (HELP) carrying Cas9 mRNA for transient Cas9 expression and two gRNAs simultaneously targeting the HSV-1 genes _UL8_ and _UL29_[47; 49]. In vitro, HELP transduction of HEK293T cells, spontaneously immortalized human keratinocyte (HaCaT) cells, and murine primary corneal stromal cells, as well as in tissue cultures of human corneas, followed by HSV-1 infection, partially inhibited HSV-1 production. Next, in vivo experiments were conducted on murine HSK models via intrastrand injection of HELP into the corneas. Interestingly, HELP was detected in trigeminal ganglia, indicating that these vectors can be retrogradely transported along axons from neurons innervating the cornea. A treatment prevention model showed that HELP injection prior to HSV-1 infection was able to reduce viral loads in eye, trigeminal ganglion, and brain samples and thus, inhibit viral transmission. Consequently, HELP treatment inhibited disease progression, corneal expression of inflammatory molecules, and infiltration of inflammatory cells. Also, in a therapeutic murine model, in which mice received HELP treatment after HSV-1 infection, HSV-1 replication in the corneas and trigeminal ganglia, as well as lesion formation in the eyelids, and disease progression were diminished. Additionally, HELP treatment decreased HSV-1 loads in both the eyes and the trigeminal ganglia of HSV-1 latently infected mice, which received a reactivation stimulus prior to HELP administration [49]. Finally, HELP treatment was tested in a clinical trial in which three patients suffering from severe refractory HSK and acute corneal perforation were intrastromally injected with HELP during penetrating keratoplasty (NCT04560790) [47]. All examined tear swabs and corneal button samplesremained negative for HSV-1 in the average 18-month follow-up, and no patient showed HSK recurrence. Adverse effects were primarily associated with the elevated risk of penetrating keratoplasty or the use of glucocorticoids post-surgery. Additionally, the HELP treatment seemed to be tolerated by the host immune system, as no vector-specific IgG antibodies were detected [47]. A fourth approach for potential HSK treatment implies targeting host gene expression instead of viral gene expression. Specifically, the _nectin cell adhesion molecule 1_ (_NECTIN-1_) gene is an interesting target as it encodes a gD receptor on the cornea important for the invasion of the virus by interaction with its glycoprotein D [62]. Lentiviral transduction of human corneal epithelial cells with a CRISPR-Cas9 system targeting exon two of _NECTIN-1_, followed by HSV-1 infection, resulted in strongly decreased HSV-1 infection and HSV-1 DNA load [62]. 2.2 CRISPR Systems Targeting the Lytic Reproduction Cycle and Reactivation of Varicella-Zoster Virus  
One study [48] observed partial inhibition of VZV lytic replication in adult retinal pigment epithelial clone 19 (ARPE-19) cells and human embryonic stem cell (hESC)-derived neurons following AAV2-mediated transduction of the CRISPR-Cas9 system targeting the duplicated IE _open reading frame (ORF)62/71_ gene encoding the major viral transcription factor ICP4 homolog protein. Applying the same CRISPR strategy on latently infected hESC-derived neuron cultures similarly resulted in reduced VZV replication upon virus reactivation [48].