*3.1. Letermovir*

Letermovir is a novel antiviral drug which has been approved by the USA Food and Drug Administration (FDA) through a fast track procedure and granted as an orphan drug by the European Medicines Agency (EMA). The drug was tested in CMV infected patients and likely be useful for other patients who had organ transplantation or human immunodeficient virus (HIV) infections [39]. It has been clinically applied for CMV prophylaxis or treatment in hematopoietic stem cell recipients, thoracic organ recipients and lung transplantation recipients [40–42]. Letermovir has several advantages over conventional CMV antiviral agents as follows. Firstly, it can be given orally, so hospitalization and intravenous injection are not needed. Secondly, it is mild in toxicity, not related to myelotoxicity and nephrotoxicity [43,44]. Thirdly, it targets the CMV terminase complex instead of CMV DNA polymerase, so there is no risk to induce cross-resistance with existing anti-CMV drugs [44]. However, the CMV antiviral therapy will finally fail and acquired antiviral drug resistance is not avoidable if there is no immune control [45]. It should be noted that more data are required to provide insights of the mutations detected in vivo, interpretation of genotyping results, and outcomes of the clinical correlation. To provide useful information, it would be recommended to establish databases for the surveillance and interpretation of resistance for CMV [36].

#### *3.2. Maribavir*

Maribavir is a promising anti-HCMV compound which is administered orally; however, it is still under advanced clinical trials. The drug targets the viral kinase UL97 which is crucial for the formation of viral teguments and assembly complexes for virion releasing [36]. However, it is not recommended to co-administer maribavir and ganciclovir both in that maribavir is an inhibitor of the UL97 enzyme which is required for the assimilation of ganciclovir. Maribavir potentially substitutes for other traditional anti-HCMV drugs because of its reduced haematotoxicity and nephrotoxicity compared with ganciclovir and valganciclovir [36].

Maertens et al. used maribavir (dose-blinded) to treat cytomegalovirus reactivation preemptively for recipients of hematopoietic cell or solid organ transplants (SOT) (≥18 years old) with CMV reactivation in a phase II and open-label clinical trial [46]. The results showed that maribavir at a dose of at least 400 mg twice daily had efficacy like that of valganciclovir for removing CMV viremia. Though a higher incidence of gastrointestinal adverse events were found in the maribavir -treated group, the neutropenia incidence was lower [46].

Papanicolaou et al. used dose-blinded maribavir 400, 800, or 1200 mg twice-daily for up to 24 weeks to treat hematopoietic-cell or SOT recipients (≥12 years old) with refractory or resistant CMV infections in a phase II and double-blind clinical trial [47]. The result revealed that it was active to against refractory or resistant CMV infections using maribavir more than 400 mg twice daily in transplant recipients and no new safety signals were identified in this trial [47].

#### **4. CMV Inhibition by Nucleic Acid-Based Therapeutic Approaches**

The treatment of diseases caused by CMV is quite challenging because of high mutation rates and latency. Thus, infection is still a serious threat to humans. Fortunately, external guide sequences (EGSs), transcription activator-like effectors nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated 9 (Cas9) nuclease system might provide effective therapeutic strategies to treat diseases caused by CMV through designing a specific DNA or RNA sequence that target essential genes for viral growth. However, the effective modification of the viral genome avoiding off-target effects and the option of escape variants ignoring the editing of these approaches are required for successful clinical application.

#### *4.1. EGS-RNase*

Ribonuclease P (RNase P) is a unique RNases in that it is a ribozyme – an RNA that acts as a catalyst which is somewhat like a protein enzyme. Its function is to cut an extra or precursor sequence of RNA on transfer RNA (tRNA) molecules; that is, to catalyze the cleavage of precursor tRNA into active tRNA without any protein component. RNase P has the activity in cleaving the 5' leader sequence of precursor tRNA. EGSs signify the short RNAs that induce RNase P to specifically cleave a target mRNA by forming a precursor tRNA-like complex. Therefore, EGS technology probably acts as an effective strategy for gene-targeting therapy.

Deng et al. reported that engineered EGS variants induced RNase P to efficiently hydrolyze target mRNAs which code for HCMV major capsid protein [48]. In vitro, the engineered EGS variant was more efficient in inducing human RNase P-mediated cleavage of the target mRNA than a natural tRNA-derived EGS by about 80-fold. In cells infected with HCMV, the EGS variant and natural EGSs resulted in HCMV gene expression reduction rate by about 98% and 73%, and the viral growth was inhibited by about 10,000 and 200-fold, respectively. The results showed that the EGS variant has higher efficiency in blocking the expression of HCMV genes and viral growth, compared with the natural EGS [48].

Li et al. explored the antiviral effects of an engineered EGS variant in targeting the shared mRNA sequence which codes for capsid scaffolding proteins (mCSP) and assemblins of murine CMV (MCMV) in the animals [49]. In vitro, the EGS variant was more active in directing RNase P cleavage of the target mRNA than a natural tRNA by 60-fold. In MCMV-infected cells, the EGS variant decreased mCSP expression by about 92% and inhibited viral growth by about 8000-fold. In MCMV-infected mice, the EGS variants were more effective in reducing mCSP expression, decreasing viral production, and increasing animal survival, compared with the natural EGS. The results demonstrated that the EGS variant with higher targeting activity in vitro are also more effective in inhibiting MCMV gene expression in mice [49].

#### *4.2. CRISPR*/*Cas9*

In CRISPR/Cas9 system, CRISPR is used to build RNA-guided genes drives to target a specific DNA sequence. By the Cas proteins and a specifically designed single-guiding RNA (sgRNA), the genome can be cut at most locations with only the limitation of a protospacer adjacent motif (PAM) sequence (NGG) existing in the target site [12]. CRISPR/Cas9 has been extensively used as an effective technique of gene editing for engineering or modifying specific genes. It was shown to successfully work as an efficient genome editing tool in a wide range of organisms including HCMV [50]. Consequently, it hints that the CRISPR/Cas9 can be a potential antiviral agent for the treatment of CMV infections.

Gergen et al. designed two CRISPR/Cas9 systems which contain three sgRNAs to target the HCMV UL122/123 gene crucial for the regulation of lytic replication and reactivation from latency [51]. Both systems caused mutations in the target gene and an accompanying reduction of immediate

early gene expression in primary fibroblasts. The singleplex strategy caused 50% of insertions and/or deletions (indels) in the viral genome to appear in further detailed analyses in U-251 MG cells, resulting in a reduction in immediately early protein production. The multiplex strategy cleaved the immediate early gene in 90% of viral genomes and thereby inhibited immediate early gene expression. Therefore, viral genome replication and late protein expression were reduced by 90%. The multiplex CRISPR/Cas9 system can target the HCMV UL122/123 gene efficiently and prevent viral replication significantly [51].

van Diemen et al. observed that the clear depletion emerged in the anti-HCMV sgRNA expressing cells targeting essential genes UL57 and UL70 (1.3% and 4.6% mutants with frameshifts, respectively), compared with the sgRNAs targeting the nonessential genes US7 and US11 (83.5% and 85.8% mutants with frameshifts, respectively) [52]. The sequence complexity of the mutants selected upon UL57 and UL70 targeting was low, this suggested the selection of few suitable variants and subsequent expansion of infectious mutants need a lot of time. The results showed that the CRISPR/Cas9 is a promising strategy to restrict HCMV replication [52].
