Herpes simplex virus-1 (HSV-1) can invade the central nervous system (CNS). However, antiviral drugs used to treat HSV-1 have significant toxicity and resistance. An alternative approach involves the use of the CRISPR/Cas9 complex as a viral replication inhibitor. Editing the UL39 gene with CRISPR/Cas9 results in >95% inhibition of HSV-1 replication in vitro; however, few studies have investigated alternative therapies in in vivo models. This study aimed to investigate the efficacy of CRISPR/Cas9 targeting the UL39 region, which was administered via the ocular route, to reduce the HSV-1 viral count in the CNS of BALB/c mice. Mice were inoculated with HSV-1 and treated using CRISPR/Cas9. The kinetics of CNS infection were assessed, and the effects of CRISPR/Cas9 were compared with those of topical acyclovir treatments. The brain viral load was analyzed, and histopathology and immunofluorescence of the nervous tissue were performed. The group treated with CRISPR/Cas9 showed a reduced viral load on the seventh day post-infection, and no brain inflammation or chromatin compaction was observed in animals that received CRISPR/Cas9 therapy. These findings suggest that CRISPR/Cas9 anti-UL39 therapy can reduce the HSV-1 viral load in brain tissue. Therefore, investigating viral detection and evaluating antiviral treatments in the brain is essential.

Background/Objectives: Simplexvirus humanalpha1 (HSV-1) can cause herpetic keratitis, which is the most common cause of infectious blindness in developed countries. Some patients can develop toxicity or resistance to available treatments and may require keratoplasty. Methods: As an alternative therapy, the CRISPR/Cas9 anti-HSV-1 activity was assessed in an experimental model of BALB/c mice. Results: The results showed that the viral load in the eyes of mice inoculated with HSV-1 at 10⁷ PFU/mL was 4.5 ± 0.2 log₁₀ copies/mL. In contrast, mice inoculated with 10⁹ PFU/mL exhibited a high viral load of 8.1 ± 0.4 log₁₀ copies/mL. The detection of HSV-1 DNA and lesions in the eye was consistent with the viral inoculum of the infection. Next, antiviral activity showed that 200 ng/µL of CRISPR/Cas9 reduced the viral load by 2 logs (p ≤ 0.0001), as well as the lesion scores, compared to the untreated group. Conclusions: Together, the data suggest that CRISPR/Cas9 could be investigated as an alternative therapy for ocular herpes.