Transient gene editing of the herpes simplex virus type 1 (HSV-1) using mRNA-based CRISPR was shown for the first time to be effective against herpetic stromal keratitis (HSK) in mice and blocked HSV-1 replication in human corneas, Chinese researchers reported in the January 11, 2021, online edition of Nature Biotechnology.

After primary HSV-1 infection and replication in the cornea, HSV-1 is transported via ophthalmic nerves to the trigeminal ganglia (TG), where a latent reservoir persists, which can be reactivated in immunocompromised patients, leading to disease recurrence, aggravation and blinding HSK.

Critically, the new study also demonstrated that eliminating latent HSV-1 in the TG, using newly developed HSV-1-erasing lentiviral particles (HELP) via retrograde transport from the cornea to the TG, prevented the recurrence of HSK in mice.

HSK is the leading cause of infectious blindness worldwide, with an estimated 1.5 million cases of ocular HSV-1 occurring each year and 40,000 people developing blinding HSK.

However, despite this high prevalence, no HSV-1 vaccine has been developed, possibly due to the complexity of the virus and its infectious mechanism.

"HSV-1 encodes for more than 80 viral proteins, many of which enable the virus to escape recognition by the immune system," said study leader Yujia Cai.

In addition, "HSV-1 uses multiple receptors to enter host cells and may remain silent in sensory neurons," said Cai, the principal investigator in the Laboratory of Gene Therapy at the Shanghai Center for Systems Biomedicine at Shanghai Jiao Tong University.

This propensity for forming latent infectious reservoirs is particularly problematic, because currently available HSV-1 treatments fail to clear the latent TG reservoir, resulting in disease recurrence.

Because most tissue damage occurring in human corneas during HSK is known to be immune-mediated, this has important implications for treatment.


The first-line HSV-1 treatment is acyclovir and its analogues, all of which target viral DNA polymerase, but drug resistance often occurs in the immunocompromised and those receiving chronic antiviral prophylaxis.

Alternative strategies include small molecules that inhibit the viral helicase-primase complex, antibodies and peptides, but no treatments can eliminate active or latent HSV-1, thus they cannot prevent recurrence, necessitating other approaches such as CRISPR.


CRISPR (clustered regularly interspaced short palindromic repeats) targets genomes and was recently approved by the FDA for phase I/II trials to treat beta-thalassemia and sickle cell disease.

In infectious diseases, combining CRISPR with antiviral prodrugs has been shown to clear infectious latent HIV-1 reservoirs in HIV-1-infected humanized mice.

Recently, elimination of latent HSV-1 genomes and therapeutic efficacy have been shown using an adeno-associated virus (AAV) vector and two meganucleases targeting the viral genome.

However, as yet the anti-HSV activity of CRISPR has only been characterized in vitro, and no studies have shown its therapeutic efficacy against HSK in vivo.

In the new Nature Biotechnology study, researchers led by Cai and Jiaxu Hong, a clinician in the Shanghai Eye, Ear, Nose and Throat Hospital at Fudan University, developed HELP, which targets HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (gRNAs).

"Derived from Streptococcus pyogenes (Sp), SpCas9 (CRISPR-associated protein 9) was the first characterized and remained the most widely used version of CRISPR," said Cai.

"SpCas9 is delivered as mRNA packaged in HELP, which was shown not only to eliminate HSV-1 at injection sites in the cornea, but also in the TG via retrograde transportation," he added.

HELP significantly blocked HSV-1 replication and HSK occurrence in three different infection models, and eliminated the TG viral reservoir, "as shown using qPCR [quantitative polymerase chain reaction], a plaque forming units assay and confocal imaging," noted Cai.

Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing.

Regarding safety, "we did not observe any significant side effects in any of the [HELP]-treated mice," Cai said.

Moreover, corneas maintained a healthy status after intracorneal HELP injection, as demonstrated using various clinically relevant assays, "including sodium fluorescein corneal staining, phenol red thread testing, and tests of mechnosensory and electrophysiology function."

Cas9 expression from HELP lasted for only 3 days in vivo, and no off-target effects were detected in the coding regions of the mouse and human genomes.

This further indicates the safety of HELP, said Cai, "as the brief duration of Cas9 expression after HELP will reduce the likelihood of off-target effects within the genomes."

Taken together, these findings support the clinical translation of HELP for treating refractory HSV-1 and, because HELP gRNAs target the viral rather than the human genome, this may speed clinical translation.

Looking forward, said Cai, "our study may facilitate the development of CRISPR therapeutics targeting other viruses, including HSV-2, human papillomavirus (HPV) and hepatitis B virus (HBV)."