Nat Biotechnol. 2021 Jan 11.
DOI: 10.1038/s41587-020-00781-8.
Background
HSV-1 is among the most common human viruses, with 50–80% of the world population being seropositive. It belongs to the alpha subfamily of herpesviruses, which are enveloped viruses carrying double-stranded DNA and are capable of establishing latent infections in sensory neurons. Despite the high prevalence, there is no vaccine currently available for HSV infection. One study delivered an HSV-1-targeting endonuclease using adeno-associated virus (AAV) in a mouse model of latent HSV infection; however, this study revealed neither a detectable loss of viral genome nor therapeutic efficacy25. Recently, the same group showed detectable elimination of latent genomes and therapeutic efficacy by using an improved AAV vector and two mega nucleases targeting the HSV genome26. So far, the anti-HSV activity of CRISPR has only been characterized in vitro, and no studies have shown the therapeutic efficacy of CRISPR against HSK in vivo. In this study, the authors developed HSV-1-erasing lentiviral particles (HELP) and showed its therapeutic efficacy in three different HSK models and in human-derived corneas. Furthermore, we found that HELP was capable of modulating the HSV-1 reservoir in the trigeminal ganglia (TG). Corneas maintained a healthy status after intracorneal injection of HELP, as shown by a variety of clinically relevant assays. Cas9 expression from HELP only lasted for 3 d in vivo, and no off-target effects were detected in the coding regions of the mouse and human genomes.
Methods
1. Cell cultures and HSV-1 propagation.2. Infection and transduction of cells.3. Immunofluorescence imaging.
Results
In this study, the authors designed a guide RNA (gRNA) expression cassette simultaneously targeting two essential genes of HSV-1, UL8 and UL29, and co-packaged it with SpCas9 mRNA in an mRNA-carrying lentiviral particle (mLP) via the specific binding of pac site-containing SpCas9 mRNA to bacteriophage-derived MS2 coat protein located at the N terminus of lentiviral Gag and GagPol polyproteins. To demonstrate the antiviral effect of HELP, the authors used three different mouse infection models: prevention model, therapeutic model and recurrent model. Help effectively prevented HSV-1 replication and the development of herpetic stromal keratitis in all three different models of HSV infection, the researchers found. The authors found evidence that HELP can be transported retrograde from the cornea to the trigeminal ganglion, clearing the library of HSV-1 virus. In contrast, the conventional drug acyclovir (ACV), although it also inhibited viral replication in the cornea, did not do anything to limit the virus in the trigeminal ganglion. In addition, the researchers also observed that HELP effectively cleared the human cornea of HSV-1 virus using donor corneas.Conclusion
In this study, the authors show that transient gene editing via mRNA-based CRISPR delivery is sufficient to achieve therapeutic efficacy against HSK in vivo and blocks HSV-1 replication in human corneas. Modulating the viral reservoir is essential to prevent HSK from recurrence. Further, their study provides evidence of HSV elimination in the reservoir by HELP via retrograde transport from the cornea to the TG.