HSV Infections Are the Latest Genome Editing Targets

A new technological approach to genome editing that can clear latent and productive herpesvirus infections from human cells in vitro could eventually lead to a potent prophylactic, as well as an anti-viral therapy, by targeting these viruses and impairing their ability to replicate, according to the results of a study published recently in the PLoS Pathogens.¹

Because herpesviruses, a family of large DNA viruses, can establish a quiescent, latent state,² they can persist in human hosts and evade immune system-mediated clearance. As a consequence, they can reactivate, causing widespread and/or lifelong infections including herpes simplex keratitis, genital herpes, herpes zoster, and Epstein-Barr virus (EBV). This is a significant clinical challenge, as most adults are known to carry multiple herpesviruses². Although available treatments for productive infections target the viral DNA polymerase,^3,4 these viruses are not susceptible to antiviral agents in their latent state.

A potential approach to targeting quiescent and productive herpesviruses involves exploiting the adaptive immune system components that participate in the degradation of viral nucleic acids, referred to as the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats [CRISPR]—CRISPR—associated [Cas]) system. By genetically engineering, or "rewiring", the CRISPR/Cas9 system through coexpression of a bacterial Cas9 nuclease with a guideRNA (gRNA), RNA-guided genome modifications have been achieved in human cells.⁵ For example, applications of this genomic editing technology have allowed for a wide array of biological manipulations including the selective modification of several types of viruses. However, its effectiveness against members of the herpesvirus family had yet to be assessed.

In describing the study rationale, first author Ferdy R. van Diemen from the department of medical microbiology at the University Medical Center Utrecht in The Netherlands said, "... there is a need for a direct and potent strategy to limit herpesvirus infections by specifically targeting the viral genome or its maintenance." Towards addressing this need, van Diemen and colleagues conducted a proof-of-concept study involving a variety of experiments designed to explore whether genetic engineering of the CRISPR/Cas9 system could be used to induce efficient editing of three members of the human herpesvirus family associated with serious health-related consequences (herpes simplex virus [HSV]-1 and 2, human cytomegalovirus [HCMV], and EBV) during both latent and productive viral life cycle stages.

The large number of experiments yielded a battery of important findings regarding the utility of this technology as applied to members of the herpesvirus family. The results from one set of experiments indicated that the CRISPR/Cas9 system could be used in the direct editing of EBV genomes. It was also determined that CRISPR/Cas9-mediated targeting of essential EBV genetic elements cleared EBV from latently infected lymphoma cells, resulting in a >95% loss of EBV genomes when using a double targeting approach with two different gRNAs. Furthermore, CRISPR/Cas9 targeting of essential HCMV protein-encoding genes impaired the ability of the virus to replicate. It is important to note, however, that inefficient targeting of HCMV by single gRNAs was shown to select for viral escape mutants after prolonged replication. Similar to the results obtained for EBV, CRISPR/Cas9 targeting of essential HSV-1 protein-encoding genes impaired virus replication, and a double targeting approach with two gRNAs completely impaired the ability of HSV-1 to replicate. Although off-target editing has been raised as a concern associated with the use of this technology, off-target editing at predicted off-target sites within the human genome was not detected. Lastly, the use of anti-HSV-1 CRISPR gRNAs was shown to abolish the replication of HSV-1 that was reactivated from a quiescent state.

In summarizing the results of their study, the authors stated, "Here, we performed proof-of-concept studies to show that the CRISPR/Cas9 genome engineering system can be efficiently used to limit or eradicate three of the most prevalent herpesviruses from human cells: HSV-1, HCMV, and EBV." As for the broader implications of the study results, van Diemen and colleagues stated, "The findings presented in this study open new avenues for the development of therapeutic strategies to combat pathogenic human herpesviruses using novel genome-engineering technologies."

William Perlman, PhD, CMPP is a former research scientist currently working as a medical/scientific content development specialist. He earned his BA in Psychology from Johns Hopkins University, his PhD in Neuroscience at UCLA, and completed three years of postdoctoral fellowship in the Neuropathology Section of the Clinical Brain Disorders Branch of the National Institute of Mental Health.

References

1. van Diemen FR, Kruse EM, Hooykaas MJG, et al. CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections. PLoS Pathog 2016;12:e1005701.
2. Pellett PE, Roizman B. Herpesviridae. In: Knipe D, Howley P, eds. Fields Virology 2. 6th ed. Philadelphia, PA: Lippincott, Williams & Wilkins; 2013:1802—22.
3. Piret J, Boivin G. Antiviral drug resistance in herpesviruses other than cytomegalovirus. Rev Med Virol 2014;24:186—218.
4. Andrei G, De Clercq E, Snoeck R. Viral DNA Polymerase Inhibitors. In: Raney KD, Gotte M, Cameron CE, eds. Viral Genome Replication. New York, NY: Springer US; 2009:481—526.
5. Sternberg SH, Doudna JA. Expanding the Biologist's Toolkit with CRISPR-Cas9. Mol Cell 2015;58:568—74.