Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Targeting herpes simplex virus with CRISPR–Cas9 cures herpetic stromal keratitis in mice

Nature Biotechnology volume 39, pages 567–577 (2021)Cite this article

Subjects

Abstract

Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus from the site of infection or latent reservoirs in the trigeminal ganglia. Here, we target HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (designated HSV-1-erasing lentiviral particles, termed HELP). We show that HELP efficiently blocks HSV-1 replication and the occurrence of herpetic stromal keratitis (HSK) in three different infection models. HELP was capable of eliminating the viral reservoir via retrograde transport from corneas to trigeminal ganglia. Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing. These results support the potential clinical utility of HELP for treating refractory HSK.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: HELP blocks HSV-1 replication in vitro.
Fig. 2: HELP blocks HSV-1 infection of corneas and neurons in a prevention model.
Fig. 3: HELP suppresses HSV-1-associated disease pathologies in the prevention model.
Fig. 4: Eye health after HELP treatment in the prevention model.
Fig. 5: HELP cures HSK in the therapeutic and recurrent models.
Fig. 6: HELP eliminates HSV-1 in tissue culture of human corneas.

Similar content being viewed by others

Data availability

Data generated or analyzed during this study are available from the corresponding author on reasonable request. The deep-sequencing and whole-genome sequencing data are available at NCBI BioProject. The BioProject IDs are PRJNA668071 and PRJNA668060, respectively. Source data are provided with this paper.

References

  1. Liesegang, T. J. Herpes simplex virus epidemiology and ocular importance. Cornea 20, 1–13 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Paludan, S. R., Bowie, A. G., Horan, K. A. & Fitzgerald, K. A. Recognition of herpesviruses by the innate immune system. Nat. Rev. Immunol. 11, 143–154 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bradshaw, M. J. & Venkatesan, A. Herpes simplex virus-1 encephalitis in adults: pathophysiology, diagnosis, and management. Neurotherapeutics 13, 493–508 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Farooq, A. V. & Shukla, D. Herpes simplex epithelial and stromal keratitis: an epidemiologic update. Surv. Ophthalmol. 57, 448–462 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Crumpacker, C. S. & Schaffer, P. A. New anti-HSV therapeutics target the helicase–primase complex. Nat. Med. 8, 327–328 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Remeijer, L. et al. Prevalence and clinical consequences of herpes simplex virus type 1 DNA in human cornea tissues. J. Infect. Dis. 200, 11–19 (2009).

    Article  PubMed  Google Scholar 

  7. Wang, L., Wang, R., Xu, C. & Zhou, H. Pathogenesis of herpes stromal keratitis: immune inflammatory response mediated by inflammatory regulators. Front. Immunol. 11, 766 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Awasthi, S. et al. Nucleoside-modified mRNA encoding HSV-2 glycoproteins C, D, and E prevents clinical and subclinical genital herpes. Sci. Immunol. 4, eaaw7083 (2019).

  9. Bolland, S. & Pierce, S. K. Ups and downs in the search for a herpes simplex virus vaccine. eLife 4, e06883 (2015).

    Article  PubMed Central  Google Scholar 

  10. Vadlapudi, A. D., Vadlapatla, R. K. & Mitra, A. K. Update on emerging antivirals for the management of herpes simplex virus infections: a patenting perspective. Recent Pat. Antiinfect. Drug Discov. 8, 55–67 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jiang, Y. C., Feng, H., Lin, Y. C. & Guo, X. R. New strategies against drug resistance to herpes simplex virus. Int. J. Oral Sci. 8, 1–6 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Schaeffer, H. J. et al. 9-(2-hydroxyethoxymethyl) guanine activity against viruses of the herpes group. Nature 272, 583–585 (1978).

    Article  CAS  PubMed  Google Scholar 

  13. Koganti, R., Yadavalli, T. & Shukla, D. Current and emerging therapies for ocular herpes simplex virus type-1 infections. Microorganisms 7, 429 (2019).

    Article  CAS  PubMed Central  Google Scholar 

  14. Crute, J. J. et al. Herpes simplex virus helicase–primase inhibitors are active in animal models of human disease. Nat. Med. 8, 386–391 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Kleymann, G. et al. New helicase–primase inhibitors as drug candidates for the treatment of herpes simplex disease. Nat. Med. 8, 392–398 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Jaishankar, D. et al. An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye. Sci. Transl. Med. 10, eaan5861 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Nelson, C. E. et al. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat. Med. 25, 427–432 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019).

    Article  CAS  PubMed  Google Scholar 

  19. Beyret, E. et al. Single-dose CRISPR–Cas9 therapy extends lifespan of mice with Hutchinson–Gilford progeria syndrome. Nat. Med. 25, 419–422 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Santiago-Fernandez, O. et al. Development of a CRISPR/Cas9-based therapy for Hutchinson–Gilford progeria syndrome. Nat. Med. 25, 423–426 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lee, B. et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat. Biomed. Eng. 2, 497–507 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gao, X. et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553, 217–221 (2018).

    Article  CAS  PubMed  Google Scholar 

  23. Dash, P. K. et al. Sequential LASER ART and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice. Nat. Commun. 10, 2753 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  24. de Buhr, H. & Lebbink, R. J. Harnessing CRISPR to combat human viral infections. Curr. Opin. Immunol. 54, 123–129 (2018).

    Article  PubMed  Google Scholar 

  25. Aubert, M. et al. In vivo disruption of latent HSV by designer endonuclease therapy. JCI Insight 1, e88468 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Aubert, M. et al. Gene editing and elimination of latent herpes simplex virus in vivo. Nat. Commun. 11, 4148 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. van Diemen, F. R. et al. CRISPR/Cas9-mediated genome editing of herpesviruses limits productive and latent infections. PLoS Pathog. 12, e1005701 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Oh, H. S. et al. Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9. eLife 8, e51662 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Weerasooriya, S., DiScipio, K. A., Darwish, A. S., Bai, P. & Weller, S. K. Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication. Proc. Natl Acad. Sci. USA 116, 1033–1042 (2019).

    Article  CAS  PubMed  Google Scholar 

  30. Weller, S. K. & Coen, D. M. Herpes simplex viruses: mechanisms of DNA replication. Cold Spring Harb. Perspect. Biol. 4, a013011 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Reinert, L. S. et al. Sensing of HSV-1 by the cGAS–STING pathway in microglia orchestrates antiviral defence in the CNS. Nat. Commun. 7, 13348 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Herpetic Eye Disease Study Group. Oral acyclovir for herpes simplex virus eye disease: effect on prevention of epithelial keratitis and stromal keratitis. Arch. Ophthalmol. 118, 1030–1036 (2000).

    Article  Google Scholar 

  33. Kennedy, D. P. et al. Ocular herpes simplex virus type 1: is the cornea a reservoir for viral latency or a fast pit stop? Cornea 30, 251–259 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Newell, C. K., Martin, S., Sendele, D., Mercadal, C. M. & Rouse, B. T. Herpes simplex virus-induced stromal keratitis: role of T-lymphocyte subsets in immunopathology. J. Virol. 63, 769–775 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Stuart, P. M., Summers, B., Morris, J. E., Morrison, L. A. & Leib, D. A. CD8+ T cells control corneal disease following ocular infection with herpes simplex virus type 1. J. Gen. Virol. 85, 2055–2063 (2004).

    Article  CAS  PubMed  Google Scholar 

  36. Jeon, S., Rowe, A. M., Carroll, K. L., Harvey, S. A. K. & Hendricks, R. L. PD-L1/B7-H1 inhibits viral clearance by macrophages in HSV-1-infected corneas. J. Immunol. 200, 3711–3719 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Biswas, P. S. & Rouse, B. T. Early events in HSV keratitis—setting the stage for a blinding disease. Microbes Infect. 7, 799–810 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Bae, S., Park, J. & Kim, J.-S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sarah, B., Ibtissam, H., Mohammed, B., Hasna, S. & Abdeljalil, M. Intrastromal injection of bevacizumab in the management of corneal neovascularization: about 25 eyes. J. Ophthalmol. 2016, 6084270 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Berrozpe-Villabona, C. et al. Intrastromal bevacizumab injection for corneal neovascularization in herpetic stromal keratitis. J. Fr. Ophtalmol. 38, 776–777 (2015).

    Article  CAS  PubMed  Google Scholar 

  41. Sharma, N. et al. Management algorithm for fungal keratitis: the TST (topical, systemic, and targeted therapy) protocol. Cornea 38, 141–145 (2019).

    Article  PubMed  Google Scholar 

  42. Narayana, S. et al. Mycotic antimicrobial localized injection: a randomized clinical trial evaluating intrastromal injection of voriconazole. Ophthalmology 126, 1084–1089 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Koelle, D. M. et al. Tegument-specific, virus-reactive CD4 T cells localize to the cornea in herpes simplex virus interstitial keratitis in humans. J. Virol. 74, 10930–10938 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Maertzdorf, J., Verjans, G. M., Remeijer, L., van der Kooi, A. & Osterhaus, A. D. Restricted T cell receptor β-chain variable region protein use by cornea-derived CD4+ and CD8+ herpes simplex virus-specific T cells in patients with herpetic stromal keratitis. J. Infect. Dis. 187, 550–558 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Okinaga, S. Shedding of herpes simplex virus type 1 into tears and saliva in healthy Japanese adults. Kurume Med. J. 47, 273–277 (2000).

    Article  CAS  PubMed  Google Scholar 

  46. Kobayashi, K. et al. Pseudotyped lentiviral vectors for retrograde gene delivery into target brain regions. Front. Neuroanat. 11, 65 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Arriagada, G. Retroviruses and microtubule-associated motor proteins. Cell. Microbiol. 19, e12759 (2017).

    Article  Google Scholar 

  48. Kato, S. et al. Enhancement of the transduction efficiency of a lentiviral vector for neuron-specific retrograde gene delivery through the point mutation of fusion glycoprotein type E. J. Neurosci. Methods 311, 147–155 (2019).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank F. Zhang (MIT, USA) for reading and commenting on our manuscript. The work was supported by grants from the National Natural Science Foundation of China (31971364), the Pujiang Talent Project of Shanghai (18PJ1404500), the Natural Science Foundation of Shanghai (18ZR1419300) and startup funding from the Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University (WF220441504) to Y.C. and by the National Natural Science Foundation of China (81970766 and 81670818), the Shanghai Rising-Star Program (18QA1401100), the Shanghai Innovation Development Program (2020779) and the Shanghai Key Clinical Research Program (SHDC2020CR3052B) to J.H. S.R.P. is supported by the European Research Council (ERC-AdG ENVISION; 786602).

Author information

Author notes

  1. These authors contributed equally: Di Yin, Sikai Ling, Dawei Wang.

Authors and Affiliations

  1. Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China

    Di Yin, Sikai Ling, Yao Dai & Yujia Cai

  2. National Research Center for Translational Medicine, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Dawei Wang

  3. Department of Ophthalmology and Vision Science, Shanghai Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, China

    Hao Jiang, Xujiao Zhou & Jiaxu Hong

  4. Department of Ophthalmology, The Affiliated Hospital of Guizhou Medical University, Guiyang, China

    Hao Jiang & Jiaxu Hong

  5. Department of Biomedicine, Aarhus University, Aarhus, Denmark

    Soren R. Paludan

Authors
  1. Di Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sikai Ling
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dawei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yao Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hao Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xujiao Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Soren R. Paludan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jiaxu Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yujia Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.Y., S.L., J.H. and Y.C. conceptualized the study and designed the experiments; D.Y., S.L., D.W., Y.D., H.J. and X.Z. performed the experiments; S.R.P. provided the HSV-1 strains and facilitated building the mouse HSK model; all the authors analyzed the data; D.Y., S.L. and Y.C. wrote the manuscript with help from all the authors.

Corresponding authors

Correspondence to Jiaxu Hong or Yujia Cai.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Biotechnology thanks Paul R. Kinchington and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–20 and Tables 1–5.

Reporting Summary

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 6

Unprocessed western blots.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yin, D., Ling, S., Wang, D. et al. Targeting herpes simplex virus with CRISPR–Cas9 cures herpetic stromal keratitis in mice. Nat Biotechnol 39, 567–577 (2021). https://doi.org/10.1038/s41587-020-00781-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-020-00781-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research