Developing oncolytic Herpes simplex virus type 1 through UL39 knockout by CRISPR-Cas9

Document Type : Original Article

Authors

1 Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2 Abadan Faculty of Medical Science, Abadan, Iran

3 Department of Virology, Pasteur Institute of Iran, Tehran, Iran

4 Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

Abstract

Objective(s): Oncolytic Herpes simplex virus type 1 (HSV-1) has emerged as a promising strategy for cancer therapy. However, development of novel oncolytic mutants has remained a major challenge owing to low efficiency of conventional genome editing methods. Recently, CRISPR-Cas9 has revolutionized genome editing.
Materials and Methods: In this study, we aimed to evaluate the capability of CRISPR-Cas9 to manipulate the UL39 gene to create oncolytic HSV-1. Herein, three sgRNAs were designed against the UL39 gene and transfected into HEK-293 cell line followed by infection with HSV-1 KOS.
Results: After three rounds of plaque purification, several HSV-1 mutants were identified by PCR analysis and sequencing. One of these mutations in which 55 nucleotides were deleted resulted in a frameshift mutation that in turn produced a truncated protein with only 167 amino acids from 1137 amino acids. Functional analysis in Vero and primary fibroblast cells revealed that viral replication was significantly lower and plaque size was smaller in the HSV-1 mutant compared with HSV-1 KOS. Moreover, the relative amount of viral genome present in the supernatants of infected cells (Vero and primary fibroblast cells) with HSV-1 mutant was significantly decreased compared with those of HSV-1 KOS.
Conclusion: Our data revealed that targeting UL39 with CRISPR-Cas9 could develop oncolytic HSV-1.

Keywords

References

1. Kaur B, Chiocca EA, Cripe TP. Oncolytic HSV-1 virotherapy: clinical experience and opportunities for progress. Curr Pharm Biotechnol 2012; 13:1842-1851.
2. Peters C, Rabkin SD. Designing herpes viruses as oncolytics. Mol Ther Oncolytics 2015; 2:15010.
3. Nishiyama Y. Herpes virus genes: Molecular basis of viral replication and pathogenicity. Nagoya J Med Sci 1996; 59:107-119.
4. Aghi M, Visted T, Depinho RA, Chiocca EA. Oncolytic herpes virus with defective ICP6 specifically replicates in quiescent cells with homozygous genetic mutations in p16. Oncogene 2008; 27:4249-4254.
5. Currier MA, Gillespie RA, Sawtell NM, Mahller YY, Stroup G, Collins MH, et al. Efficacy and safety of the oncolytic herpes simplex virus rRp450 alone and combined with cyclophosphamide. Mol Ther 2008; 16:879-885.
6. Sokolowski NA, Rizos H, Diefenbach RJ. Oncolytic virotherapy using herpes simplex virus: how far have we come? Oncolytic Virother 2015; 4:207-219.
7. Varghese S, Rabkin SD. Oncolytic herpes simplex virus vectors for cancer virotherapy. Cancer Gene Ther 2002; 9:967-978.
8. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014; 346:1258096.
9. Ebrahimi S, Teimoori A, Khanbabaei H, Tabasi M. Harnessing CRISPR/Cas 9 System for manipulation of DNA virus genome. Rev Med Virol 2019; 29:e2009.
10. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157:1262-1278.
11. Rath D, Amlinger L, Rath A, Lundgren M. The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie 2015; 117:119-128.
12. Wang D, Wang XW, Peng XC, Xiang Y, Song SB, Wang YY, et al. CRISPR/Cas9 genome editing technology significantly accelerated herpes simplex virus research. Cancer Gene Ther 2018; 25:93-105.
13. Xiao-Jie L, Hui-Ying X, Zun-Ping K, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. J Med Genet 2015; 52:289-296.
14. Yuan M, Webb E, Lemoine NR, Wang Y. CRISPR-Cas9 as a powerful tool for efficient creation of oncolytic viruses. Viruses 2016; 8:72.
15. Bi Y, Sun L, Gao D, Ding C, Li Z, Li Y, et al. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases. PLoS Pathog 2014; 10:e1004090.
16. Finnen RL, Banfield BW. CRISPR/Cas9 mutagenesis of UL21 in multiple strains of herpes simplex virus reveals differential requirements for pUL21 in viral replication. Viruses 2018; 10:258.
17. Li J, Zeng W, Huang Y, Zhang Q, Hu P, Rabkin SD, et al. Treatment of breast cancer stem cells with oncolytic herpes simplex virus. Cancer Gene Ther 2012; 19:707-714.
18. Lin C, Li H, Hao M, Xiong D, Luo Y, Huang C, et al. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing of HSV-1 virus in human cells. Sci Rep 2016; 6:34531.
19. Roehm PC, Shekarabi M, Wollebo HS, Bellizzi A, He L, Salkind J, et al. Inhibition of HSV-1 Replication by Gene Editing Strategy. Sci Rep 2016; 6:23146.
20. Suenaga T, Kohyama M, Hirayasu K, Arase H. Engineering large viral DNA genomes using the CRISPR-Cas9 system. Microbiol Immunol 2014; 58:513-522.
21. Brandt CR, Kintner RL, Pumfery AM, Visalli RJ, Grau DR. The herpes simplex virus ribonucleotide reductase is required for ocular virulence. J Gen Virol 1991; 72 ( Pt 9):2043-2049.
22. Jacobson JG, Leib DA, Goldstein DJ, Bogard CL, Schaffer PA, Weller SK, et al. A herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells. Virology 1989; 173:276-283.
23. Vangipuram M, Ting D, Kim S, Diaz R, Schule B. Skin punch biopsy explant culture for derivation of primary human fibroblasts. J Vis Exp 2013:e3779.
24. Kennedy EM, Bassit LC, Mueller H, Kornepati AVR, Bogerd HP, Nie T, et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology 2015; 476:196-205.
25. Lin SR, Yang HC, Kuo YT, Liu CJ, Yang TY, Sung KC, et al. The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo. Mol Ther Nucleic Acids 2014; 3:e186.
26. Song Y, Lai L, Li Z. Large-scale genomic deletions mediated by CRISPR/Cas9 system. Oncotarget 2017; 8:5647.
27. Bhattacharya D, Van Meir EG. A simple genotyping method to detect small CRISPR-Cas9 induced indels by agarose gel electrophoresis. Sci Rep 2019; 9:4437.
28. Chen D, Tang JX, Li B, Hou L, Wang X, Kang L. CRISPR/Cas9-mediated genome editing induces exon skipping by complete or stochastic altering splicing in the migratory locust. BMC Biotechnol 2018; 18:60.
29. Ren Z, Li S, Wang QL, Xiang YF, Cui YX, Wang YF, et al. Effect of siRNAs on HSV-1 plaque formation and relative expression levels of RR mRNA. Virol Sin 2011; 26:40-46.
30. Idowu AD, Fraser-Smith EB, Poffenberger KL, Herman RC. Deletion of the herpes simplex virus type 1 ribonucleotide reductase gene alters virulence and latency in vivo. Antiviral Res 1992; 17:145-156.
31. Mostafa HH, Thompson TW, Konen AJ, Haenchen SD, Hilliard JG, Macdonald SJ, et al. Herpes simplex virus 1 mutant with point mutations in UL39 is impaired for acute viral replication in mice, establishment of latency, and explant-induced reactivation. J  Virol 2018; 92:e01654-01617.
32. Perkins D. Targeting apoptosis in neurological disease using the herpes simplex virus. J Cell Mol Med 2002; 6:341-356.

Iranian Journal of Basic Medical Sciences
Volume 23, Issue 7
July 2020
Pages 937-944

Files

Share

How to cite

Statistics