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:: Volume 35, Issue 4 (winter 2025) ::
MEDICAL SCIENCES 2025, 35(4): 426-435 Back to browse issues page
Simultaneous targeting of three human papillomavirus type 16 oncoproteins using gene editing in a C57BL/6 mouse model of C3 epithelial tumor
Ali Anvar1 , Azam Bolhassani2 , Iman Salahshoorifar1 , Shiva Irani1
1- Faculty of Converging Sciences and Technologies, Science and Research Branch, Islamic Azad University, Tehran, Iran
2- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran , azam.bolhassani@yahoo.com
Abstract:   (280 Views)
Background: Human papillomavirus type 16 (HPV16) is a leading cause of cervical cancer, with its oncoproteins E5, E6, and E7 playing pivotal roles in tumor development. This study aimed to investigate the effect of simultaneous targeting of these three oncogenes using CRISPR/Cas9 technology delivered via the HR9 peptide in a mouse model of solid epithelial tumors.
Materials and methods: Female C57BL/6 mice (4–6 weeks old, weighing approximately 15–20 g) were divided into five groups. Subcutaneous injection of HPV16-positive C3 tumor cells was performed. After tumor formation, intratumoral administration of PX460-based CRISPR/Cas9 vectors containing E5, E6, or E7 sgRNAs, complexed with HR9 peptide, was carried out on days 7, 14, and 21. On day 60, apoptosis was assessed using H&E staining, immunohistochemistry (IHC), and caspase-3 expression analysis.
Results: Mice treated with E6-PX460 + E7-PX460 nanoparticles exhibited a statistically significant reduction in tumor size and an increased rate of apoptosis compared to those treated with the triple combination (E5+E6+E7-PX460) or E5-PX460 alone (p<0.0001). These findings indicate the superior therapeutic efficacy of dual targeting of E6 and E7.
Conclusion: Simultaneous targeting of E6 and E7 oncogenes using CRISPR/Cas9 technology delivered via the HR9 peptide effectively induced apoptosis and suppressed tumor growth in C57BL/6 mice. This approach shows promise as a novel targeted strategy for HPV16-related tumor therapy and warrants further investigation.
 
Keywords: HPV16, Oncoproteins, CRISPR/Cas9, Gene editing, C3 tumor cells
Full-Text [PDF 1319 kb]   (148 Downloads)    
Semi-pilot: Semi-pilot | Subject: Genetic
Received: 2025/02/4 | Accepted: 2025/06/9 | Published: 2025/12/1
References
1. Choi S, Ismail A, Pappas-Gogos G, Boussios S. HPV and cervical cancer: A review of epidemiology and screening uptake in the UK. Pathogens 2023;12:298. [DOI:10.3390/pathogens12020298]
2. Na J, Li Y, Wang J, Wang X, Lu JL, Han S. The correlation between multiple HPV infections and the occurrence, development, and prognosis of cervical cancer. Front Microbiol 2023;14:1220522. [DOI:10.3389/fmicb.2023.1220522]
3. Münger K, Baldwin A, Edwards KM, Hayakawa H, Nguyen CL, Owens M, et al. Mechanisms of human papillomavirus-induced oncogenesis. J Virol 2004;78:11451-60. [DOI:10.1128/JVI.78.21.11451-11460.2004]
4. Moody CA, Laimins LA. Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer 2010;10:550-60. [DOI:10.1038/nrc2886]
5. Torres AD, Spurgeon ME, Bilger A, Blaine-Sauer S, Uberoi A, Buehler D, et al. The human papillomavirus 16 E5 gene potentiates MmuPV1-Dependent pathogenesis. Virology 2020;541:1-12. [DOI:10.1016/j.virol.2019.12.002]
6. Collins NB, Al Abosy R, Miller BC, Bi K, Zhao Q, Quigley M, et al. PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment. J Immunother Cancer 2022;10:e003402. [DOI:10.1136/jitc-2021-003402]
7. Scott ML, Woodby BL, Ulicny J, Raikhy G, Orr AW, Songock WK, et al. Human Papillomavirus 16 E5 Inhibits Interferon Signaling and Supports Episomal Viral Maintenance. J Virol 2020;94:e01582-19. [DOI:10.1128/JVI.01582-19]
8. Brea EJ, Oh CY, Manchado E, Budhu S, Gejman RS, Mo G, et al. Kinase Regulation of Human MHC Class I Molecule Expression on Cancer Cells. Cancer Immunol Res 2016;4:936-47. [DOI:10.1158/2326-6066.CIR-16-0177]
9. Bonami JR, Widada JS. Viral diseases of the giant freshwater prawn Macrobrachium rosenbergii: a review. J Invertebr Pathol 2011;106:131-42. [DOI:10.1016/j.jip.2010.09.007]
10. Jendoubi-Ferchichi M, Satouri L, Ghoul F, Malek-Mellouli M, Derbel AM, Makni MK, et al. Phylogeny and classification of human papillomavirus (HPV) 16 and HPV18 variants based on E6 and L1 genes in Tunisian women with cervical lesions. Asian Pac J Cancer Prev 2018;19:3361. [DOI:10.31557/APJCP.2018.19.12.3361]
11. Sausen DG, Shechter O, Gallo ES, Dahari H, Borenstein R. Herpes simplex virus, human papillomavirus, and cervical cancer: Overview, relationship, and treatment implications. Cancers 2023;15(14):3692. [DOI:10.3390/cancers15143692]
12. Zhang A, Zheng X, Chen S, Duan G. In vitro study of HPV18-positive cervical cancer HeLa cells based on CRISPR/Cas13a system. Gene 2024;921:148527. [DOI:10.1016/j.gene.2024.148527]
13. Zhan X, Zhou J, Jiang Y, An P, Luo B, Lan F, et al. DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging. Biosens Bioelectron 2023;229:115229. [DOI:10.1016/j.bios.2023.115229]
14. Zhao Y, Chen D, Xu Z, Li T, Zhu J, Hu R, et al. Integrating CRISPR-Cas12a into a microfluidic dual-droplet device enables simultaneous detection of HPV16 and HPV18. Anal Chem 2023;95:3476-85. [DOI:10.1021/acs.analchem.2c05320]
15. Öktem M, Mastrobattista E, de Jong OG. Amphipathic cell-penetrating peptide-aided delivery of Cas9 RNP for in vitro gene editing and correction. Pharmaceutics 2023;15:2500. [DOI:10.3390/pharmaceutics15102500]
16. Liu Q, Yang J, Xing Y, Zhao Y, Liu Y. Development of delivery strategies for CRISPR‐Cas9 genome editing. BMEMat 2023;1:e12025. [DOI:10.1002/bmm2.12025]
17. Chong ZX, Yeap SK, Ho WY. Transfection types, methods and strategies: a technical review. PeerJ 2021;9:e11165. [DOI:10.7717/peerj.11165]
18. Liu MJ, Chou JC, Lee HJ. A gene delivery method mediated by three arginine-rich cell-penetrating peptides in plant cells. Adv Stud Biol 2013;5:71-88. [DOI:10.12988/asb.2013.13007]
19. Hasannejad-Asl B, Pooresmaeil F, Takamoli S, Dabiri M, Bolhassani A. Cell penetrating peptide: A potent delivery system in vaccine development. Front Pharmacol 2022;13:4834. [DOI:10.3389/fphar.2022.1072685]
20. Sinclair F, Begum AA, Dai CC, Toth I, Moyle PM. Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing. Drug Deliv Transl Res 2023;13:1500-19. [DOI:10.1007/s13346-023-01320-z]
21. Azadbakht N, Doosti A, Jami MS. CRISPR/Cas9-mediated LINC00511 knockout strategies, increased apoptosis of breast cancer cells via suppressing antiapoptotic genes. Biol Proced Online 2022;24:8. [DOI:10.1186/s12575-022-00171-1]
22. Moret I, Esteban Peris J, Guillem VM, Benet M, Revert F, Dasi F, et al. Stability of PEI-DNA and DoTAP-DNA complexes: Effect of alkaline pH, heparin and serum. J Control Release 2001;76:169-82. [DOI:10.1016/S0168-3659(01)00415-1]
23. Sadeghian F, Hosseinkhani S, Alizadeh A, Hatefi A. Design, engineering and preparation of a multi-domain fusion vector for gene delivery. Int J Pharm 2012;427:393-9. [DOI:10.1016/j.ijpharm.2012.01.062]
24. Zhen S, Li X. Oncogenic human papillomavirus: application of CRISPR/Cas9 therapeutic strategies for cervical cancer. Cell Physiol Biochem 2018;44:2455-66. [DOI:10.1159/000486168]
25. Borrelli A, Tornesello AL, Tornesello ML, Buonaguro FM. Cell penetrating peptides as molecular carriers for anti-cancer agents. Molecules 2018;23:295. [DOI:10.3390/molecules23020295]
26. Hooshmand SE, Sabet MJ, Hasanzadeh A, Kamrani Mousavi SM, Haeri Moghaddam N, Hooshmand SA, et al. Histidine‐enhanced gene delivery systems: The state of the art. J Gene Med 2022;24:e3415. [DOI:10.1002/jgm.3415]
27. Alizadeh S, Irani S, Bolhassani A, Sadat SM. HR9: An important cell penetrating peptide for delivery of HCV NS3 DNA into HEK-293T cells. Avicenna J Med Biotechnol 2020;12:44.
28. Sahab Z, Sudarshan SR, Liu X, Zhang YY, Kirilyuk A, Kamonjoh CM, et al. Quantitative measurement of human papillomavirus type 16 e5 oncoprotein levels in epithelial cell lines by mass spectrometry. J Virol 2012;86:9465-73. [DOI:10.1128/JVI.01032-12]
29. Paolini F, Amici C, Carosi M, Bonomo C, Di Bonito P, Venuti A, et al. Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors. J Exp Clin Cancer Res 2021;40:1-11. [DOI:10.1186/s13046-021-01841-w]
30. Li LL, Wang HR, Zhou ZY, Luo J, Wang XL, Xiao XQ, et al. C3-Luc cells are an excellent model for evaluation of cellular immunity following HPV16L1 vaccination. PLoS One 2016;11:e0149748. [DOI:10.1371/journal.pone.0149748]
31. Jubair L, Lam AK, Fallaha S, McMillan NAJ. CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice. PLoS One 2021;16:e0223288. [DOI:10.1371/journal.pone.0223288]
32. Liu BR, Lin MD, Chiang HJ, Lee HJ. Arginine-rich cell-penetrating peptides deliver gene into living human cells. Gene 2012;505:37-45. [DOI:10.1016/j.gene.2012.05.053]
33. Anderson SD, Hobbs RJ, Gwenin VV, Ball P, Bennie LA, Coulter JA, et al. Cell-penetrating peptides as a tool for the cellular uptake of a genetically modified nitroreductase for use in directed enzyme prodrug therapy. J Funct Biomater 2019;10:45. [DOI:10.3390/jfb10040045]
34. Liu BR, Chiou SH, Huang YW, Lee HJ. Bio-membrane internalization mechanisms of arginine-rich cell-penetrating peptides in various species. Membranes 2022;12:88. [DOI:10.3390/membranes12010088]
35. Huang YW, Lee HJ, Tolliver LM, Aronstam RS. Delivery of nucleic acids and nanomaterials by cell-penetrating peptides: opportunities and challenges. Biomed Res Int 2015;2015:834079. [DOI:10.1155/2015/834079]
36. Liu BR, Chen HH, Lo S, Huang YW, Lee HJ. Effects of surface charge and particle size of cell-penetrating peptide/nanoparticle complexes on cellular internalization. Cell Membranes 2013:43-57.
37. Laufer S, Restle T. Peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems. Curr Pharm Des 2008;14:3637-55. [DOI:10.2174/138161208786898806]
38. Namazi F, Bolhassani A, Sadat SM, Irani S. In vitro delivery of HIV-1 Nef antigen by histidine-rich nona-arginine and latarcin 1 peptide. J Med Microbiol Infect Dis 2019;7:107-15. [DOI:10.29252/JoMMID.7.4.107]
39. Mattern J, Volm M. Imbalance of cell proliferation and apoptosis during progression of lung carcinomas. Anticancer Res 2004;24:4243-6.
40. Khairkhah N, Bolhassani A, Rajaei F, Najafipour R. Systemic delivery of specific and efficient CRISPR/Cas9 system targeting HPV16 oncogenes using LL-37 antimicrobial peptide in C57BL/6 mice. J Med Virol 2023;95:e28934. [DOI:10.1002/jmv.28934]
41. Lei J, Sun L, Huang S, Zhu C, Li P, He J, et al. The antimicrobial peptides and their potential clinical applications. Am J Transl Res 2019;11:3919-31.
42. Manteghi M, Can O, Kocagoz T. Peptosome: A New Efficient Transfection Tool as an Alternative to Liposome. Int J Mol Sci 2024;25:6918. [DOI:10.3390/ijms25136918]
43. Kim JH, Park HS, et al. CRISPR/Cas9-mediated targeting of HPV16 E6 and E7 induces apoptosis and growth inhibition in cervical cancer cells via p53 reactivation. Int J Mol Sci 2019;20:4763.
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Anvar A, Bolhassani A, Salahshoorifar I, Irani S. Simultaneous targeting of three human papillomavirus type 16 oncoproteins using gene editing in a C57BL/6 mouse model of C3 epithelial tumor. MEDICAL SCIENCES 2025; 35 (4) :426-435
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Volume 35, Issue 4 (winter 2025) Back to browse issues page
فصلنامه علوم پزشکی دانشگاه آزاد اسلامی واحد پزشکی تهران Medical Science Journal of Islamic Azad Univesity - Tehran Medical Branch
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