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Disruption of Human Papillomavirus 16 E6/E7 Genes Using All-in-One Adenovirus Vectors Expressing Eight Double-Nicking

Megumi Yamaji1, Tomomi Nakahara2, Tomoko Nakanishi1

  • 1Center of Biomedical Research Resources, Juntendo University School of Medicine, Tokyo 113-8421, Japan.

International Journal of Molecular Sciences
|September 13, 2025
PubMed

Insights

This study used a double-nicking CRISPR/Cas9 technique to target human papillomavirus (HPV) oncogenes, significantly reducing off-target effects for safer cancer therapy.

Area of Science:

  • Genomic Medicine
  • Oncology
  • Viral Oncology

Background:

  • Human papillomavirus (HPV) oncoproteins E6 and E7 are critical for cervical cancer development.
  • CRISPR/Cas9 genome editing offers a potential therapeutic strategy against HPV-induced cancers.
  • Minimizing off-target effects is essential for the clinical safety of CRISPR/Cas9 therapies.

Purpose of the Study:

  • To develop a precise genome-editing strategy targeting HPV16 E6 and E7 oncogenes.
  • To assess the efficacy and specificity of a double-nicking CRISPR/Cas9 approach.
  • To evaluate the therapeutic potential of an adenovirus-delivered system for HPV-positive cancers.

Main Methods:

  • Designed and selected double-nicking (DN)-gRNA pairs targeting HPV16 E6/E7 oncogenes.
  • Delivered DN-gRNA pairs and Cas9 nickase (Cas9n) to HPV16-positive cells using lentiviral vectors.
  • Utilized an all-in-one adenovirus (AdV) system for in vivo delivery of gRNAs and Cas9n.
  • Quantified cell killing, off-target effects, and tumor growth inhibition in preclinical models.

Main Results:

  • Multiple DN-gRNA pair combinations effectively eliminated HPV16-positive cancer cells while sparing normal cells.
  • Off-target mutations were reduced by nearly three orders of magnitude compared to standard CRISPR/Cas9.
  • The AdV-based system demonstrated significant inhibition of tumor growth in HPV16-positive cancer models.
  • The approach showed high specificity for HPV-infected cells.

Conclusions:

  • The double-nicking CRISPR/Cas9 technique offers enhanced safety and specificity for targeting HPV oncogenes.
  • Adenovirus-mediated delivery of this system presents a promising therapeutic strategy for HPV-induced malignancies.
  • This genome-editing approach holds potential for developing safer and more effective treatments for HPV-related cancers.
Keywords:
CRISPR/Cas9adenovirus vectorall-in-onecancer therapygene therapygenome editinghuman papillomavirusmultiplex guide RNAnickase

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