CRISPR/Cas9‑mediated cervical cancer treatment targeting human papillomavirus E6

Yoshiba,Takahiro; Saga,Yasushi; Urabe,Masashi; Uchibor,Ryosuke; Matsubara,Shigeki; Fujiwara,Hiroyuki; Mizukami,Hiroaki

doi:10.3892/ol.2018.9815

CRISPR/Cas9‑mediated cervical cancer treatment targeting human papillomavirus E6

Authors:
- Takahiro Yoshiba
- Yasushi Saga
- Masashi Urabe
- Ryosuke Uchibor
- Shigeki Matsubara
- Hiroyuki Fujiwara
- Hiroaki Mizukami
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Affiliations: Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi 329‑0498, Japan, Department of Obstetrics and Gynecology, Jichi Medical University, Shimotsuke, Tochigi 329‑0498, Japan
Published online on: December 10, 2018 https://doi.org/10.3892/ol.2018.9815
Pages: 2197-2206
Copyright: © Yoshiba et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

High‑risk human papillomavirus (HPV) is a common cause of cervical cancer. HPV E6 oncoprotein promotes the degradation of host tumor suppressor gene p53, leading to the development of tumors. Therapeutic strategies that specifically target E6, which is constitutively expressed in tumors and is not present in normal tissues, may be highly effective and safe. CRISPR‑CRISPR associated protein 9 (Cas9) is one of the genome editing technologies that has recently garnered attention, and is used to knockout target gene expression. By combining cervical cancer cell lines engineered to constitutively express Cas9 and an adeno‑associated virus (AAV) vector carrying a single guide (sg) RNA targeting E6 (AAV‑sgE6), the present study sought to investigate the effects of this novel therapeutic approach on cervical cancer. The Cas9 gene was transfected into three high‑risk HPV‑positive cervical cancer cell lines (HeLa, HCS‑2, and SKG‑I) to establish cell lines that constitutively expressed Cas9. Using these cell lines, genetic mutations and their frequencies, as well as the levels of protein expression, apoptosis and cell proliferation were examined in vitro. In addition, the effects of AAV‑sgE6 were examined in a mouse model of cervical cancer in vivo by a single administration of AAV‑sgE6 directly into subcutaneous tumors. The results demonstrated that multiple mutations occurred frequently in the targeted E6 genomic sequence in cervical cancer cells transduced with AAV‑sgE6. In addition, these AAV‑sgE6‑transduced cells had reduced expression of E6, increased expression of p53, increased apoptosis and their growth was suppressed in a concentration‑dependent manner. Furthermore, subcutaneous tumor growth was significantly suppressed in vivo following intratumoral administration of AAV‑sgE6, and adverse events due to AAV‑sgE6 administration were not observed. Collectively, the present results indicated that targeting E6 expression in high‑risk HPV by CRISPR‑Cas9 is a highly specific and effective strategy that may be effective in treating patients with cervical cancer.

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