Development of a Gene Therapy Method for Cervical Cancer Using Attenuated Coxsackievirus B3 as a Vector System

Seung-Hyun Shim; Yeon-Jung Kim; Dae-Sun Kim; Jae-Hwan Nam

doi:10.4167/jbv.2011.41.2.123

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Shim, Kim, Kim, and Nam: Development of a Gene Therapy Method for Cervical Cancer Using Attenuated Coxsackievirus B3 as a Vector System

Original Article

J Bacteriol Virol 2011;41(2):123-130.

Published online: 18 January 2011

DOI: https://doi.org/10.4167/jbv.2011.41.2.123

Development of a Gene Therapy Method for Cervical Cancer Using Attenuated Coxsackievirus B3 as a Vector System

Seung-Hyun Shim^†, Yeon-Jung Kim^†, Dae-Sun Kim, Jae-Hwan Nam^∗

Department of Biotechnology, The Catholic University, Gyeonggi-do, Korea

^† These authors contributed equally to this work

^∗Corresponding author: Jae-Hwan Nam, Ph.D. Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, Gyeonggi-do, 420-743, Korea. Phone: +82-2-2164-4852, Fax: +82-2-2164-4917 e-mail: jhnam@catholic.ac.kr

^∗∗ This study was supported by a grant from GRRC of the Catholic University of Korea, the Next-generation Biogreen21 Program (PJ007186) of Rural Development of Administration, MKE and KOTEF through the Human Resource Training Project for Strategic Technology.

Revised 13 April 201130 May 2011 Accepted 8 June 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

We previously reported the development of an attenuated coxsackievirus B3, known as YYFF, which functioned as a viral vector system for foreign gene expression. In this study, we demonstrated the potential use of YYFF as a gene therapy vector. Recombinant YYFF was constructed to express the human papillomavirus 16 (HPV16) E7 gene, referred to as YYFF-HPV16-E7. Growth of YYFF-HPV16-E7 resembled the wild type, YYFF, and it expressed HPV16-E7 in cell culture. When YYFF-HPV16-E7 was directly injected into TC-1-transplanted C57/BL6 mice, there was no reduction in tumor size, because of the non-growth of YYFF in C57/BL6 mice. However, when YYFF-HPV16-E7-induced immune cells/serum that originated from BALB/c mice was passively delivered into BALB/c background TC-1-transplanted nude mice, it reduced the size of cervical tumors in the nude mice. This study indicates the potential use of YYFF-HPV16-E7 as a gene therapy agent for treating HPV-induced cervical cancer.

Keywords: Attenuated coxsackievirus B3, YYFF, Gene therapy, Vector system, Cervical cancer

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Figure 1.

Construction of recombinant coxsackievirus expressing HPV16 E7 flanked by viral protein 4 (VP4). The black box in viral protein 2 (VP2) indicates the conserved amino acid sequence in the C-terminal region of the VP2 protein of coxsackievirus B3 (CVB3). The VP2 protein contains tyrosines at positions 240 and 254 in the wild-type CVB3 (WT), whereas both tyrosines are replaced by phenylalanine in YYFF VP2.

Figure 2.

The growth of the viruses YYFF and YYFF-HPV16-E7 in HeLa cells (MOI = 10). (A) Intracellular viral titer. (B) Extracellular viral titer at each time point as determined by plaque assay (described in Materials and Methods). YYFF (○) and YYFF-HPV16-E7 (▪).

Figure 3.

Expression of E7 and VP1 in HeLa cells infected with YYFF and YYFF-HPV16-E7. The HPV16 E7 mRNA was detected by RT-PCR using the specific primer set (A). The E7 protein was detected by Western blot analysis using anti-HPV E7 and anti-VP1 (B), and IFA using anti-HPV E7 (C). Mock indicates uninfected HeLa cells.

Figure 4.

Antitumor therapeutic effects of YYFF-HPV16-E7 in C57/BL6. PBS (○) and YYFF-HPV16-E7 (▪) were injected into C57/BL6 mice that carried TC-1 tumor cells. Each group contained three mice.

Figure 5.

Antitumor therapeutic effects of PBS (○) and YYFF-HPV16-E7 (▪) in BALB/c nude mice. Passive immunization (↓) was conducted three times, with a three day interval. ∗P < 0.05, compared to control. (A) Tumor size. (B) Tumor morphology 15 days after passive immunization.

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