J Bacteriol Virol.  2011 Jun;41(2):123-130. 10.4167/jbv.2011.41.2.123.

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

• Shim SH
• Kim YJ
• Kim DS
• Nam JH

• KMID: 2168630
• DOI: http://doi.org/10.4167/jbv.2011.41.2.123

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.

Keyword

Attenuated coxsackievirus B3; YYFF; Gene therapy; Vector system; Cervical cancer

MeSH Terms

Animals
Cell Culture Techniques
Gene Expression
Genes, vif
Genetic Therapy
Human papillomavirus 16
Mice
Mice, Nude
Uterine Cervical Neoplasms

Figure

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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.

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  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.

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  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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