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Fusion of the HSV-1 tegument protein vp22 to cytosine deaminase confers enhanced bystander effect and increased therapeutic benefit

Gene Therapy volume 13, pages 127–137 (2006)Cite this article

Abstract

A major limitation in cancer gene therapy, specifically gene-dependent enzyme prodrug therapy (GDEPT), is inefficient gene delivery and expression. The suicide gene cytosine deaminase (CD) and its substrate, 5-fluorocytosine (5-FC), have been extensively explored due to the inherent ‘bystander’ effect achieved through diffusion of the toxic metabolite 5-fluorouracil (5-FU). In this study, we aimed to enhance this ‘bystander’ effect by fusing the Saccharomyces cerevisiae CD to the HSV-1 tegument protein vp22, a novel translocating protein. Two constructs were created: one with vp22 fused to CD (vp22CD) and a second wherein a truncated vp22, lacking the necessary residues for trafficking, fused to CD (delvp22CD). The generated 9L stable lines exhibited similar growth rates, enzyme expression, CD activity, and sensitivity to 5-FC and 5-FU. However, mixed population colony formation assays demonstrated greater bystander effect with the vp22CD fusion as compared to delvp22CD. This enhancement was maintained in vivo where 9L tumors expressing 20 or 50% vp22CD exhibited increased growth delay compared to the respective delvp22CD tumors. Moreover, adenoviral transduction of established wild-type 9L tumors showed increased growth delay with vp22CD (Ad-EF_vp22CD) as compared to equivalent CD (Ad-EF_CD) transduced tumors. Finally, confirming the increased efficacy, 19F magnetic resonance spectroscopy (MRS) of vp22CD-expressing tumors demonstrated increased 5-FU levels as compared to tumors expressing the nontranslocating CD. These results together demonstrated that fusion of vp22 to CD resulted in CD translocation, which in turn amplified conversion of 5-FC to 5-FU in vivo and enhanced the therapeutic benefit of this GDEPT strategy.

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Acknowledgements

Financial support for this work was provided by NIH Grants P01CA85878, P50CA01014, and R24CA83099, The University of Michigan SPORE in Head and Neck Cancer.

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Authors and Affiliations

  1. Department of Biological Chemistry, The University of Michigan Medical Center, Ann Arbor, MI, USA

    K C Lee & B D Ross

  2. Department of Radiation Oncology, The University of Michigan Medical Center, Ann Arbor, MI, USA

    D A Hamstra, S Bullarayasamudram, M S Bhojani, K J Dornfeld & A Rehemtulla

  3. Department of Radiology, The University of Michigan Medical Center, Ann Arbor, MI, USA

    B A Moffat, B D Ross & A Rehemtulla

  4. The Center for Molecular Imaging, The University of Michigan Medical Center, Ann Arbor, MI, USA

    B A Moffat, B D Ross & A Rehemtulla

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  1. K C Lee
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Correspondence to A Rehemtulla.

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Lee, K., Hamstra, D., Bullarayasamudram, S. et al. Fusion of the HSV-1 tegument protein vp22 to cytosine deaminase confers enhanced bystander effect and increased therapeutic benefit. Gene Ther 13, 127–137 (2006). https://doi.org/10.1038/sj.gt.3302631

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