title: Fusion of the HSV-1 tegument protein vp22 to cytosine deaminase confers enhanced bystander effect and increased therapeutic benefit.
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, (19)F 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.
authors.affiliation: Department of Biological Chemistry, The University of Michigan Medical Center, Ann Arbor, 48109, USA.
authors.forename: K C
authors.initials: KC
authors.lastname: Lee
authors.forename: D A
authors.initials: DA
authors.lastname: Hamstra
authors.forename: S
authors.initials: S
authors.lastname: Bullarayasamudram
authors.forename: M S
authors.initials: MS
authors.lastname: Bhojani
authors.forename: B A
authors.initials: BA
authors.lastname: Moffat
authors.forename: K J
authors.initials: KJ
authors.lastname: Dornfeld
authors.forename: B D
authors.initials: BD
authors.lastname: Ross
authors.forename: A
authors.initials: A
authors.lastname: Rehemtulla
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use*; D024201:Bystander Effect; D002460:Cell Line; D043525:Cytosine Deaminase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005437:Flucytosine / Q000627:therapeutic use; D005472:Fluorouracil / Q000627:therapeutic use*; D050939:Gene Fusion; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051379:Mice; D008819:Mice, Nude; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods; D014178:Translocation, Genetic; D015678:Viral Structural Proteins / Q000235:genetics*
chemical_list: D000964:Antimetabolites, Antineoplastic; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D005437:Flucytosine; D043525:Cytosine Deaminase; D005472:Fluorouracil