Cancer-specific targeting of an adenovirus-delivered herpes simplex virus thymidine kinase suicide gene using translational control.
Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor-specific gene delivery was optimized by incorporation of the 5'-UTR-modified suicide gene (HSV-UTk) into an adenovirus vector (Ad-CMV-UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF-7, MDA-MB435, and ZR-75-1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify HSV-Tk mRNA for cells infected with Ad-CMV-UTk as well as with Ad-CMV-Tk (a control adenovirus in which HSV-Tk is not regulated at the level of translation). Translation of HSV-Tk in the Ad-infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro-drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell-specific expression of HSV-Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad-CMV-Tk or Ad-CMV-UTk. These data collectively showed enhanced cancer cell-specific gene expression and reduced normal tissue gene expression for the Ad-HSV-UTk compared to the Ad-CMV-Tk, leading to increased cancer cell-enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity.
Mathis
J Michael
JM
Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA. jmathi@lsuhsc.edu
Williams
B Jill
BJ
Sibley
Don A
DA
Carroll
Jennifer L
JL
Li
Jie
J
Odaka
Yoshinobu
Y
Barlow
Shayne
S
Nathan
Cherie-Ann O
CO
Li
Benjamin D L
BD
DeBenedetti
Arrigo
A
D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology; D041722:Genes, Transgenic, Suicide; D005814:Genes, Viral; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics*
D017931:DNA Primers; D039561:Eukaryotic Initiation Factor-4E; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir