Comparative evaluation of survivin, midkine and CXCR4 promoters for transcriptional targeting of glioma gene therapy.
OBJECTIVE
Transcriptional targeting is a key strategy to enhance therapeutic efficacy of gene therapy applications. In the context of oncolytic virotherapy, transcriptional promoter elements are used from genes that are over expressed in a variety of malignant cancers. In the present study, we examined the feasibility of transcriptional targeting to glioma cells by comparing the activity of survivin, midkine, and CXCR4 tumor-specific promoters.


METHODS
To evaluate the expression level of several glioma related genes, we performed quantitative RT-PCR analyses on samples obtained from cell lines and patients. To determine specific level of gene expression mediated by selective promoter elements, we measured luciferase expression in glioma samples transduced with replication deficient adenoviral vectors. Finally, we incorporated the optimal promoters into a conditionally replicative adenoviral vector, CRAd-5/3, and examined the cytopathic effect in vitro.


RESULTS
The survivin promoter demonstrated the highest level of mRNA expression in primary tumor samples and cell lines. Transcriptional targeting was confirmed by infection of glioma cells with an adenovirus expression vector containing a surviving-driven luciferase reporter gene. Of the tested promoters, minimal level of survivin activity was detected in normal human liver and brain. A novel vector, CRAd-survivin5/3, with E1a under the control of the survivin promoter, exhibited enhanced cytopathic effect in vitro.


CONCLUSIONS
Our data demonstrate that the survivin promoter element is very active in glioma samples and has low activity in normal human brain and liver. A novel oncolytic virus, CRAd-survivin-5/3, was effective against a panel of glioma cell lines in vitro. Our results suggest that employing the survivin promoter element in the context of CRAd-5/3 may present a new opportunity for the development of glioma specific oncolytic vectors.
Ulasov
Ilya V
IV
Division of Neurosurgery, The University of Chicago, Chicago, Illinois 60637, USA.
Rivera
Angel A
AA
Sonabend
Adam M
AM
Rivera
Lisa B
LB
Wang
Ming
M
Zhu
Zeng B
ZB
Lesniak
Maciej S
MS
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D045744:Cell Line, Tumor; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005910:Glioma / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics*; D000078792:Midkine; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D014158:Transcription, Genetic / Q000235:genetics*
C107489:BIRC5 protein, human; D016207:Cytokines; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D019718:Receptors, CXCR4; D000077022:Survivin; D000078792:Midkine; D008156:Luciferases