Engineering an improved cell cycle-regulatable herpes simplex virus type 1 amplicon vector with enhanced transgene expression in proliferating cells yet attenuated activities in resting cells.
We previously generated a herpes simplex virus type 1 (HSV-1)-based amplicon vector (denoted pC8-36) in which gene expression from the minimal cyclin A promoter is repressed by preventing the binding of a trans-activating protein, Gal4-NF-YA, to it through selective interaction with the transcriptional repressor protein CDF-1. Because CDF-1 is absent in actively dividing cells, transgene expression conferred by the pC8-36 vector is therefore cell cycle dependent. As gene therapy evolves to become a promising therapeutic modality for many human diseases, there is an increasing need to further improve the kinetics of gene regulation. In the present study, we examined whether the availability of more binding sites for CDF-1 repressor proteins could enhance transgene expression. Using an overlap extension polymerase chain reaction (PCR) method, the CDE and CHR elements within the minimum cyclin A promoter were multimerized to contain two, three, and six copies of the designated CDE/CHR sequence. Interestingly, our results demonstrated that six-copy CDE/CHR sequence motifs (pC8-6CC-Luc) conferred an approximately 20-fold increase in the ratio of cell cycle regulation compared with the previous reported construct. Further, the overall transcriptional activities mediated by pC8-6CC-Luc were stronger compared with the native human survivin promoter, which consists of three copies of the CDE element and one copy of the CHR element. pC8-6CC-Luc contained, in essence, only the synthetic six-copy CDE/CHR sequence motif (about 262 bp). In comparison with other native endogenous promoters, which usually contain many other transcription binding sites, pC8-6CC-Luc amplicon vectors should confer better regulated and consistent transgene expression and may be considered a gene delivery vector of choice to target actively proliferating tumor cells.
Wang
Grace Y
GY
Gene Vector Laboratory, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore 169610.
Ho
Ivy A W
IA
Sia
Kian C
KC
Miao
L
L
Hui
Kam M
KM
Lam
Paula Y P
PY
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019925:Cyclin A / Q000235:genetics; D005260:Female; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008869:Microtubule-Associated Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics; D009369:Neoplasms / Q000628:therapy*; D012097:Repressor Proteins / Q000378:metabolism; D000077022:Survivin; D020080:Tandem Repeat Sequences; D019076:Transgenes / Q000235:genetics; D014771:Virion
C107489:BIRC5 protein, human; D019925:Cyclin A; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D012097:Repressor Proteins; D000077022:Survivin; D008156:Luciferases