163. Engineered of a Highly Efficient Cell Cycle Regulatable-HSV-1 Amplicon Viral Vector

Abstract

We have demonstrated in our previous study that when cell cycle specific-regulatory elements are incorporated in cis into a single HSV-1 amplicon plasmid vector backbone (denoted pC8-36), reporter luciferase activities can be regulated in a cell cycle-dependent manner. This was not observed in the construct (pIH8GalLuc) without the cell cycle regulatory elements, which serve as the negative control. The strategy taken is based on a transcriptional repressor protein termed cell-cycle-dependent factor 1 (CDF-1), which is specifically expressed during the G0/G1 phase. In non-dividing or G1 arrested cells, the transactivation of transgene is repressed because of the occupancy of CDF-1 to its binding site (CDE/CHR) located within the minimal cyclin A promoter. Likewise, the absence of CDF-1 repressor protein in dividing cells also mean that transactivation of minimal cyclin A promoter could take place. The degree of cell cycle regulation is measured as a ratio of luciferase activity (expressed as RLU/ug) of the proliferating and G1-arrested cells. We hypothesize that the availability of more binding sites for CDF-1 repressor binding would improve the overall cell cycle regulation mediated by pC8-26 amplicon viral vector. This is supported by preliminary findings that cell cycle-dependent transgene expression can be regulated in a viral dosage dependent manner. To further increase the tightness of regulation, the binding sites (CDE/CHR elements) for CDF-1 located within cyclin A promoter were multimerized. Using overlap extension via polymerase chain reaction, two copies of the CDE/CHR was incorporated within the minimal cyclin A promoter. The recombinant amplicon plasmid was subsequently transfected into human glioma cells, and the cells were kept in standard cell culture medium or induce into G1-arrested state using lovastatin. The degree of cell cycle regulation is measured as a ratio of luciferase activity (expressed as RLU/ug) of the proliferating and G1-arrested cells and our results showed a 7-fold higher cell cycle regulation compared to pC8-36 (p= 0.008). These amplicon plasmids could also be packaged into infectious amplicon virions and will be characterize further with respect to their abilities to confer a better transgene regulation.