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A novel suicide gene therapy system for p53-mutated cells using a wild-type p53-specific promoter and Cre/loxP switch
Masaki Mizumoto1, Shigeki Arii, Masaharu Furutani
1Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Sakyoku, Japan.
Abstract:
Approximately half of all malignant tumors have a mutation or deficiency in the p53 tumor suppressor gene. The present study was designed to develop a p53-mutated cancer cell-specific suicide gene therapy system using p53 as a transcriptional activating factor. We introduced the promoter containing the wild-type p53-specific binding sequence (p53SP) and the Cre/loxP exchange system to induce specific expression of the herpes simplex viral thymidine kinase (HSV-tk) gene in p53-mutated cells. The transfection of an enhanced green fluorescent protein (EGFP) reporter gene expression vector containing p53SP resulted in selective EGFP expression in wild-type p53-producing cells, but not in p53-mutated cells. To express HSV-tk alone in the p53-mutated cells, two plasmid vectors were prepared: one regulator plasmid vector to express Cre under the control of the p53SP promoter (p53Cre), and another tk expression vector driven by the CAG promoter containing loxP sites at both ends of the HSV-tk gene (pCALtkL). The cotransfection of p53Cre with pCALtkL preferentially reduced the expression of HSV-tk in the wild-type p53-producing cells, but not in the p53-mutated cells. This cotransfection led to selective growth inhibition in the cotransfected p53-mutated cells mediated by ganciclovir, whereas a single transfection of pCALtkL caused nonselective growth inhibition in both cell lines following ganciclovir treatment. Thus, the HSV-tk expression system containing the wild-type p53-specific promoter and the Cre/loxP switch endabled the selective growth inhibition of p53-mutated cancer cells.
Insights
This study developed a novel gene therapy system targeting cancer cells with p53 mutations. The system selectively inhibits the growth of p53-mutated cancer cells using the herpes simplex viral thymidine kinase (HSV-tk) gene and ganciclovir.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- TP53 gene mutations are prevalent in approximately 50% of human malignant tumors.
- Developing targeted therapies for p53-mutated cancers is crucial for improving treatment outcomes.
Purpose of the Study:
- To engineer a suicide gene therapy system that specifically targets and eliminates cancer cells with p53 mutations.
- To utilize the p53 tumor suppressor gene as a molecular target for selective cancer cell killing.
Main Methods:
- Constructed a system using a wild-type p53-specific binding sequence (p53SP) promoter to control Cre recombinase expression.
- Developed a herpes simplex viral thymidine kinase (HSV-tk) gene expression vector with flanking loxP sites (pCALtkL).
- Co-transfected p53-mutated and wild-type p53 cancer cells with p53Cre and pCALtkL vectors, followed by ganciclovir treatment.
Main Results:
- The p53SP promoter selectively directed Cre expression in wild-type p53 cells, leading to HSV-tk gene deletion.
- Co-transfection resulted in preferential HSV-tk expression and ganciclovir-mediated cell death specifically in p53-mutated cancer cells.
- Single transfection of pCALtkL caused non-selective growth inhibition, highlighting the specificity of the co-transfection system.
Conclusions:
- The developed p53SP-Cre/loxP-HSV-tk system enables selective growth inhibition of p53-mutated cancer cells.
- This targeted approach offers a promising strategy for p53-mutated cancer therapy.
- The system leverages p53 status for precise therapeutic intervention, minimizing off-target effects.