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Insulinoma-induced hypoglycemic death in mice is prevented with beta cell-specific gene therapy
T A Tirone1, S P Fagan, N S Templeton
1Departments of Molecular Biology and Center for Gene Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA.
Objective And Summary Background Data:
Tumor-specific gene therapy can be achieved if a tumor-specific promoter can be identified. In this study the authors investigated the use of the rat insulin promoter (RIP) for insulinoma-specific expression of a reporter gene. Insulinoma-specific cytotoxicity using the suicide gene thymidine kinase (tk) was studied both in vitro and in vivo. RIPtk gene therapy, delivered by a nontoxic, noninflammatory liposomal delivery system, was used in an insulinoma ICR/SCID mouse model to prevent hypoglycemic death.
Methods:
Rat insulin promoter (0.502 kb) was ligated to the reporter gene lacZ and ligated to the tk gene. These two genes were transfected into a mouse insulinoma (NIT) cell line to ascertain insulinoma-specific expression and insulinoma-specific cytotoxicity in vitro. Reverse transcriptase-polymerase chain reaction and electrophoretic mobility-shift assays were performed on NIT-1 cell RNA and nuclear extract, respectively, to determine the transcription factors present and responsible for RIP activation in NIT-1 cells. A mouse insulinoma model was created with NIT-1 cells. These mice were treated with the RIPtk gene, and both blood sugars and animal viability were monitored.
Results:
Only NIT-1 cells stained blue after X-gal staining or had detectable levels of beta-galactosidase protein. A significant decrease in cell survival was observed in NIT-1 cells transfected with RIPtk in vitro. Messenger RNA for both BETA2 and PDX-1 was found in NIT-1 cells, and a supershift was observed for both BETA2 and PDX-1. Experimental mice treated with the RIPtk gene, delivered by a liposomal gene delivery system, maintained their blood glucose levels, and the animals did not die of hypoglycemia.
Conclusions:
The data suggest that the RIP is an insulinoma-specific promoter. An ICR/SCID mouse insulinoma model was used to show that insulinoma-specific cytotoxicity can be accomplished by RIP coupled to a suicide gene in vivo, preventing hypoglycemic death.
Insights
The rat insulin promoter (RIP) enables targeted gene therapy for insulinoma. This study demonstrated RIP-thymidine kinase (tk) gene therapy effectively reduced insulinoma cell survival and prevented hypoglycemic death in mice.
Area of Science:
- Oncology
- Gene Therapy
- Molecular Biology
Background:
- Identifying tumor-specific promoters is crucial for targeted gene therapy.
- The rat insulin promoter (RIP) was investigated for its potential in insulinoma-specific gene expression.
Purpose of the Study:
- To evaluate the RIP's specificity for insulinoma.
- To assess the efficacy of RIP-thymidine kinase (tk) gene therapy in vitro and in vivo.
- To prevent hypoglycemic death in an insulinoma mouse model.
Main Methods:
- The RIP was ligated to lacZ and tk reporter genes.
- Genes were transfected into a mouse insulinoma cell line (NIT-1) for in vitro analysis.
- Reverse transcriptase-polymerase chain reaction and electrophoretic mobility-shift assays identified transcription factors.
- An insulinoma mouse model was treated with RIPtk delivered via a liposomal system.
Main Results:
- NIT-1 cells demonstrated insulinoma-specific expression of reporter genes.
- RIPtk transfection significantly reduced NIT-1 cell survival in vitro.
- Transcription factors BETA2 and PDX-1 were identified in NIT-1 cells.
- Treated mice maintained blood glucose levels and survived hypoglycemic events.
Conclusions:
- The RIP functions as an insulinoma-specific promoter.
- RIP-coupled suicide gene therapy achieves insulinoma-specific cytotoxicity in vivo.
- Liposomal delivery of RIPtk gene therapy prevents fatal hypoglycemia in an insulinoma model.