Development of Persistent DNA Vectors for Safe and Lasting Non-Viral Gene Therapies

Overview

Scaffold/MatrixAttachment Regions (S/MARs) are specific regions of DNA that help to stabilize chromatin structure and regulate gene expression by facilitating interactions between DNA and the nuclear matrix. Non-viral plasmid vectors used for gene therapy have severe limitations such as short-term cargo gene expression due to rapid loss of the plasmid DNA in the cell. The objective of this project was to develop a non-viral plasmid vector that is able to persist and replicate at high levels to combat the short gene expression from rapid loss and silencing of plasmid DNA. It was hypothesized that inserting S/MARs into proTLx-K plasmids would cause the GFP and luciferase reporter genes in the plasmid to persist and replicate at a higher level. The project used subcloning to insert an N-DISar2 S/MAR element into a proTLx-K plasmid that contained GFP and luciferase reporter genes. The resulting plasmid was transfected into M17 cells which had GFP and luciferase intensity measured at intervals of every 5 cell passage. The cells transfected with the ​​S/MARs proTLx-K plasmids were compared to a control group of cells transfected with proTLx-K plasmids that didn’t contain any S/MARs elements. From passage 5 to passage 10, the GFP expression from the cell with a S/MAR containing plasmid increased by 104 RLU while the cell without a S/MAR decreased by 4322 RLU. S/MAR elements aided plasmids in tethering to and entering the nucleus, to transcribe and replicate the genes at higher levels.

Competition history

• AJAS 2025 Category not listed

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