Efficient episomal gene transfer to human hepatic cells using the pFAR4-S/MAR vector

Area of Science:

• Molecular Biology
• Gene Therapy
• Hepatology

Background:

• Liver-directed gene therapy holds promise for genetic and metabolic liver diseases.
• Current approaches often utilize viral vectors like AAV, which have known benefits and limitations.
• There is a need for alternative, non-viral gene transfer systems for liver applications.

Purpose of the Study:

• To develop and evaluate a novel non-viral episomal gene transfer system for liver-directed gene therapy.
• To investigate the role of the Scaffold/Matrix Attachment Region (S/MAR) element in enhancing gene transfer efficiency.
• To assess the stability and episomal nature of the gene transfer in liver cells.

Main Methods:

• Construction of pFAR4 miniplasmid vectors with and without the S/MAR element, incorporating the eGFP gene under a liver-specific promoter.
• Lipofection of Huh7 liver cells with experimental vectors.
• Evaluation of transfection efficiency and eGFP expression using fluorescent microscopy and flow cytometry.
• Assessment of stable transfection, plasmid persistence, and integration using colony expansion, FISH analysis, and plasmid rescue assays.

Main Results:

• The pFAR4 derivative vector containing the S/MAR element in an upstream configuration (pFAR4-S/MAR-IN) demonstrated significantly higher transfection efficiency and eGFP expression compared to control vectors.
• Stable transfections were achieved only with the pFAR4-S/MAR-IN vector, showing sustained GFP expression for at least 3 months.
• Analysis confirmed the presence of free, circular plasmids and no vector integration events, indicating stable episomal maintenance.

Conclusions:

• The S/MAR element significantly enhances the efficiency of episomal gene transfer using the pFAR4 miniplasmid vector.
• This S/MAR-containing miniplasmid vector is a promising tool for liver-directed gene therapy applications.
• The developed system offers a non-viral alternative for stable, episomal gene delivery to liver cells.