Plasmid DNA size does affect nonviral gene delivery efficiency in stem cells.

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Genetic modification of stem cells enhances their therapeutic potential in cell therapy.
• Mesenchymal stem cells (MSC) and neural stem cells (NSC) are promising candidates for cell-based gene therapy.
• Nonviral gene delivery methods often face low efficiencies, particularly in stem cells.

Purpose of the Study:

• To investigate the impact of plasmid size on gene delivery efficiency in human MSC and mouse NSC.
• To evaluate the role of minimized plasmids and polyA signals in stem cell gene transfer.
• To understand how plasmid characteristics influence nuclear uptake and gene expression in stem cells.

Main Methods:

• Utilized minimized plasmids encoding a fluorescent protein, lacking antibiotic resistance genes.
• Compared gene delivery efficiency using plasmids of varying sizes in human MSC and mouse NSC.
• Analyzed intracellular plasmid copy number, cell fluorescence, and transgene mRNA levels.

Main Results:

• Smaller plasmids resulted in up to 2.6-fold higher intracellular plasmid copy number.
• The number of fluorescent cells was twice as high with smaller plasmids compared to larger ones.
• Differences in plasmid efficiency were largely dependent on transgene mRNA levels, influenced by polyA signals.

Conclusions:

• Plasmid size is a critical factor affecting gene delivery efficiency into stem cells.
• Smaller plasmids enhance nuclear uptake and gene expression in MSC and NSC.
• Plasmid size may also influence heterochromatin-associated gene silencing in stem cells, impacting therapeutic outcomes.