An enhanced site-specific transcription efficiency of DNA/polypeptide-vector polyplexes

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy

Background:

• Gene delivery systems are crucial for therapeutic applications.
• Efficient and targeted plasmid DNA release remains a challenge.
• Intracellular enzymes offer potential triggers for controlled release.

Purpose of the Study:

• To establish a novel gene delivery system utilizing intracellular proprotein convertase (PC), furin, for enhanced plasmid DNA release.
• To synthesize and evaluate polypeptides containing a furin recognition sequence as gene vectors.
• To assess the efficiency of this signal-responsive system in gene transfer.

Main Methods:

• Synthesis of polypeptides with a specific furin cleavage site (Arg-X-Arg/Lys-Arg) using Fmoc solid-phase method.
• Formation and characterization of Fur-polypeptide/plasmid DNA polyplexes.
• In vitro assessment of polyplex disassembly and DNA release upon furin treatment via agarose gel electrophoresis.
• Evaluation of transgene expression in COS-1 cells using Fur-polypeptide vectors compared to controls.

Main Results:

• Polypeptides with the recognition sequence were successfully synthesized and formed polyplexes.
• Furin treatment induced polyplex disassembly and plasmid DNA release in vitro.
• Fur-polypeptide vectors demonstrated significantly higher transgene expression in COS-1 cells compared to control polypeptides.
• The cationic nature of polypeptides alone was insufficient for effective gene delivery.

Conclusions:

• A novel intracellular signal-responsive gene transfer system was successfully established.
• Furin-mediated cleavage of engineered polypeptides enhances plasmid DNA release and subsequent gene expression.
• This strategy offers a promising approach to improve the transcription efficiency of delivered plasmid DNA for gene therapy applications.