Sustained, high transgene expression in liver with plasmid vectors using optimized promoter-enhancer combinations

Area of Science:

• Molecular Biology
• Gene Therapy
• Biotechnology

Background:

• Plasmid-based gene therapy often suffers from short-term transgene expression due to vector silencing or loss.
• Nonsilenced promoters combined with strong enhancers can enhance long-term gene expression.

Purpose of the Study:

• To develop a novel gene therapy vector with improved long-term transgene expression.
• To evaluate the efficacy of a synthetic promoter combining CMV and EF1α elements.

Main Methods:

• Constructed plasmids with murine or human cytomegalovirus (CMV)-derived enhancers and the human elongation factor 1α (EF1α) promoter.
• Delivered plasmids hydrodynamically into mouse liver and monitored luciferase transgene activity.
• Developed a synthetic CMV-EF1α hybrid promoter (SCEP) for enhanced expression.

Main Results:

• CMV-promoter driven plasmids showed rapid decline in luciferase activity.
• EF1α-driven plasmids maintained high activity for 2 weeks (murine enhancer) and over 80 days (human enhancer).
• The novel SCEP demonstrated a three-fold increase in constitutive expression compared to EF1α alone, correlating with higher plasmid copy numbers.

Conclusions:

• Optimized plasmid backbones with novel enhancer-promoter combinations offer more efficient nonviral gene therapy.
• The SCEP represents a promising tool for achieving sustained transgene expression in gene therapy applications.