Optimization of plasmid vectors for high-level expression in lung epithelial cells

Area of Science:

• Molecular Biology
• Gene Therapy
• Biotechnology

Background:

• Nonviral gene therapy utilizes plasmid vectors for transgene expression.
• Optimizing regulatory elements in these vectors is crucial for effective gene delivery.

Purpose of the Study:

• To systematically evaluate regulatory elements within plasmid vectors for enhanced gene expression.
• To identify optimal elements for high-level, consistent transgene expression in vitro and in vivo for gene therapy applications.

Main Methods:

• Construction of a series of plasmids differing in specific regulatory elements (promoter, enhancer, intron, polyadenylation signal).
• In vitro and in vivo assessment of gene expression levels driven by various element combinations.
• Comparative analysis of transient expression profiles in mouse lung models.

Main Results:

• The human cytomegalovirus (CMV) immediate early gene promoter, bovine growth hormone (BGH) or rabbit beta-globin polyadenylation signals, and a hybrid intron significantly enhanced in vitro expression.
• CMV enhancer elements and co-transfection with a chimeric transcription factor further boosted expression levels.
• Selected elements (CMV promoter, hybrid intron, BGH polyadenylation signal) provided high expression in vitro and in mouse lung, but expression was transient in vivo, with >60% loss within 7 days.

Conclusions:

• Optimization of plasmid vector regulatory elements, including the CMV promoter, hybrid intron, and BGH polyadenylation signal, can achieve high-level transgene expression.
• Transient in vivo expression remains a significant challenge for nonviral gene therapy vectors, irrespective of the promoter used.
• These findings provide a foundation for developing improved expression plasmids for lung disease gene therapy.