Construction of pVAX-1-based linear covalently closed vector with improved transgene expression

Area of Science:

• Molecular Biology
• Gene Therapy
• Biotechnology

Background:

• Linear covalently closed (LCC) vectors offer potential advantages over traditional circular plasmids for gene expression.
• Optimizing vector design is crucial for enhancing transfection efficiency and transgene expression in mammalian systems.
• Evaluating in vivo performance and associated immune responses is essential for therapeutic vector development.

Purpose of the Study:

• To develop and characterize a Mammalian Linear Expression System (MLES) based on the pVAX-1 vector.
• To assess the efficacy of MLES for transient transfection and transgene expression in vitro and in vivo.
• To evaluate the inflammatory potential of MLES compared to its parental vector.

Main Methods:

• MLES was constructed by modifying pVAX-1 and its integrity confirmed via gel electrophoresis.
• Transfection efficiency and gene expression were evaluated in HEK-293, CHO-K1, and NIH-3T3 cell lines using Lipofectamine®2000.
• In vivo studies in mice involved injecting MLES/EGFP to analyze transfection, expression, and inflammatory markers.

Main Results:

• MLES demonstrated superior transfection efficiency and expression levels in cell lines compared to pVAX-1.
• In vivo, MLES/EGFP resulted in higher gene expression in the heart, kidney, liver, and spleen.
• MLES induced a significantly reduced inflammatory response in mice relative to pVAX-1.

Conclusions:

• MLES provides enhanced gene expression and reduced inflammation, marking a significant advancement for gene therapy applications.
• The improved vector performance suggests potential for increased recombinant protein production.
• Further investigation into MLES-mediated gene expression and immunomodulation can refine gene therapy strategies.