Highly efficient multicistronic lentiviral vectors with peptide 2A sequences.

Area of Science:

• Molecular biology
• Gene therapy
• Biotechnology

Background:

• Gene discovery and therapy require reliable gene expression assessment.
• Efficient coupling of transgene expression to reporter genes is crucial for monitoring.
• Noninvasive molecular imaging tracks biological processes in animal models.

Purpose of the Study:

• To evaluate strategies for linking transgene expression to reporter gene expression.
• To compare the efficiency of internal ribosomal entry sites (IRES), 2A-like peptides, and bidirectional promoters for co-expression.
• To develop a multimodality reporter system for in vivo applications.

Main Methods:

• Lentiviral vectors were engineered with IRES, 2A-like peptides, or bidirectional promoters.
• Co-expression efficiency of multiple reporter genes was assessed in vitro and in vivo.
• A triple reporter lentiviral vector (LV-3R) was constructed using peptide 2A sequences.

Main Results:

• Encephalomyocarditis virus (EMCV) IRES enabled bicistronic vectors but with lower downstream reporter expression.
• Peptide 2A constructs demonstrated superior performance in vitro and in vivo.
• Reporter gene expression levels from peptide 2A constructs matched single reporter levels.
• LV-3R facilitated efficient multimodality readouts in vivo.

Conclusions:

• Peptide 2A sequences are highly effective for achieving proportional co-expression of multiple proteins.
• This approach enables noninvasive monitoring of transgene expression.
• The developed triple reporter system supports multimodality imaging for functional genomics and gene therapy.