A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• Adeno-associated virus (AAV) vectors have limited DNA packaging capacity.
• Co-expression of multiple genes in AAV vectors is challenging.
• Efficient delivery of therapeutic and marker genes is crucial for gene therapy applications.

Purpose of the Study:

• To develop a novel dicistronic AAV vector for efficient co-delivery of genes.
• To utilize a hepatitis C virus (HCV) internal ribosome entry site (IRES) for gene expression.
• To enable packaging of both therapeutic and selectable marker genes within a single AAV vector.

Main Methods:

• Construction of a dicistronic AAV vector containing a 650 bp HCV IRES-bsr cassette.
• Cloning the HCV IRES-bsr construct downstream of a luciferase reporter gene.
• Transfection and transduction of 293 cells with the engineered AAV vector.
• Assessment of luciferase activity and blasticidin S resistance.

Main Results:

• The novel dicistronic AAV vector successfully co-expressed luciferase and blasticidin S-resistance genes.
• Expression was confirmed in 293 cells following both transfection and AAV transduction.
• The vector supports therapeutic gene expression up to 3.6 kb alongside a selectable marker.
• The vector facilitates co-delivery of two therapeutic genes if a marker is not required.

Conclusions:

• A novel HCV IRES-based dicistronic AAV vector enables efficient co-expression of multiple genes.
• This vector design overcomes AAV packaging limitations for gene therapy applications.
• The developed AAV vector is versatile for delivering therapeutic cDNAs and selectable markers.