Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids

Area of Science:

• Gene Therapy
• Virology
• Molecular Biology

Background:

• Adeno-associated virus (AAV) vectors are crucial for gene therapy, but current production methods are difficult to scale.
• Plasmid transfection methods limit the efficiency and scalability of recombinant AAV (rAAV) production.

Purpose of the Study:

• To develop a simpler, more efficient, and scalable method for producing recombinant AAV (rAAV) vectors.
• To utilize recombinant herpes simplex virus (HSV) vectors for improved rAAV production, bypassing traditional transfection methods.

Main Methods:

• Constructed disabled and nondisabled recombinant HSV vectors encoding AAV rep and cap genes.
• Integrated an rAAVGFP construct into HSV vectors, with and without rep/cap expression.
• Optimized double-infection ratios for HSV vector pairs to maximize rAAV production.

Main Results:

• Achieved high yields of rAAV particles (up to 1.55 x 10^12) and expression units (4 x 10^8) from BHK cells.
• Confirmed transgene incorporation and expression via Southern and Western blots.
• Demonstrated vector functionality through successful green fluorescent protein (GFP) expression in rat brain injections.

Conclusions:

• Developed a simple, efficient, transfection-free rAAV production process using HSV vectors.
• The method does not require a proviral cell line, offering a scalable alternative for clinical-grade rAAV production.