A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors...

Area of Science:

• Biotechnology
• Gene Therapy
• Molecular Biology

Background:

• Adeno-associated virus (AAV) vectors are crucial for preclinical gene therapy research.
• Producing high-yield, high-purity AAV vectors is essential for in vivo studies.
• Traditional HEK293T cell culture methods are labor-intensive and inefficient.

Purpose of the Study:

• To present a simplified and efficient in-house method for AAV vector production.
• To improve AAV vector yield and purity for research applications.
• To establish a robust cell factory platform for scalable AAV production.

Main Methods:

• Utilized a cell factory (cell stacks, CF10) platform for HEK293T cell cultivation.
• Integrated polyethylene glycol/aqueous two-phase partitioning for initial purification.
• Employed iodixanol gradient ultracentrifugation for enhanced AAV vector purification.
• Verified purity using SDS-PAGE and silver staining.
• Determined full to empty particle ratio via transmission electron microscopy (TEM).

Main Results:

• The cell factory platform significantly simplified the AAV production process.
• The integrated purification method markedly improved AAV vector yield and purity.
• SDS-PAGE, silver staining, and TEM confirmed high purity and quality of the produced AAV vectors.

Conclusions:

• The developed cell factory approach offers an efficient method for producing high-yield AAV vectors.
• The enhanced purification strategy meets the stringent quality requirements for in vivo gene therapy studies.
• This method provides a scalable and reliable platform for preclinical AAV vector production.