Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

In this work, we intend to develop an improved protocol for the production, purification, and characterization of mosaic adeno-associated viral vectors that could prove their worth in preclinical studies. Despite efforts to establish stable producer cell lines, transient transfection in mammalian cells is still the predominant workflow for AAV production. Then the AAVs are purified by ultra-high speed density gradient centrifugation, or by chromatography techniques that rely on the biochemical properties of viral particles.

The commonly used methods to purify AAVs make use of cesium chloride, or iodixanol density gradient ultracentrifugation. Despite their advantages, they have some limitations. They are time-consuming, they have limited scalability, and they are often giving results to some vectors with low purity.

Now, to overcome these constraints, several researchers have turned their attention to chromatography techniques. We describe a step-by-step protocol for the generation of mosaic rAAVs based on the heparin-binding ability of AAV2. This method renders highly pure and biologically active rAAVs ready to use in six days, presenting itself as a semi-automated, scalable, and cost-effective strategy to generate rAAVs for preclinical studies.

Having demonstrated the applicability of this method for the generation of mosaic rAAVs, the production system can now be fine-tuned to achieve a better scalability and cost effectiveness by establishing a producer cell line. Additionally, we aim to remove empty particles by including a second polishing purification step.