This purification of recombinant adeno-associated virus serotype three (rAAV3), eight (rAAV8) and nine (rAAV9) uses inexpensive, efficient reagents and is easily adaptable to large-scale manufacturing. An AAV is a small nonenveloped virus that packages a linear single-stranded DNA genome. rAAV vectors have become popular for their versatile and successful use as gene therapy delivery vehicles. rAAV3, rAAV8 and rAAV9 particles are often used in pharmaceuticals, vaccines, screening for phenotypes, and understanding the pharmacokinetics of a drug. All of these uses require a highly pure rAAV product. Current processes to purify AAV vectors require the use of buoyant density gradients, such as CsCl, or iso-osmotic medium iodixanol discontinuous gradients. While these are useful laboratory practices, they are not easily adapted for good manufacturing process (GMP) protocols. This purification of rAAV3, rAAV8, rAAV9 and other AAVs, however, is easily adaptable to large scale manufacturing because the reagents used are inexpensive, safe, and work quickly.
Production of small scale rapid purification kits for rAAV3, rAAV8 and rAAV9 as well large-scale production of clinical grade vector.
This versatile, efficient purification of rAAV3, rAAV8 and rAAV9 particles is rapid, cost-effective and more effective than available methods. Researchers selected AAV9, as it is one of the most challenging AAV serotypes to purify. The efficient, reproducible protocol takes advantage of two general biochemical properties of all characterized AAV serotypes: the low isoelectric point of a capsid and the relative biological stability of the viral particle in an acidic environment. Researchers completed a simple and rapid clarification of cell lysate to remove the bulk of proteins and DNA by using off-the-shelf reagents such as sodium citrate and citric acid. Centrifuging the mixture isolates the supernatant, which is subjected to cation exchange to elute the virus. Other spinning or filtration yields material of biochemical purity. This purification method works well, if not better, for purification of AAV serotypes 3 and 8 as well.
