Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 10¹² viral particles/mL and are suitable for pre-clinical in vivo use.

We use a directed evolution approach to identify adeno-associated viral capsids with higher tropism for target human cell types. Through this research, we aim to produce recombinant adeno-associated viral vectors with improved target cell transduction and clinical relevance within the field of gene therapy. Our group recently detargeted several AAV serotypes from the liver by incorporating capsid residues original to an AAV serotype that naturally possesses decreased liver tropism.

If successfully expanded to additional serotypes, this provides the groundwork to decrease necessary rAAV clinical dose by peripheral injection and reduce adverse patient outcomes. The iodixanol purification method, while being cost-effective, is difficult to scale up. Additionally, in downstream applications, the directed evolution approach is performed with animal models or in vitro.

So capsids isolated by directed evolution may not transduce in vivo targets in humans to the same extent as in preclinical models. This protocol is affordable and accessible for smaller labs. Additionally, this method yields high purity recombinant adeno-associated virus that can be used for downstream preclinical in vivo applications.

To begin, obtain clarified virus from cultured cells. Place the ultracentrifuge tubes in a rack on the output side of the multichannel peristaltic pump. Using a serological pipette, carefully dispensed 10 milliliters of clarified virus into each ultracentrifuge tube.

Then transfer 22 milliliters of the 15%iodixanol fraction into a clean 50 milliliter conical tube. Place the capillaries on the right side of the pump into the tube and start the pump. Stop the pump when the iodixanol fraction reaches the end of the capillaries on the output side of the pump.

Insert an output capillary into each ultracentrifuge tube with clarified virus and start the pump. Stop the pump when the last of the 15%fraction is about to be taken into the input capillaries. Next, transfer 22 milliliters of the 25%iodixanol fraction into the 50 milliliter conical tube.

After turning the pump on, let the entire fraction be taken into the input capillaries and stop the pump. If required, add more of the 60%fraction and fill each tube until the lysate makes a dome over the top but does not overflow. Stop the pump and carefully remove the output capillary.

Cap the ultracentrifuge tube with a spacer and load it into a type 70 Ti rotor. After centrifugation, clamp the ultracentrifuge tube to a support stand. Using a lint-free wipe, remove the cap from the ultracentrifuge tube.

Then attach a 20 gauge needle to a five milliliter syringe and penetrate the wall of the ultracentrifuge tube with the needle about three milliliters below the 40 and 60%iodixanol interface. Slowly aspirate the interface and part of the 40%fraction. Holding one finger over the open top of the ultracentrifuge tube, pull out the syringe and transfer the aspirated AAV fraction to a 50 milliliter conical tube.

Finally, dilute the aspirated virus fraction twofold in AAV lysis buffer to a volume of 40 milliliters. After flushing the peristaltic tubing, load 20 milliliters of the diluted AAV fraction into a new ultracentrifuge tube for the second round of fractionation.