Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

This protocol for production of AAV viral vectors is cost-effective and yields high purity, high titer, research-grade AAV for use in preclinical, large animal models. This technique uses adherent HEK293 cells in cell culture chambers, which is time efficient and less hands-on, allowing for fewer disruptions to the cells. This protocol can greatly aid in viral vector production for the field of gene therapy, and can also be used for the production of other AAV serotypes that also bind heparan sulfate.

To begin, collect HEK293 cells from the culture when it reaches 80%confluency. Aspirate the media before gently washing the cell culture plate with three milliliters of PBS. Then aspirate PBS and add three milliliters of trypsin.

Incubate the plates for two minutes at 37 degrees Celsius. After incubation, neutralize trypsin, by adding seven milliliters of complete DMEM to the plate, and collect the supernatant in 50 milliliter conical tubes. Gently invert the tubes to ensure the cells are homogenous.

To determine the cell density, mix 10 microliters of the cell samples with 10 microliters of trypan blue. And add the mixture to a cell counting slide to analyze in the cell counter. Mix the cell suspension with one liter of pre warmed complete DMEM to see the culture chamber.

Gently rotate the chamber to distribute the cells evenly. Incubate the chamber at 37 degrees Celsius with 5%carbon dioxide. In addition to the cell culture chamber, culture cells at 10 to the fourth cells per centimeter squared in a 15 centimeter plate as a reference for confluency.

After 65 hours of incubation, when the cells are 80 to 90%confluent, slowly add the polyethyleneimine-DNA and DMEM mixture into the cell culture chamber port. Distribute the liquid evenly to all the rows before incubating at 37 degrees Celsius, and 5%carbon dioxide for 72 hours. After incubation, shake the cell culture chamber vigorously to dislodge the cells until the media appears cloudy, and pour it into four or 500 milliliter centrifuge tubes.

Pellet the cells by centrifugation at 18, 000 times G for 30 minutes at four degrees Celsius. Pour the clarified supernatant into a one-liter PETG bottle, and resuspend the cell pellets in 50 milliliters of licensed buffer in 500 milliliter centrifuge tubes. Incubate the tubes for 60 minutes at 37 degrees Celsius.

After incubation, centrifuge the tubes at 18, 000 times G for 30 minutes, and transfer the supernatant into the same one-liter PETG bottle. Store at minus 80 degrees Celsius. Thaw the crude lysate at four degrees Celsius overnight.

Once thawed, filter it using a 0.22 micron filter. Immediately before the purification steps, passivate the centrifugal concentrator by adding four milliliters of filter pretreatment buffer for each heparin-Sepharose column being used. Place the tubing in a peristaltic pump.

Sequentially run the solutions and media. Prepare for chromatography by attaching a five milliliter heparin-Sepharose column to the tubing, and running 25 milliliters of Bazell DMEM through the column. Then load 0.2 micromolar of the filtered crude lysate onto the column at a flow rate of one to two drops per second.

Wash the column sequentially to elute five milliliters five times with 300 millimolar of sodium chloride HBSS, with magnesium and calcium, and label the five elutions as E1 to E5.When ready, spin the centrifugal concentrator containing the pretreatment buffer at 900 times G for two minutes. Discard the flow-through. Wash the centrifugal concentrator filter with four milliliters of HBSS with magnesium and calcium by spinning at 1000 times G for two minutes.

Then add the elution E2 to the centrifugal concentrator, and spin at 1000 times G for five minutes. Discard the flow-through. Similarly spin the elution E3 into the centrifugal concentrator until the concentrated virus is approximately one milliliter.

Remove the concentrated virus from the centrifugal concentrator using a P200 filtered tip, and collect it into a sterile 1.5 milliliter centrifuge tube. After collection of the virus, rinse the centrifugal concentrator with 200 microliters of HBSS with magnesium and calcium. Pipette up and down vigorously for 30 seconds to dislodge any virus adhered to the membrane, and collect the concentrated virus in the same 1.5 milliliter centrifuge tube.

Mix the tube well. Wash the column and saturate with 20%ethanol before storing at four degrees Celsius. Store the pump tubing in one molar sodium hydroxide.

The transduction efficiency of the AAV6.2FF capsid was compared in mice, hamsters, and lambs for 28 days upon intramuscular administration. Mice administered five times 10 to the 12th vector genomes per kilogram of AAV6.2FF human IgG, expressed between 171 to 237 micrograms per milliliter of human IgG in the serum. Hamster is administered at two times 10 to the 13th vector genomes per kilogram of AAV6.2FF human IgG, expressed much higher levels of human IgG than the mice.

While the large animal model was able to express an average 35 micrograms per milliliter of human IgG levels in the plasma. Large animal models can determine the expression of the AAV transgene in vivo, and determine if the transgenes have therapeutic effect against the disorder or disease of interest. The study has showcased the great transduction ability of AAV6.2FF, a novel AAV 6 serotype vector in the skeletal muscle, as well as the low immunogenicity of AAV viral vectors in vivo.