By following this protocol, the user will be able to produce AAV vectors of high purity and yield suitable for in vitro or in vivo applications. This protocol can be used to produce and purify a range of AAV serotypes with differences in configurations. Combining these two elements can be useful to obtain cell type and tissue specific expression.
Production of some serotypes can give to lower yield. This is production where there are close serotypes and should be evaluated on an individual basis. Demonstrating these procedures will be Shelly Fripont, a technician from our laboratory.
After growing the HEK cells, mix 360 micrograms of helper plasmid, 180 micrograms of plasmid encoding the vector capsid, and 180 micrograms of plasmid containing the transgene in 18 milliliters of 150 millimolar sodium chloride to prepare the DNA mix. Distribute this mix over three 50 milliliter conical tubes. In a new conical tube, mix 840 micrograms of PEI and six milliliters of 150 millimolar sodium chloride to prepare the PEI mix for six cell culture dishes.
Then, add six milliliters of the PEI mix, drop by drop, to one of the conical tubes containing the DNA mix. Incubate at room temperature for 20 minutes. Next, remove six cell culture dishes from the incubator.
In a laminar flow hood, completely aspirate the medium from each dish. Rinse the dishes with five milliliters of pre-warmed DPBS to remove traces of the medium. Gently tilt each dish to ensure DPBS is evenly distributed over the entire surface.
Then, gently aspirate the DPBS. Add 12 milliliters of DMEM one to each dish slowly, with a pipette placed at the edge of the dish. Mix the PEI and DNA mixture by pipetting it up and down three to five times.
Add two milliliters of this mixture to each of the six cell culture dishes in a drop by drop fashion, making sure to carefully distribute it over the entire surface. Repeat this process using six cell culture dishes at a time until reaching 18 cell culture dishes. Gently shake the cell culture dishes to distribute the PEI DNA mixture.
Incubate the transfected cells at 37 degrees Celsius with 95 percent humidity and five percent carbon dioxide for five hours. After this, add an additional 12 milliliters of DMEM 10 to each of the 18 dishes without removing the preexisting medium. Continue incubating the transfected cells in the same conditions.
At 72 hours post-transfection, use a cell scraper to carefully detach the cells from each culture dish. Collect the medium and the cells of two culture dishes in a 15 milliliter conical tube, kept on ice. Centrifuge the tubes at 420 times G and at four degrees Celsius for 10 minutes with the acceleration and deceleration of the centrifuge set to maximum.
Carefully discard the supernatant from each tube by gently pouring it into a waste disposal container. Then, place the tubes containing the cell pellets onto ice. We suspend each pellet in two milliliters of lysis buffer and mix by pipetting up and down five to 10 times.
Pull the AAV from three tubes, together. To begin, freeze the re-suspended cells by placing them in a bucket containing dry ice mixed with ethanol. Then thaw the cells by immediately placing them in a water bath set at 37 degrees Celsius.
Repeat this freeze and thaw cycle three times to lyse the cells and release the AAV particles. After the third thawing step, centrifuge at 1167 times G and at four degrees Celsius for 15 minutes. Carefully transfer the supernatants to clean 50 milliliter conical tubes.
Add nuclease to each tube to a final concentration of 50 units per milliliter of supernatant. Incubate at 37 degrees Celsius for 30 minutes, while swirling the tubes every 10 minutes to ensure that the nuclease is thoroughly mixed with the supernatant. Centrifuge at 13490 times G and at four degrees Celsius for 20 minutes to clarify the supernatant.
After this, remove the plunger of a 10 milliliter syringe, attach a 0.45 micrometer filter to the syringe and place it on top of a clean 50 milliliter conical tube. Carefully fill the syringe with the clarified supernatant. Use the plunger to force the lysate through the filter.
Next, prepare each of the iodixanol fractions in four separate 50 milliliter conical tubes, as outlined in table one of the text protocol. Pipette eight milliliters of 15 percent iodixanol into each of the three ultracentrifuge tubes. Pipette 5.5 milliliters of 25 percent iodixanol into a clean 50 milliliter conical tube.
Using a non-graduated Pasteur pipette, carefully layer 5.5 milliliters of 25 percent iodixanol solution below the 15 percent iodixanol solution. Add the 40 percent and 60 percent solutions as described in the text protocol. Iodixanol fractions should not intermix on layering.
Using a Pasteur pipette, layer the crude lysate on top of the 15 percent iodixanol gradient, drop by drop, to avoid disturbing the interface between the crude lysate and the iodixanol solution. Fill each ultracentrifugation tube with lysis buffer until the meniscus reaches the base of the tube neck, to ensure the tube does not collapse under the very high forces generated during ultracentrifugation. Close the tubes using appropriate lids.
Using a digital scale, make sure all three ultracentrifugation tubes have the same weight. Adjusting the weight as necessary, by adding more lysis buffer on top of the crude lysate. Use a fixed angle titanium rotor to centrifuge the tubes at 301580 times G and at 12 degrees Celsius for one hour and 40 minutes using maximum acceleration and deceleration.
Next, attach a needle to a five milliliter syringe. Remove the spacer from the tubes in the rotor, and recover the tubes after centrifugalization. Aspirate only the 40 percent iodixanol fraction which contains the vector particles.
Either process the sample or store it at four degrees Celsius. Here, a protocol is demonstrated that can be used to produce AAV vectors with a variety of capsids, genome configurations, promoter types, and transgene cargos. In this example, we produced and purified two different AAV vectors that expressed the green fluorescent protein from a self complimentary genome.
The two vectors are distinguished by different capsids:PHPB and AAV9. They were delivered, via tail vein injection, into adult C57 black six mice. To evaluate the levels of transgene expression three weeks post-injection, sections are stained with primary antibodies against GFP with detection using secondary antibodies conjugated to a fluorescent dye.
Fluorescence intensity measurements show a significant increase in GFP expression when the PHPB vector is used relative to AAV9. Increases in GFP were observed in the cerebrum, the cerebellum, and in the brainstem. The accurate collection of the vector containing fraction is crucial for this protocol.
In case this is not done properly, the vector yield as well as the vector purity are adversely affected. Being able to produce AAV vectors, small labs will be in a position to take advantage of numerous experimental possibilities to deliver genes in the central nervous system. This protocol requires the use of an ultracentrifuge and it's important to receive proper training before handling this in order to avoid accidents.
Furthermore, AAV vectors are often classified as biohazards and, therefore, you should consult local regulations before handling and managing them.
