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 AV production. Then the AVs 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 AVs 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. To begin, plate HEK 293T cells by adding 22 milliliters of supplemented culture medium in 10 treated culture dishes of 15-centimeter diameter.

Incubate the plates for 24 hours to reach 70%to 80%confluency. For cell transfection, prepare a mixture by combining the reagents in a microcentrifuge tube and mix by tapping. Add the transfection mixture to 4.56 milliliters of non-supplemented DMEM in a 50-milliliter centrifuge tube, and mix by tapping.

Then add 1.37 milliliters of sterile polyethylene amine solution, drop by drop. Mix by tapping and incubate at room temperature for 10 minutes to form DNA-polyethylene amine complexes. Add this mixture into 231 milliliters of pre-warmed supplemented DMEM.

Replace the culture medium in each culture dish with 22 milliliters of this transfection mixture, and incubate the cells for 48 hours. If the PITR encodes a fluorescent reporter, observe the transfected cells under a fluorescence microscope. Next, collect the medium from each dish, centrifuge at 800 G for 10 minutes and remove the supernatant.

Then add 10 milliliters of pre-warmed PBS to the plate and use a cell scraper to remove the cells. Collect the cell suspension into the centrifuge tubes containing the cell pellet. Ensure all cells are collected by washing five dishes at a time with 10 milliliters of PBS.

Centrifuge again to pellet the cells. For rAAV extraction, thaw the transfected cell pellets and resuspend in 100 milliliters of sterile buffer and pipette to ensure a homogenous suspension. Then add 12.5 milliliters of a freshly-prepared, sterile 10%sodium deoxycholate solution to induce cell lysis and mix by pipetting.

Add 27 microliters of Benzonase nuclease and mix thoroughly until the mixture is no longer viscous. Incubate at 37 degrees Celsius, vortexing every 10 minutes for one hour. Centrifuge at 3000 G for 60 minutes at 25 degrees Celsius.

Filter the supernatant using a 0.45-micron PVDF syringe filter into a new sterile container. To begin, prepare the FPLC system for purification. Thoroughly rinse the liquid flow path with sterile, ultra pure water using manual instructions, or a customized method.

Connect a one-milliliter pre-packed heparin column. Adjust the pressure alarm. And wash with five column volumes of ultrapure water at a flow rate of one milliliter per minute.

Then switch the solutions for system pumps to prepare for sample application. Wash the system pump B with buffer B and fill the rest of the liquid flow path with buffer A.Insert the sample tubing of the sample pump into the viral preparation. Alternatively, use a super loop for the sample.

Insert the outlet tubing into a new container. When performing the purification for the first time, define a customized method for automatic purification. After enabling the general purification settings, the method will prime the flow path from sample inlet S1 to the injection valve with the sample solution.

Then equilibrate the column with a total of five column volumes, using 12.5%of buffer B at a rate of one milliliter per minute. Apply the sample to the column at 0.5 milliliters per minute and collect the flow through using the outlet port. Wash the column with 20 column volumes of buffer A at one milliliter per minute.

Then elute the sample at one milliliter per minute with this scheme. Select to collect the eluted sample in one-milliliter fractions using a fraction collector. Then re-equilibrate the column at one milliliter per minute with 12.5%of buffer B for five column volumes.

Open the created method and wait for the elution step. Collect the fractions using a fraction collector. Also collect an aliquot of the flow through and store the fractions at minus 20 degrees Celsius.

Evaluate the automatically-saved chromatogram of all the purification steps, including the elution profile. After completion, switch the inlets from the buffer solutions to ultrapure water and wash the column at one milliliter per minute for five column volumes. Restoring the column, switch the inlets from ultrapure water to 20%ethanol and wash the column for five column volumes.

To concentrate rAAVs, load the desire FPLC fractions into a 15-milliliter centrifugal filter unit with a 100-kilodalton molecular weight cutoff. Centrifuge at 2000 G for two minutes at room temperature to reach a concentrated volume of approximately 500 microliters. Continue with buffer exchange by adding one milliliter of sterile PBS to the centrifugal filter unit.

Wash the filter by pipetting and centrifuge in one-minute intervals until reaching a volume of 500 microliters. Further concentrate the rAAVs by transferring this 500-microliter sample to a 0.5-milliliter centrifugal filter unit with a 100-kilodalton cutoff and centrifuging at 6000 G for one minute intervals until the volume is less than 100 microliters. Recover the concentrated rAAV by inverting the filter device into a new microcentrifuge tube and centrifuging to transfer the rAAVs into the tube.

Aliquot the rAAVs into low-retention microcentrifuge tubes and store at minus 80 degrees Celsius. The purity of rAAV preparations was analyzed using SDS-PAGE, revealing distinct capsid protein bands. Visualization of viral particles by TEM showed empty particles with an electron-dense center and full particles.

Assessment of physical impurity properties of rAAVs using the Stunner platform revealed intact capsid particles around 30 nanometers, overall capsid titer, free and aggregated protein, and single-stranded DNA. Infectivity assays in neuro 2A cells showed high infectivity levels. Primary neuronal cultures infected with the viral preparations demonstrated preserved gene transfer properties.

In vivo transduction of mice cerebellum with rAAV vectors resulted in prolonged GFP expression, indicating successful transgene expression in the mammalian brain.