Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Summary

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer (≥1 x 10¹³ vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Abstract

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Introduction

Over the past 30 years, wild-type AAVs have been engineered to create recombinant AAV vectors, which have proven to be exceptionally useful tools for gene transfer into the CNS^1,2,3,4,5,6 and gene therapy approaches to disease (including FDA- and EMA-approved therapies)^4,7. Their suitability for use in the CNS largely derives from their ability to infect non-dividing, post-mitotic cells, typically found in the CNS⁸. However, AAV-based vectors also have the advantage of allowing a long-term expression of any given therapeutic transgene^4,9 while eliciting a milder immune response compared to other viral vectors^7,8,10,11,12.

The main elements of any AAV vector are the genome and the capsid. Wild-type AAVs are single-stranded (ss) DNA viruses with a genome of approximately 5 kilobases (kb)¹³. For the production of recombinant AAV vectors, the rep and cap genes (necessary for genome replication and the assembly of the viral capsid) are deleted from the genome of the wild-type AAV and provided in trans, leaving room for an expression cassette containing the transgene^14,15. The inverted terminal repeat sequences (ITRs) of the original viral genome are the only retained elements in an AAV vector, as these are essential for replication and packaging^3,10,14. AAV vectors can be engineered to enhance transgene expression; a mutation in one of the ITRs leads to the formation of a hairpin loop which effectively allows the generation of a complementary DNA strand^3,7,15. The main advantage of this configuration, termed a self-complementary (sc) genome, is that it bypasses the need for second-strand synthesis typical in the conventional life cycle of AAVs, considerably increasing the speed and levels of transgene expression¹. Nevertheless, using an scAAV genome reduces the cargo capacity of the vector to approximately 2.4 kb. This includes transgene sequences, as well as any regulatory sequences such as promoters or microRNA-binding sites, to limit expression to specific cell types¹⁶.

The AAV capsid determines the vector-host cell interaction and confers a degree of cell type or tissue tropism for an AAV serotype, which can also be exploited to limit transgene expression to specific locations. Several AAV serotypes are found in nature, whereas others have been produced in laboratories through recombinant approaches (i.e., PHP.B). In addition, some capsids also bestow other useful characteristics, such as the ability to cross the BBB, resulting in the delivery of transgenes throughout the CNS after systemic administration. This has been shown for AAV9, as well as for the recently described PHP.B capsid¹⁷. As a consequence, these serotypes are proving to be particularly relevant for new gene therapy approaches for neurodegenerative disorders^1,17,18.

The aim of this protocol is to describe a cost-effective method for the small-scale production of AAV vectors with high titer and purity. Although the results presented here use the PHP.B capsid and a scAAV expression cassette, the protocol is suitable for the production of several AAV vector serotypes and genome configurations, allowing maximum experimental flexibility. However, the vector yield and final purity can vary depending on the chosen serotype.

The protocol itself is a variant of the classical tri-transfection method for viral vector production and incorporates the use of an iodixanol gradient for vector clean-up, which, in comparison to the classical use of cesium chloride (CsCl) gradients, has been reported to produce AAV vectors of higher purity in a more time-efficient manner^19,20,21.

The transfection, purification and concentration steps are intended to be performed according to good laboratory practice (GLP) in a tissue culture laboratory rated for viral vector work. Each task needs to be performed in compliance with the relevant local and national legislation concerning viral vector production and use. Work must be carried out under a laminar flow hood and in sterile conditions. Inside the vector facility, it is recommended to wear a lab apron, in addition to the regular tissue culture lab coat. Moreover, a double pair of gloves, as well as plastic overshoes, should be worn at all times.

Before starting the vector production, ensure all necessary equipment and plasmids are available. 1) pCapsid plasmid contains the rep gene that encodes four non-structural proteins necessary for replication, namely Rep78, Rep68, Rep52, and Rep40, and the cap gene that encodes three structural capsid proteins, namely VP1, VP2, and VP3. 2) pHelper plasmid contains the genes E4, E2A, and VA from adenovirus, which facilitate AAV production in HEK293T cells. 3) pTransgene plasmid contains the transgene expression cassette, flanked by two ITRs. These plasmids can be synthesized de novo in the laboratory using sequences available online²². For plasmids made de novo, especially those containing novel transgenes, sequencing is required, to make sure that the transgene and ITRs are correct. Alternatively, premade plasmids can be directly acquired through online plasmid repositories. When necessary, plasmids can be amplified and purified using standard kits, according to the manufacturer's instructions²³.

Vector titer and purity can adversely affect the transduction ability of the vector. Additional protocols are supplied to evaluate the quality of the produced vector. The final vectors will be useful for studies of the CNS cell function in both in vitro and in vivo applications.

Protocol

Solution	Composition
Cell culture and transfection
DMEM1	DMEM 1x
	1% FBS (v/v)
	1% GlutaMAX (v/v)
DMEM10	DMEM 1x
	10% FBS (v/v)
	1% GlutaMAX (v/v)
150 mM NaCl	4.380 g NaCl
	Up to 500 mL Ultrapure water
AAV purification and desalting
5 M NaCl	146.1 g NaCl
	Up to 500 mL Ultrapure water
1 M Tris HCl (pH 8.5)	12.11 g Tris base	CAUTION
	Up to 100 mL Ultrapure water
	Add 1 M HCl using a Pasteur pipette to reduce the pH to 8.5
Lysis buffer	15 mL of 5 M NaCl
	25 mL of 1 M Tris HCl (pH 8.5)	CAUTION
	Up to 500 mL Ultrapure water
10x Phosphate-buffered saline (PBS)	80 g NaCl
	2 g KCl	CAUTION
	14.4 g Na2HPO4
	2.4 g KH2PO4
	Up to 1 L ddwater
1 M MgCl2	20.33 g MgCl2-6H2O
	Up to 100 mL Ultrapure water
1 M KCl	7.45 g KCl	CAUTION
	Up to 100 mL Ultrapure water
5x PBS Magnesium-Potassium (PBS-MK) stock solution	250 mL of 10x PBS
	2.5 mL of 1 M MgCl2
	6.25 mL of 1 M KCl	CAUTION
	Up to 500 mL Ultrapure water H2O
15% Iodixanol	12.5 mL of Optiprep density gradient medium	CAUTION
	10 mL of 5 M NaCl
	10 mL of 5x PBS-MK
	17.5 mL of Ultrapure water
25% Iodixanol	20.8 mL of Optiprep density gradient medium	CAUTION
	10 mL of 5x PBS-MK
	19.2 mL of Ultrapure water
	100 µL of phenol red
40% Iodixanol	33.3 mL of Optiprep density gradient medium	CAUTION
	10 mL of 5x PBS-MK
	6.7 mL of Ultrapure water
60% Iodixanol	50 mL of Optiprep density gradient medium	CAUTION
	100 µL of phenol red	CAUTION
AAV purity control
10x Tris acetate EDTA (TEA) buffer	44.8 g Tris base	CAUTION
	11.4 mL glacial acetic acid (17.4M)
	3.7 g EDTA
	Up to 1 L Ultrapure water
Agarose gel	0.8 g Ultrapure agarose
	Up to 100 mL 1x TEA buffer
Gel buffer	181.7 g Tris base	CAUTION
	1.5 g SDS	CAUTION
	Adjust pH to 8.45 with 1 M HCl	CAUTION
	Up to 500 mL Ultrapure water
Cathode buffer 10x	121.14 g Tris base	CAUTION
	179.2 g Tricine	CAUTION
	1% SDS (w/w)	CAUTION
	Up to 1 L Ultrapure water
Anode buffer 10x	242.3 g Tris base	CAUTION
	Up to 1 L Ultrapure water
	Adjust pH to 8.9 with 1 M HCl	CAUTION
Sample buffer 5x	For 20 mL:
	605 mg Tris base	CAUTION
	4 g SDS	CAUTION
	10 mg Serva Blue G
	12 g Glycerol
	Adjust pH to 6.8 with 1 M HCl, aliquot and store at -20 °C	CAUTION
Stacking gel	For 2 gels:
	400 µL acrylamide	CAUTION
	750 µL gel buffer
	1.85 mL Ultrapure water
	4 µL TEMED	CAUTION
	20 µL 10% APS (v/v)	CAUTION
	Add TEMED and 10% APS immediately before pouring the gel. Use both chemicals under a chemical hood.
Resolving gel	For 2 gels:
	3.32 mL acrylamide	CAUTION
	3.35 mL gel buffer
	1.14 mL Ultrapure water
	2.12 mL 50% glycerol
	6 µL TEMED	CAUTION
	50 µL 10% APS (w/v)	CAUTION
	Add TEMED and 10% APS immediately before pouring the gel. Use both chemicals under a chemical hood.
Water-saturated butanol	10 mL n-butanol	CAUTION
	1 mL Ultrapure water
CAUTION: Refer to the Materials Table for guidelines on the use of dangerous chemicals.

Table 1: Composition of the required solutions.

1. Tri-transfection of HEK293T Cells

NOTE: Please refer to Table 1 for the composition of the buffers and solutions used in the protocol.
NOTE: Performance of this section of the protocol takes approximately 4 days.

1. Thaw a vial of human embryonic kidney (HEK) 293T cells in a water bath set at 37 °C.
   NOTE: Use only cells that have been passaged less than 20x to guarantee optimal transfection efficiency.
2. Seed HEK293T cells at a density of 2 x 10³ to 6 x 10³ cells/cm² in DMEM10 in 15 cm diameter cell culture dishes.
3. Grow the cells to 70–80% confluence in a standard incubator set at 37 °C, with 95% humidity and 5% CO₂.
   NOTE: The production of one batch of AAV vectors using this protocol requires 18 cell culture dishes (15 cm diameter). A cell confluence of 70% - 80% corresponds to 6 x 10³ to 7 x 10³ cells/cm², maintained in 17–20 mL of DMEM10 culture medium.
4. Prepare polyethylenimine (PEI)/DNA mix at a concentration ratio of 1/3.5 (w/w).
   1. Prepare the DNA mix for 18 cell culture dishes in one 50 mL conical tube by mixing 360 µg of pΔF6, 180 µg of pCapsid, and 180 µg of pTransgene in 18 mL of 150 mM NaCl.
   2. Distribute the DNA mix over three 50 mL conical tubes (6 mL of DNA mix per conical tube).
   3. Prepare the PEI mix for six cell culture dishes in a new 50 mL conical tube by mixing 840 µg of PEI (1 µg/µL) in 6 mL of 150 mM NaCl.
   4. Prepare the PEI/DNA mix by adding 6 mL of the PEI mix (prepared in step 1.4.3), drop by drop, to one of the conical tubes containing the DNA mix (prepared in step 1.4.2) and incubate for 20 min at room temperature.
      NOTE: After 20 min of incubation, the PEI/DNA mix will become slightly turbid.
5. Take six cell culture dishes out of the incubator and completely aspirate the medium from each culture dish in a laminar flow hood. Remove the traces of the medium by rinsing the dishes with 5 mL of prewarmed Dulbecco’s phosphate-buffered saline (DPBS).
6. Ensure the distribution of DPBS over the entire surface by gently tilting the dish.
7. Gently aspirate the DPBS and add 12 mL of DMEM1 to each dish.
   NOTE: Avoid detaching the cells by adding the medium slowly, from a pipette placed at the edge of the dish.
8. Mix the PEI/DNA by pipetting up and down 3x - 5x. Add 2 mL of the PEI/DNA mix to each of the six cell culture dishes in a drop-by-drop fashion, carefully distributing it over the entire surface. Once the mix is added to each dish, place the dishes back in the incubator. Repeat steps 1.4.3– 1.8 for the remaining culture dishes.
9. Incubate the transfected cells for 5 h at 37 °C, with 95% humidity and 5% CO_2.
10. Add an additional 12 mL of DMEM10 to each culture dish without removing the pre-existing medium (total medium volume = 25 mL).
11. Incubate the transfected cells up to 72 h post-transfection at 37 °C, with 95% humidity and 5% CO₂.

Supplementary Figure 1: HEK 293T cell morphology visualized by phase contrast microscopy (left) with confirmation of GFP expression visualized by fluorescence imaging (right). (A) Successful transfection of HEK293T cells with a GFP-encoding pTransgene is confirmed by fluorescence imaging. (B) HEK293T cells treated solely with transfection reagents show no GFP expression. Please click here to view a larger version of this figure.

12. Harvest the medium and the cells 72 h post-transfection. Use a cell scraper to carefully detach the cells from the culture dish. Collect the medium and the cells in a 50 mL conical tube kept on ice.
    NOTE: The contents of two cell culture dishes can be collected into a single 50 mL conical tube. At the end of this step, each of the nine tubes will contain approximately 50 mL of medium.
13. Centrifuge the conical tubes at 420 x g for 10 min at 4 °C with the acceleration and deceleration of the centrifuge set to maximum.
14. Carefully discard the supernatant from each tube. Do not use pipettes to prevent the loss of material. Instead, gently pour the supernatant into a waste disposal container and place the tubes containing the cell pellets back on the ice.
15. Resuspend each cell pellet in 2 mL of lysis buffer directly in the 50 mL conical tube by pipetting up and down 5–10x. Do not vortex. Pool the lysates from three tubes together.
    NOTE: At this point, there will be three 50 mL conical tubes, each one containing 6 mL of the resuspended cells in lysis buffer.

2. AAV Vector Purification

NOTE: Performance of this protocol section takes approximately 1 day. Perform the following steps simultaneously on each of the three 50 mL conical tubes containing the cells resuspended in lysis buffer (see the previous note).

1. Freeze and thaw the resuspended cells 3x to lyse them and release the AAV particles. Perform the freezing step by placing the tubes in a bucket containing dry ice mixed with ethanol. Perform thawing by immediately placing the cells in a water bath set at 37 °C.
2. After the third thawing step, centrifuge at 1,167 x g for 15 min at 4 °C.
   NOTE: A noticeable pellet composed of cell debris will be formed. When handling the tubes, avoid sudden movements as these can cause the detachment of the pellet into the supernatant, which will compromise the purity of the final vector.
3. Carefully transfer the supernatants to clean 50 mL conical tubes and then add nuclease to each tube, to a final concentration of 50 U/mL supernatant.
4. Incubate for 30 min at 37 °C. Swirl the 50 mL conical tubes by hand every 10 min to ensure that the nuclease is thoroughly mixed with the supernatant.
5. Clarify the supernatant by centrifugation at 13,490 x g for 20 min at 4 °C.
6. Attach a 0.45 µm filter to a 10 mL syringe and place it on top of a clean 50 mL conical tube. Carefully remove the plunger and fill the syringe with the supernatant from step 2.5.
7. Use the plunger to force the lysate through the filter. Use a new filter and syringe for each tube of supernatant obtained in step 2.5.
   NOTE: The obtained fraction is known as ‘crude lysate’.
8. Prepare each of the 15%, 25%, 40%, and 60% iodixanol fractions in four separate 50 mL conical tubes, according to the instructions in Table 1.
9. Prepare the iodixanol gradients in three ultracentrifugation tubes using the following order of iodixanol fractions: 8 mL of 15% iodixanol, 5.5 mL of 25% iodixanol, 5 mL of 40% iodixanol, and 4.5 mL of 60% iodixanol.
   CAUTION: Iodixanol can cause irritation to eyes, skin, and the gastrointestinal and respiratory tracts. When handling iodixanol gradients, wear gloves and work under a laminar flow hood.
   1. Pipette 8 mL of 15% iodixanol into each ultracentrifugation tube.
   2. Pipette 5.5 mL of 25% iodixanol solution into a clean 50 mL conical tube (Figure 1A).
   3. Use a non-graduated Pasteur pipette (as the neck of the ultracentrifugation tube is too narrow for conventional graduated pipettes) to carefully layer 5.5 mL of the 25% iodixanol solution below the 15% iodixanol solution (Figure 1B).
      NOTE: This can be successfully achieved by adding the iodixanol in three steps since the Pasteur pipette can only hold around 2 mL.
   4. Add the 40% and 60% iodixanol solutions as described in step 2.9.3. Do not disturb the different iodixanol interfaces during layering.
      NOTE: The proper preparation of the different iodixanol fractions can be ensured by visual confirmation thanks to the phenol red added to the iodixanol fractions (see the instructions in Table 1). Since each fraction has a specific density, they will not intermix during the layering step, if it is performed properly (check Figure 1C).
10. Layer the crude lysate on top of the 15% iodixanol gradient with a Pasteur pipette. Proceed drop by drop to avoid disturbing the interface between the crude lysate and the iodixanol solution.
11. Fill the ultracentrifugation tube up 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 the ultracentrifugation (Figure 1C).
12. Close the ultracentrifugation tubes using appropriate lids. Use a digital scale to make sure all three ultracentrifugation tubes have the same weight. Adjust the weight, if necessary, by adding more lysis buffer on top of the ‘crude lysate’.
    NOTE: The weight difference between the ultracentrifugation tubes must be below 0.1 g to ensure the safe operation of the ultracentrifuge. Ultracentrifuges are potentially dangerous pieces of equipment and should only be used by properly trained personnel.
13. Centrifuge the tubes at 301,580 x g, using a fixed-angle titanium rotor, for 1 h and 40 min at 12 °C, using maximum speed of acceleration and deceleration.
14. Carefully insert a stainless-steel blunt needle at the 40% and 60% iodixanol interface.
    1. Attach the needle to a 5 mL syringe (Figure 1E).
    2. Aspirate only the clear fraction, containing the vector particles.
       NOTE: The total volume collected is approximately 2.5–3 mL. Avoid the collection of material from the 25–40% interface, since this will increase the level of contamination in the final vector batch, due to the presence of non-desirable proteins.
    3. Process the collected fraction (desalting and concentration) or store it overnight at 4 °C.

Figure 1: Setup for iodixanol gradient purification and subsequent vector collection. (A) Before pipetting the different iodixanol gradients into the ultracentrifugation tube, pipette an adequate volume of each iodixanol solution into a separate conical tube. (B) Then use a Pasteur pipette to sequentially transfer each iodixanol solution to the ultracentrifugation tube: layers of an increasingly high iodixanol concentration should be added at the bottom of the tube underneath the previous layer(s). (C) Layer the crude vector lysate on top once the gradient has been prepared. This vector collection system does not use sharp needles, which present a risk of 'needlestick' injuries. (D) A stainless-steel 316-syringe needle is inserted through the iodixanol gradient up to the 40%/60% iodixanol interface. (E) Vector particles are found in the 40% iodixanol phase and are collected. Please click here to view a larger version of this figure.

3. Desalting and Concentration of the AAV Vector

NOTE: Performance of this section of the protocol takes approximately 2 h.

1. Prerinse the filter membrane of the centrifugal filter by adding 5 mL of 1x PBS-MK and centrifuge for 5 min at 4,000 x g.
2. Add 5 mL of 1x PBS-MK to the collected AAV vector fraction, mix and then add the total volume to the filter. Upon addition of 1x PBS-MK, turbidity is observed. Centrifuge at 4,000 x g until the volume present on the cone-shaped filter has been reduced to 1 mL. Discard the flow-through accumulated in the outer collection tube.
3. Add 13 mL of 1x PBS-MK to the cone-shaped filter and repeat the centrifugation step as many times as necessary (minimum 3x), until there is no more turbidity observed when fresh 1x PBS-MK is added.
4. In the final wash step, add 13 mL of PBS + 0.01% (v/v) non-ionic surfactant and centrifuge at 4,000 x g until the volume is reduced to 300–350 µL.
5. Collect the remaining fraction present on the filter by pipetting the whole volume into a sterile skirted microcentrifuge tube.
   NOTE: This fraction contains the desalted and concentrated AAV vector. In the following steps of this protocol, this fraction is referred to as the ‘primary’ fraction. Make sure to handle the tube under the hood and store it at 4 °C.
6. Rinse the filter with 200 µL of 1x PBS-MK to collect the residual vector particles retained on the filter. Collect in a sterile microcentrifuge tube.
   NOTE: This is a diluted vector stock, which may be suitable for some applications requiring lower vector titers. In future steps, this fraction is referred to as the ‘secondary’ fraction.
7. For short-term storage, store the vector fraction(s) at 4 °C (less than 2 weeks). Aliquot and store the vector at ‑20 °C when long-term storage is required.
8. Perform quality control as described in section 4.

4. Titration of the Vector by Quantitative Polymerase Chain Reaction

NOTE: Performance of this section of the protocol takes approximately 3 h.

1. Standard curve
   1. Linearize the transgene-expressing plasmid (pTransgene) used for the transfection of HEK-293T cells in section 1.
   2. Prepare the restriction digest mix in a 0.5 mL microcentrifuge tube (refer to Table 2 for the composition of the restriction digest mix).
      NOTE: Adjust the composition of the restriction digest mix according to the enzyme used and the related guidelines from the manufacturer.
   3. Incubate the restriction digest mix for 1 h at 37 °C.
   4. Check the efficiency of the restriction digestion by running the linearized plasmid on a 0.8% (w/v) agarose gel at 100 V for 1 h. Complete digestion of the plasmid should produce a single fragment of defined size.
   5. Purify the linear plasmid DNA using a PCR purification kit, following the manufacturer’s instructions²⁴, and measure the DNA concentration with a spectrophotometer by measuring the absorption at 260 nm. After the titration, store the remaining aliquot of the linear plasmid at ‑20 °C for further use.
   6. Calculate the molecules (i.e. DNA copies) of the plasmid stock per microliter as follows.
      1. First, calculate the molecular weight of the plasmid. Assuming that the average weight of a DNA base pair (bp) is 650 Daltons (Da) and that one mole of a bp weighs 650 g, the molecular weight of any double-stranded DNA template can be estimated by taking the product of its length (in bp) and weight per base pair.
         ​Plasmid molecular weight [g/mol] = plasmid size [bp] x 650 [Da/bp]
      2. Next, calculate the number of moles of plasmid per microliter.
         Moles of plasmid per microliter = plasmid concentration [g/µL] / plasmid molecular weight [g/mol]
         ​NOTE: The inverse of the molecular weight is the number of moles of plasmid present in 1 g of the material.
      3. Then, calculate the number of plasmid molecules per microliter, using Avogadro’s number (6.022 x 10²³ molecules/mole).
         Molecules of plasmid per microliter = moles of plasmid per microliter [moles/µL] x Avogadro’s number [molecules/mole]
      4. Finally, dilute the plasmid stock (molecules/µL) to obtain a 100 µL solution with the desired concentration of 1 x 10⁹ molecules (vector genomes (vg) per microliter).
         Plasmid stock (100 µL) = (desired concentration [molecules/µL] x 100 µL) / plasmid molecules [molecules/µL]
   7. Make serial dilutions of the plasmid stock (1 x 10⁹ vg/µL) in triplicates:
      10 µL of 1 x 10⁹ vg/µL plasmid stock + 90 µL of H₂O = 1 x 10⁸ vg/µL solution
      10 µL of 1 x 10⁸ vg/µL dilution + 90 µL of H₂O = 1 x 10⁷ vg/µL solution
      10 µL of 1 x 10⁷ vg/µL dilution + 90 µL of H₂O = 1 x 10⁶ vg/µL solution
      10 µL of 1 x 10⁶ vg/µL dilution + 90 µL of H₂O = 1 x 10⁵ vg/µL solution
      ​Continue to obtain a 1x10¹ vg/µL solution.
   8. Keep the serial dilutions of the standard plasmid stock on ice until loading them on the qPCR plate (section 4.3).

Component	Amount
10x Restriction enzyme buffer	5 µL
Restriction enzyme	2,5 µL
Plasmid	5 µg
H2O	up to 50 µL

Table 2: Restriction digest mix composition.

Table 3: Stock plasmid volume calculator. Please click here to download this table.

2. DNA extraction from the AAV vector
   1. Mix 2 µL of the AAV vector stock (the primary fraction from step 3.5) with 198 µL of DNase I buffer (1x) in strip tubes (PCR tubes) and add 2 µL of DNase I.
      NOTE: DNase I will degrade any genetic material that is not contained inside a vector capsid (which would distort the qPCR results). This solution is referred to as dilution ‘dil1 x 10^-2’.
   2. Incubate for 30 min at 37 °C, followed by 10 min at 95 °C.
      NOTE: The protocol can be stopped at this point and the material can be stored indefinitely at 4 °C, to avoid product deterioration.
   3. Add 2 µL of proteinase K to the dil1 x 10^-2 solution (step 4.2.1) and incubate for 60 min at 50 °C, followed by 20 min at 95 °C.
      NOTE: This step will disassemble the AAV vector capsid and release the AAV vector genome into the solution. Add proteinase K in excess as the protein (capsid) content of the sample is not known. Note, it is essential to ensure that all proteinase K activity is removed by denaturation, prior to qPCR, to avoid issues with (partial) polymerase degradation influencing the final result.
   4. Prepare 1:10 serial dilutions of the proteinase K treated dil1 x 10^-2 solution (step 4.2.3) in 1.5 mL microcentrifuge tubes as follows:
      10 µL of dil1 x 10^-2 + 90 µL of H₂O = dil1 x 10^-3 dilution
      10 µL of dil1 x 10^-3 + 90 µL of H₂O = dil1 x 10^-4 dilution
      ​10 µL of dil1 x 10^-4 + 90 µL of H₂O = dil1 x 10^-5 dilution
   5. Keep the serial dilutions of DNA extracted from the vector on ice until loading them on the qPCR plate (section 4.3).
3. Titration by SYBR Green detection-based qPCR
   1. Prepare the qPCR master mix in a 1.5 mL microcentrifuge tube, for both the sample and the standards. Use 10 µL of SYBR Green Master Mix, 1 µL of forward primer (10 µM stock), 1 µL of reverse primer (10 µM stock), and 3 µL of H₂O per reaction. See the protocol in Table 4 for primer sequences.
   2. Pipette the qPCR master mix up and down, but do not vortex.
   3. Add 15 µL of the qPCR master mix, followed by either 5 µL of the standard curve prepared in section 4.1 or the DNA extracted from the AAV vector in section 4.2, into each well. Include three wells containing only the qPCR master mix, as a negative control. Refer to Figure 2 for the plate layout.
   4. Seal the plate with a sealing film and briefly centrifuge the qPCR plate at 1,500 x g for 30 s at 4 °C.
   5. Run the qPCR reaction on a plate-based real-time PCR amplification and detection instrument, using the conditions suggested in Table 5.

Primer name	Sequence
Forward primer	5’-CCCACTTGGCAGTACATCAA-3’
Reverse primer	5’-GCCAAGTAGGAAAGTCCCAT-3’

Table 4: Primer sequences designed against the CBA promoter.

Figure 2: Plate layout for qPCR-based vector titration. The samples are color-coded: green = standard curve; blue = H₂O control; grey = primary fraction; orange = secondary fraction. Please click here to view a larger version of this figure.

Step	Time	Temperature	Cycles	Aim
Pre-incubation	5 min	95 °C	x1	DNA denaturation and polymerase activation (hot-start reaction).
Amplification	10 min	95 °C	x1	Amplification of the DNA. Settings may be optimized if alternative primers with different annealing temperature are used.
	10 s	95 °C	x40
	40 s	60 °C
	1 s	72 °C
Cooling	10 s	40 °C	x1	Plate cooling. End of the PCR.

Table 5: Thermal cycling protocol for SYBR green-based qPCR titration.

4. Data analysis to determine the AAV vector titer
   1. Fill the spreadsheet data cells (Table 6A) with the Ct values obtained for the different dilutions of the standard sample prepared in section 4.1 to generate a standard curve.
      ​NOTE: The equation of the standard curve will be shown (y = a ln(x) + b) together with the R² efficiency (Table 6B). A qPCR must have an efficiency close to 100% and R² close to 1.0 (≥ 0.96).
   2. Use the calculated values of a and b to fill in the corresponding data cells in the spreadsheet (Table 6C).
   3. Complete the spreadsheet with the Ct values obtained for the different dilutions of the AAV sample prepared in section 4.2.
   4. Calculate the average AAV vector titer in vector genomes per microliter of the 'primary fraction' by using the following formula:
      
      NOTE: The factor 400 is used for single stranded genomes, while sc genomes use a multiplication factor of 200²⁵. In fact, as DNA quantities double during each qPCR cycle, sc DNA is detected 2x in comparison to an ss genome. The aim of the titration is to calculate the concentration in terms of vector genomes per microliter. A correction is necessary for running the qPCR when using 2 µL of starting material (step 4.2.1). dil represents the dilution factors from step 4.2.4. The titer of the 'secondary' AAV vector fraction (step 3.6) can be calculated simultaneously.

Table 6: Template for qPCR data analysis. Please click here to download this file.

5. Purity Control by SDS-PAGE and Silver Staining

NOTE: Performance of this section of the protocol takes approximately 5 h.

1. Use 70% (v/v) ethanol to thoroughly clean the glass plates used for casting the gels.
2. Assemble the gel casting system. Ensure that the bottoms of the glass plates are not chipped, to prevent leakage of the acrylamide mixture when casting the gel.
3. Prepare the stacking and the resolving gel solutions in two separate 50 mL conical tubes. Omit the tetramethylethylenediamine (TEMED) and the 10% (w/v) ammonium persulfate (APS), as they are responsible for polymerization of the gel. Add them immediately before casting the gel.
   NOTE: The final percentage of acrylamide in the gel influences the separation profile of the proteins: typically, a 10% (v/v) final concentration of acrylamide will be sufficient to test the purity of an AAV vector preparation.
4. Mix gently by swirling the tube containing the gel components. Do not vortex, since excessive oxygenation can impair polymerization.
5. Add TEMED and 10% (w/v) APS to the resolving gel solution. Mix and then pour into the gel holder until it reaches 1-2 cm below the top of the small glass plate.
6. Place a layer of water-saturated butanol on the top of the acrylamide mixture. This will ensure the formation of a flat surface during polymerization. Do not leave the gel under alcohol for longer than 30 min as this will dehydrate the gel and impair its function.
   CAUTION: Butanol is hazardous in case of skin contact. It also presents a fire risk. Handle with care.
7. Wait for the gel to polymerize and then pour off the butanol. Tip: Check to see whether the excess gel mix in the tube has solidified. Polymerization occurs in less than 20 min. Wash the surface of the gel with H₂O and then dry it using paper towels, taking care not to disturb the surface of the gel.
8. Add TEMED and 10% (w/v) APS to the stacking gel and pour the gel into the gel holder on top of the separating gel.
9. Place the provided comb in the gel. Perform this action with one steady movement to avoid the formation of air bubbles inside the wells.
10. Wait for at least 20 min for the gel to polymerize. Tip: Check if the excess gel mix in the conical tube has solidified.
11. Prepare the two mixes (Table 7) for both the primary and the secondary AAV vector fractions (steps 3.5 and 3.6, respectively).

	High Amount	Low Amount
AAV vector stock	5 µL	1 µL
5x Sample buffer	3 µL	3 µL
H2O	7 µL	11 µL

Table 7: Composition of the sample mixes required for silver staining.

12. Fully denature the sample mixes by heating them for 5 min at 95 °C. Assemble the electrophoresis tank, paying attention to the electrode orientation.
13. Fill the tank with 1x cathode buffer on the inside of the electrode assembly and 1x anode buffer on the outside.
    NOTE: Anode buffer can be recycled from run-to-run. Fresh cathode buffer must always be used.
14. Remove the comb from the gel and clean the wells of the gel with 1x cathode buffer.
15. Load 1 µL of protein ladder in a well. Load the sample mixes in different wells on the same gel (Figure 3). Run the gel at 50 V until the samples enter the resolving gel. Then increase the voltage to 100 V until the dye front reaches the lower limit of the gel.
16. Extract the gel carefully from the glass plates and use the silver staining kit (according to the manufacturer’s instructions²⁶) to visualize the viral proteins (VP1, VP2, and VP3 subunits) that comprise the AAV capsid, as well as to check for possible protein contamination (Figure 3).

Figure 3: Evaluation of vector purity using SDS-PAGE and silver staining. Using a Tricine-SDS gel, 5 µL of various vector preparations were separated. Proteins were subsequently detected by silver staining. Vectors are considered pure when VP1 (82 kDa), VP2 (67 kDa), and VP3 (60 kDa) are visible in a 1:1:10 ratio (lane 1), without excessive background (lane 2) or non-specific bands (lane 3). Please click here to view a larger version of this figure.

Discussion

The production of recombinant AAV vectors described here uses materials and equipment common to most molecular biology labs and cell culture facilities. It allows the user to obtain pure, preclinical grade AAV vectors that can be used to target multiple cell and tissue types across a range of in vitro and in vivo applications. One of the greatest advantages of this protocol, compared to others (i.e., CsCl-based purification), is the shorter working time needed. Ready-to-use AAV vectors are obtainable in a maximum of 6 working days after the initial transfection of HEK293T cells.

Several factors can negatively influence the final yield or the quality of the AAV vector. Poor transfection efficiency is one of the main reasons for a low viral yield³³. A major recommendation is the use of HEK293T cells that have not been passaged more than 20 times and do not have a cell confluence greater than 90% at the time of transfection²¹. In addition, the transfection method selected has a major impact on the results. This protocol is based on the use of PEI. PEI is a cationic polymer with the ability to deliver exogenous DNA to the cell nucleus through the generation of complexes of polymer and nucleic acid, known as polyplexes, which are taken up by the cell and trafficked via endosomes³⁴. PEI-based transfection is easy and fast to perform, in contrast to other widely used methods, such as the co-precipitation of DNA with calcium phosphate³⁵. Also, PEI-based transfection is much cheaper when compared to other newly introduced methods, such as the usage of cationic lipids and magnet-mediated transfection³⁶.

The purification strategy plays a key role in the protocol. Compared to other methods, iodixanol-based purifications tend to contain a higher percentage of empty viral particles (20%)²⁰. This is offset, to a degree, by the fact that iodixanol-based purification routinely results in AAV vector preparations with a particle-to-infectivity ratio of less than 100. This represents a significant improvement in comparison to conventional CsCl-based procedures, for which substantial loss of particle infectivity is reported³⁷. Another common alternative method to purify AAV vectors is chromatography-based purification. However, this method has the major drawback that a specific column is required for each vector capsid used: for example, while AAV2 is classically isolated using heparin columns, this methodology does not work with AAV4 and AAV5, which do not possess heparin-binding sites on their capsids³⁸. Considering that chromatography purification is also expensive, iodixanol-based purification is generally more suitable for laboratories that wish to produce high-quality batches of AAV vectors on a small scale^33,39,40. However, to maximize the final yield and purity of the vector, extreme care is needed when making the iodixanol gradients. The various iodixanol fractions should be transferred to the ultracentrifugation tube using a sterile Pasteur pipette whose tip is touching the wall of the tube: iodixanol should be expelled from the pipette slowly and continuously. As the vector particles accumulate in the 40% iodixanol layer, care needs to be taken to ensure that the gradient interfaces do not mix²⁰. Finally, the fraction containing the vector should be recovered by the insertion of a stainless-steel blunt needle with a gauge not larger than 20 G. To maximize vector recovery, the clear fraction should be retrieved in its entirety. During this step, timing is critical. To avoid compromising the purity of the preparation, it is essential to stop the collection before other (contaminating) phases of the gradient are collected.

Differences in the obtained vector titer can be also attributed to the intrinsic ability of the vector to produce packaged particles. A comparison between different AAV serotypes showed that some AAV vectors are more difficult to produce at a higher titer than others (e.g., AAV2)⁴¹. Precipitation of the vector, during the desalting step, can be a possible reason for a lower titer and easily prevented by avoiding overconcentration³³. Moreover, it is also possible that the efficiency of iodixanol gradient-based purification slightly differs between serotypes and, therefore, discrepancies in the titer obtained with different serotypes can be observed⁴¹.

Finally, it needs to be pointed out that even though qPCR is a very accurate method for DNA quantification some inherent variability in the technique can be observed. Accuracy in the titration primarily depends on the precise pipetting and proper vortexing of all the solutions. To guarantee the most accurate titer reading, the qPCR can be independently repeated, and the obtained values averaged. The choice of primers presented in this protocol is based on the sequence of the CBA promoter located in the pTransgene plasmid used in our laboratory. The CBA promoter is a strong synthetic promoter that is widely used in the vector field to drive expression across multiple cell types. It incorporates multiple elements, including the cytomegalovirus (CMV) early enhancer element; the promoter, first exon, and the first intron of the CBA gene; and the splice acceptor of the rabbit β-globin gene. However, primers can be designed for virtually any element located within the expression cassette (including the promoter, transgene, and regulatory elements). The comparison of titer across batches is also possible, providing primers are used against regions common to the vectors in question.

In conclusion, this protocol can be used to produce AAV vectors with a variety of capsids, genome configurations, promoter types and transgene cargos. This will allow users to easily adapt the final characteristics of their vectors to best suit experimental needs. In the example presented in the representative results, the use of the PHP.B capsid, which efficiently crosses the BBB, gave highly efficient gene expression in the CNS, following tail vein injection³². The systemic administration of CNS penetrant vectors has considerable advantages in terms of possible side-effects^2,17,32. A possible alternative to peripheral injection, while avoiding the caveats of invasive techniques, is intrathecal delivery, which consists of delivery of the AAV vector into the cerebrospinal fluid. This delivery route is proven to be effective, showing widespread expression of transgene across the CNS, less off-target effects in peripheral organs, and low levels of immune response⁴². However, intrathecal injections are much more challenging, as they require higher technical skills than tail vein injection.

Further capsid development to refine this technology will be driven by the opportunities for AAV vector use in gene therapy applications. Such approaches offer attractive possibilities to treat currently incurable CNS disorders, such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Parkinson’s and Alzheimer’s disease¹⁸.

Materials

Name	Company	Catalog Number	Comments
Plasmid production
pTransgene plasmid	De novo design or obtained from a plasmid repository	N/A	See step 1 of main protocol for further details
pCapsid	De novo design or obtained from a plasmid repository	N/A	See step 1 of main protocol for further details
pHelper	Agilent	240071
Plasmid Plus maxi kit	Qiagen	12963
QIAquick PCR purification Kit	Qiagen	28104
AAV Helper-Free System	Agilent	240071
Cell culture and transfection
Dulbecco’s Modified Eagle Medium (DMEM), high glucose, no glutamine	Life technologies	11960-044	Supplement DMEM with FBS (1% or 10% v/v) and GlutaMAX 200 mM (1% v/v) then filter sterilize the medium using a 0.22 mm filter
Fetal bovine serum (FBS)	GIBCO	10500-064
GlutaMAX supplement	GIBCO	35050038
(200 mM)
Corning bottle-top vacuum filter system	Sigma-Aldrich	CLS430769
Dulbecco’s Phosphate Buffered Saline (DPBS) with no calcium and no magnesium	GIBCO	14190094
HEK293T cells	American Tissue Culture Collection	CRL3216	Upon receipt, thaw the cells and culture as described in the protocol. After a minimal number of passages, freeze a subfraction for future in aliquots. Always use cells below passage number 20. Once cultured cells have been passaged more than 20 times, restart a culture from the stored aliquots
Cell culture dishes	Greiner Cellstar	639160	15 cm diameter culture dishes
Cell scrapers	VWR	10062-904
Polyethylenimine (PEI)	Polyscience	23966-2	PEI in powder form is dissolved at 1 µg/µL in deionized water (ddwater) at pH=2 (use HCl). Prepare in a beaker and stir for 2-3 h. When dissolved, bring the pH back to 7 with NaOH. Filter sterilize and store the resuspended stock solution in 1 ml aliquots at -20 °C. PEI aliquots can freeze/thawed multiple times.
Virkon solution	Fisher Scientific	NC9821357	Disinfect any material that has been in contact with assembled viral particles with Virkon solution
Mutexi long-sleeve aprons	Fisher Scientific	11735423	Wear an apron over the top of a regular lab coat
Fisherbrand maximum protection disposable overshoes	Fisher Scientific	15401952
AAV Purification and desalting
Optiprep density gradient medium	Sigma-Aldrich	D1556	Optiprep is a 60% (w/v) solution of iodixanol in water (sterile). CAUTION. Use under a laminar flow hood. Wear gloves
Phenol red	Sigma-Aldrich	P0290	CAUTION. Use under a laminar flow hood
Pasteur pipette	Sigma-Aldrich	Z627992	Sterilize before use
OptiSeal Polypropylene tubes	Beckman	361625
Benzonase (250 U/µL)	Sigma-Aldrich	E1014	Supplied as a ready-to-use solution
Acrodisc syringe filter	Pall corporation	4614
Omnifix syringe (5mL)	Braun	4617053V
Blunt syringe needle	Sigma Aldrich	Z261378	Stainless steel 316 syringe needle, pipetting blunt 90° tip gauge 16, L 4 in. Referred to in the text as a blunt-end needle
Aqua Ecotainer	B. Braun	0082479E	Sterile endotoxin-free water. Referred to as 'Ultrapure water'
Amicon ultra-15 centrifugal filter unit	Millipore	UFC910024	These filters concentrate the final product by collecting the viral particles in consecutive centrifugation steps
Pluronic F68 (100X)	Thermo Fisher	24040032	Non-ionic surfactant. Dilute in sterile PBS to use at 0,01% (v/v)
Fisherbrand Sterile Microcentrifuge Tubes with Screw Caps (2 mL)	Fisher Scientific	02-681-374	Use skirted tubes for easy handling
AAV Titration
Restriction enzyme: StuI (10 U/µL)	Promega	R6421
DNAse I (1 U/µL)	Fisher scientific	EN0521
Proteinase K	Sigma-Aldrich	3115852001	Reconstitute in ultrapure water and use at a final concentration of 10 mg/ml. Solution can be stored at -20°C
EasyStrip Plus Tube Strips (with attached caps)	Fisher scientific	AB2000
Eppendorf microtube 3810x	Sigma-Aldrich	Z606340-1000EA
LightCycler 480 SYBR Green I Master Mix	Roche	4707516001
LightCycler Multiwell Plates, 96 wells	Roche	4729692001	White polypropylene plate (with unique identifying barcode)
Microseal 'A' PCR Plate and PCR Tube Sealing Film	Bio-Rad	msa5001
AAV Purity control
Ammonium persulfate (APS)	Sigma-Aldrich	A3678	Reconstitute in ultrapure water to 10% (v/v). CAUTION. Use under laminar flow hood. Wear gloves
Tetramethylethylenediamine (TEMED)	Sigma-Aldrich	T9281	CAUTION. Use under a laminar flow hood. Wear gloves
Tris Base ULTROL Grade	Merck	648311	CAUTION. Use under a laminar flow hood. Wear gloves
UltraPure Agarose	Thermo Fisher	16500-500
Rotiphorese® Gel 30 (37,5:1)	Carl Roth	3029.3	Aqueous 30 % acrylamide and bisacrylamide stock solution at a ratio of 37.5:1. CAUTION. Use under laminar flow hood. Wear gloves
Serva Blue G	Sigma-Aldrich	6104-58-1
Precision Plus prestained marker	Bio-Rad	1610374edu
1-Butanol	Sigma-Aldrich	B7906	CAUTION. Use under a laminar flow hood. Wear gloves
Immunohistochemistry
Rabbit anti-GFP	Synaptic System	132002	1:300 dilution
Anti-rabbit Alexa Fluor 488	Invitrogen	A21206	1:1000 dilution
Equipment	Company	Catalog number	Comments
Vector production lab
Rotina 380 bench-top centrifuge *	Hettich	1701
Optima XPN 80 ultracentrifuge *	Beckmann Coulter	A95765
Type 50.2 Ti fixed-angle titanium rotor *	Beckmann Coulter	337901
Entris digital scale *	Sartorius	2202-1S
Warm water bath *			Set at 37°C
Ice bucket *	VWR	10146-290	Keep material used in the vector production lab separate from that used in standard lab areas
Pipetboy pro *	Integra	156,400
Graduated pipettes: Cell star *	Greiner bio-one	606180	Capacity of 5 ml, 10 ml and 25 ml
Graduated pipettes: Cell star *	Greiner bio-one	607180	Capacity of 5 ml, 10 ml and 25 ml
Graduated pipettes: Cell star *	Greiner bio-one	760180	Capacity of 5 ml, 10 ml and 25 ml
Co2 incubator CB150 *	Binder	9040-0038	Set at 37°C, 5% CO2 and 95% humidity
Nuaire safety cabinet NU 437-400E *	Labexchange	31324	Clean all the surfaces with 70% ethanol and Virkon before and after use
Conventional lab
T100 thermal cycler *	Bio-Rad	1861096
LightCycler 480 Instrument II *	Roche	5015278001
ThermoMixer *	Eppendorf	C 5382000015
Nanodrop *	ThermoFisher Scientific	ND 2000
Mini-Protean Tetra Cell*	Bio-Rad	1658001FC	For use with handmade or precast gels
ProteoSilver silver stain kit	Sigma-Aldrich	PROTSIL1	High sensitivity protein detection with low background
Centrifuge 5804 R *	Eppendorf	B1_022628045	High speed centrifuge for medium capacity needs (up to 250 ml)
Graduated pipettes Cell star *	Greiner bio-one	606180	5 ml, 10 ml and 25 ml
Graduated pipettes Cell star *	Greiner bio-one	607180	5 ml, 10 ml and 25 ml
Graduated pipettes Cell star *	Greiner bio-one	760180	5 ml, 10 ml and 25 ml
Filter tips *	Greiner bio-one	750257	2 µl, 20 µl, 200 µl
Filter tips *	Greiner bio-one	738257	2 µl, 20 µl, 200 µl
Filter tips *	Greiner bio-one	771257	2 µl, 20 µl, 200 µl
Ice bucket with lid *	VWR	10146-290
Mini diaphragm vacuum pump, VP 86 *	VWR	181-0065
Pipetman P2, P20, P100, P200, P1000	Gilson	F144801
Pipetman P2, P20, P100, P200, P1000	Gilson	F123600
Pipetman P2, P20, P100, P200, P1000	Gilson	F123615
Pipetman P2, P20, P100, P200, P1000	Gilson	F123601
Pipetman P2, P20, P100, P200, P1000	Gilson	F123602
* Materials marked with an asterisk are expensive vpieces of equipment and are usually central infrastructure items shared between multiple labs. These items can also be replaced by equivalents if available. Note, when a different ultracentrifuge is used, care must be taken to select the correct rotor and centrifuge tubes.