Optimization of large-scale adeno-associated virus (AAV) production

Abstract

Genetic manipulation in vivo is a critical method for mechanistically understanding gene function in disease and physiological processes. To facilitate this, embryonic transgenesis in popular animal models like mice have been developed. Compared to the longer, expensive methods of transgenesis, viral vectors, such as adeno-associated virus (AAV), have grown increasingly in popularity, due to their relatively low cost and ease of production, translating to an overall greater versatility as a biological tool. In this protocol, we describe a method for AAV production and purification, for efficient transduction, in vivo. Importantly, our method differs from others in its application of a streamlined, more cost-effective approach. From this method, as many as 2×10¹³ genome-containing viral particles (vp), or 200 units, can be produced within 3–4 weeks, with a minimal cost of $1,800-$2,000 for supplies and reagents, and less than 15 hours per week of personnel time. A unit here is defined as 1×10¹¹ vp, our standard dose of AAV per animal, injected via tail-vein. Therefore, our method provides for production of AAV in quantities capable of transducing up to 200 animals. The following basic protocols outline the method described:

INTRODUCTION:

AAV production and purification is an integral process for the production of a biological tool that facilitates mechanistic study of gene function in vivo (Blackwood, 2019). AAV production and purification for in vivo studies represents a challenge for large-scale production, due to the sheer number of reagents required to facilitate such production. This study describes an optimized approach to AAV production and subsequent purification that facilitates an organized, cost-effective, consistent method for large-scale AAV production in academia. From the described methods, as many as of 2×10¹³ genome-containing viral particles (vp) can be produced within 3–4 weeks, at a cost of $1,800-$2,000 for reagents (Table 1), and less than 15 hours of personnel time per week. By following the described methods, large-scale AAV production can be achieved consistently and economically.

Table 1.

Cost-analysis for 2 AAV preps amounting to the value of 2×10¹³vp generation.

Item	Catalog number	Price per unit	Quantity to order	Initial cost for supplies	Estimated # of preps worth	Estimated value per 2 preps = ~2×1013vp or 200 units*
AAVPro 293T Cells	Takara 632273	$387	1	$387	Infinite
DMEM F:12 (option 1)	Gibco 11330032	$42.25/500 mL	5	$226.25	2 preps	$226.25
DMEM, F:12 (option 2)	Cytiva SH30023.01	$20.42/500 mL	5	$102.10	2 preps	$102.10
Fetal Bovine Serum	Omega FB-02	$270/500 mL (price will vary greatly)	1	$270	4–5 preps	$135
150 mm surface treated tissue culture dish	Fisher FB012925	$98.72/120	1	$98.72	4 preps	$49.36
T-175 tissue culture flask, vented cap (option 1)	Corning 355001	$359.50/case of 40	3	$1078.50	2.5 preps	$862.80
T-175 tissue culture flask, plug seal (option 2)	Corning 353028	$340.50/case of 40	3	$1021.50	2.5 preps	$817.20
T-175 tissue culture flask, vented caps (option 2)	Corning 354639	$102/case of 50	2	$205	2 preps	$205
PEI MAX-Transfection Grade Linear Polyethylenimine Hydrochloride	Polysciences 24765–100	$129/100 mg	1	$129	Infinite	~0
pAdDeltaF6	Addgene #112867	Bacterial agar stab	1	$75	Infinite**	~0
pAAV2/9n	Addgene #112865	Bacterial agar stab	1	$75	Infinite**#	~0
Benzonase Nuclease	Sigma 71205–3	$273/25 KU	1	$273	2 preps	$273
OptiSeal 32.4 mL Tube Kit	Beckman 361662	$2,271/kit	1	$2,271	Tubes – 6 preps Other kit elements – infinite use (one time purchase)	$111
Optiprep	Sigma D1556	$332/250 mL	1	$332	6 preps	$111
Exel International Disposable Spinal Needles	Med Vet International 26960	$19.88/needle	1	$19.88	Infinite (one time purchase)	~0
10 mL Sterile Syringe	Fisher 14–955-459	$22.45/pack of 100	1	$22.45	25 preps	~0
Weighing Scale	Ohaus 1450-SD	$367.84/scale	1	$367.84	Infinite (one time purchase)	~0
Cast Iron Support Ring Stand	Fisher S13747	$23/stand	1	$23	Infinite (one time purchase)	~0
Three Prong Extension Clamps	Fisher 05–769-7Q	$72.80/clamp	1	$72.80	Infinite (one time purchase)	~0
Lactated Ringers	NDC 0990–7953-09	$9.99/1000 mL	1	$9.99	3 preps	$6
Vivaspin 20 MWCO 100,000	Cytiva 28–9323-63	$236.50/pack of 12	1	$236.50	3 preps	$158
					Cost for 2 preps using Option 1 selections	$1808
					Cost for 2 preps using Option 2 selections	$2092

Note: These are the costs associated with supplies/reagents unique to AAV production/purification and supplies/reagents that are consumed in high numbers in this method (See culture dishes, fetal bovine serum, and DMEM F:12). Not included are standard supplies/reagents associated with cell culture or animal work.

^*One unit is defined as 1×10¹¹ vp, our standard dose per animal.

^**These items require DNA preparation, and its associated costs are not detailed in this protocol.

^#This item will vary depending on the serotype desired.

The methods described consist of two protocols. Basic Protocol 1: AAV Production covers the methods of AAVPro-293T cell culture and growth, transfection of AAV plasmids and helper plasmids, and harvest of AAV particles from AAVPro-293T cell lysates. Basic Protocol 2: AAV purification covers the method of purification of AAV particles from other cellular contents of AAVPro-293T cell lysates, and subsequent solubilization in a solution suitable for in vivo administration.

CAUTION: AAV is a Biosafety Level 2 pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms.

STRATEGIC PLANNING:

To address the challenge of large-scale production of AAV, the following streamlined approach has been developed (Figure 1), which allows for 2 AAV preparations over the course of 3–4 weeks. A single AAV preparation should yield 80–100 units, where a unit is defined as 1×10¹¹ vp. Therefore, within 3–4 weeks, as many as of 2×10¹³ vp or 200 units of AAV can be generated with this approach.

Figure 1. Strategic planning workflow.

Top illustration: Timeline for optimal work-flow through Basic Protocol 1 (BP1) for 2 AAV preparations (prep), starting with the first AAV prep on Thursday of week 1 and the second AAV prep on Thursday of week 2. Bottom illustration: Timeline for optimal workflow through Basic Protocol 2 (BP2) for 2 AAV preps, starting with the first AAV Prep on Tuesday of week 3 and the second AAV Prep on Tuesday of week 4.

BASIC PROTOCOL 1:adeno-associated virus (AAV) Production

Introductory paragraph:

Here, we describe our Basic Protocol 1 for AAV production, which entails the use of AAVPro 293T cell culture, transfection, and cell lysis for AAV particle harvest. Specifically, this protocol encompasses the following major steps:

1. Passaging of AAVPro-293T cells

2. Plating the AAVPro-293T cells onto T-175 TC flasks

3. Transfection of AAVPro-293T cells for recombinant AAV production

4. Harvest of recombinant AAV particles from AAVPro-293T cells

By the end of Basic Protocol 1, cell lysates containing AAV should be ready for subsequent purification, which is further described in Basic Protocol 2.

CAUTION: AAV is a Biosafety Level 2 pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms.

Materials:

AAVPro 293T Cells (Takara, cat. no. 632273)

DMEM F12 (Gibco, cat. no. 11320033, or Cytiva, cat. no. SH30023.FS)

Fetal Bovine Serum (Omega, or equivalent)

Penicillin-Streptomycin-Glutamine (100X) (Gibco, cat. no. 10378016)

Amphoterecin B (10mg/ml) (Sigma, A9528)

Culturing Media (see recipe in Reagents and Solution)

TrypLE Express Enzyme (1X) (Gibco, cat. no. 12605010)

DPBS, no calcium, no magnesium (1X) (Gibco, cat. no. 14190250)

Transfection Media (see recipe in Reagents and Solution)

Polyethylenimine (5.7mg/ml = 10X) (Polysciences 24765–100)

pAAV2/9n: rep/cap gene expression construct (Addgene #112865)

pAdDeltaF6: AAV helper plasmid (Addgene #112867)

rAAV plasmid (Backbone vectors are available on Addgene)

Lysis Buffer (see recipe in Reagents and Solution)

37°C Water bath

150mm tissue culture (TC) dishes (Fisher, cat. no. FB012925, or equivalent)

Laminar flow hood (Labconco Purifier BSC Class II, or equivalent)

Motorized pipette controller (Pipet aid, VWR, or equivalent)

Serological pipets, sterile (100ml, 50ml, 25ml, 10ml, 5ml, from VWR, or equivalent)

15ml and 50ml conical centrifuge tubes (Corning, or equivalent)

Centrifuge that can hold a swinging bucket rotor (Eppendorf, or equivalent)

Swinging bucket rotor with 50ml conical inserts (Eppendorf, or equivalent)

Single channel pipet (1000µl, 200µl, 20µl)

Nalgene sterile disposable filter units with PES membranes (Thermo Scientific, cat. no. 5670020, or equivalent)

Nalgene sterile disposable 250ml or 500ml receiver (Thermo Scientific, cat. no. 455–0250 or 455–0500, or equivalent)

Tissue culture microscope (ECHO, or equivalent)

Water-jacketed CO2 incubator (Thermo Scientific, or equivalent)

Hemocytometer

175‐cm² (T‐175) cell culture flasks, vented cap (Corning, cat. no. 355001, or equivalent)

Protocol steps with step annotations:

1. Thaw 1 frozen vial consisting of 1ml of 8–10×10⁶ AAVPro-293T cells quickly in a 37°C water bath. Once thawed, spray vial with appropriate disinfectant, e.g. 70% ethanol, before entering a laminar flow hood.

   Note: Ensure vial cap is not submerged in water to prevent contamination.

   The cell number per vial is crucial in order for the subsequent steps to occur according to the schedule outlined in the Strategic Planning section (Figure 1). However, if a lower cell number is used, start this step earlier than what is outlined in Figure 1.

   If purchasing a new vial from a vendor, consider growing the cells according to vendor instructions, and passaging until several 150mm dishes are confluent. These cells can then be frozen, wherein 1 confluent 150mm TC dish yields sufficient cells for 2 cryovials consisting of 8–10×10⁶ of AAVPro 293T cells each. We recommend passaging and freezing cells to build an adequate supply of frozen vials for future AAV preparations.

2. Add 1ml of 37°C culturing media (See reagents and solutions) drop-wise to thawed cells in vial, then collect with a serological pipet and add, drop-wise, to 3ml of culturing media in a 50ml conical tube.

3. Centrifuge at 1500xg for 5 minutes.

4. During that time, add 10ml of culturing media to one 150mm TC dish.

5. After centrifugation of the cells, aspirate the supernatant, and resuspend the cell pellet in 5ml of culturing media, using a 5ml serological pipet to resuspend the cell pellet gently but thoroughly, pipetting up and down 3–4 times.

6. Add the 5ml of cells to the 150mm TC dish drop wise. Rock 3–4 times and incubate in a 37°C water-jacketed incubator with 5% CO₂ for 16–24 hours.

   Apart from centrifugation and microscopic visualization, ensure all cell culture steps are performed in a laminar flow hood.

7. Visually inspect cells the next day to ensure that they are at least ~70% confluent, then proceed to the next step. If they have not reached the required confluence, maintain them in the incubator until ~70% confluency is reached (Figure 2), after which you may proceed to the next step.

   Passage cells in a laminar flow hood by performing a 1:8 split

8. using the following procedure: Aspirate media, add 10ml of dPBS to the dish, rock 1–2 times, aspirate dPBS, add 1ml TrypLE, and incubate at room temperature for 2–3 minutes.

9. Then, rock 3–4 times to ensure that the cells are no longer adherent to the dish. At this step, you should visually see cells lift off the dish.

10. Add 4ml of culturing media to collect trypsinized cells into a 50ml conical, and centrifuge at 1500xg for 5 minutes.

11. During centrifugation of the cells, wash the 150mm TC dish with 7–10ml of dPBS, and aspirate. This ensures complete removal of TrypLE from the dish.

12. To this dish and 3 new 150mm dishes, add 14ml of culturing media to each. After centrifugation of cells, aspirate supernatant from the 50ml conical and resuspend cell pellet in 8ml of culturing media, using a 5ml or 10ml serological pipet to resuspend the cell pellet gently but thoroughly, pipetting up and down 3–4 times.

13. Add 1ml of cells to each of the four 150mm TC dishes, and rock 3–4 times for even plating. Place in a 37°C water-jacketed incubator with 5% CO₂ for 72 hours. The remaining 4ml of cells can be frozen in cryovials or discarded.

    To reduce plastic use and for cost-effectiveness, we re-use previously used 150mm TC dishes, after a wash with dPBS.

    A 72-hour incubation is used here for efficiency, where 1 frozen vial is thawed and plated on one 150mm TC dish on Thursday and cells are passaged for a 1:8 split on Friday, resulting in four 150mm TC dishes that are ~70% confluent on Monday (Figure 1). The scheduling paradigm in Figure 1 is an example, and a recommendation to streamline the protocol for a Monday to Friday workweek but can be designed according to the research personnel’s interest. No more than a 1:8 split is recommended.

    Note that the volume used to resuspend the cell pellet can be adjusted, as long as one 150mm dish has a total volume of 15ml.

14. After 72 hours, visually inspect cells to ensure all four 150mm TC dishes are at least 70% confluent. Passage the cells by trypsinization, as described in Steps 8 to 12 but for a 1:2 split, i.e. cells from four 150mm TC dishes should be resuspended in 8ml of culturing media, then plated 1ml each onto eight 150mm TC dishes. Incubate 16–24 hours in a 37°C water-jacketed incubator with 5% CO₂.

    To reduce plastic use and for cost-effectiveness, we re-use previously used 150mm TC dishes, as was done in Step 4. For example, after trypsinization and collection of cells from the four 150mm TC dishes, wash the used 150mm TC dishes by adding 7–10ml of dPBS, then rock, and aspirate before adding culturing media and cells. Obtain 4 new 150mm TC dishes for the other 4ml of cells.

15. Repeat Step 14 every day for 2 days, until you have thirty-two 150mm TC dishes. This completes the first major step of Basic Protocol 1: Passaging AAVPro-293T cells. The next step, which is the second major step in Basic Protocol 1, is to plate the AAVPro-293T cells on T-175 flasks.

    According to the Strategic Planning outline (Figure 1), at this point you should have thirty-two 150mm TC dishes on Thursday.

16. Trypsinize all the cells, as previously described in Steps 8 to 12, but after centrifugation, resuspend the cells in 30ml of culturing media.

17. Perform a 1:10 dilution by adding 100µl of cells to 900µl of Culturing Media and perform a cell count by adding 10µl of the diluted cells to a hemocytometer. The total cell count from 32 confluent 150mm TC dishes should be between 520–560×10⁶ cells.

18. Resuspend the cells to a concentration of 11×10⁶ cells/ml with culturing media, i.e. should be about 50ml, depending on the exact count.

19. Add 17ml of culturing media to 48 T-175 cell culture flasks. Then, add 1ml of cells (i.e. 11×10⁶ cells from Step 7) to each flask. Rock, and incubate in a 37°C water-jacketed incubator with 5% CO₂ for 16–24 hours.

20. If another AAV preparation is desired, as outlined in Figure 1, proceed to Steps 21 and 22. If not, skip Steps 21 and 22. This step completes the second major step of Basic Protocol 1: Plating on T-175 flasks.

    The cell density has been crucial for high AAV production (Figure 3). If less than the expected total number of cells is achieved, use fewer flasks. Next time, adjust the number of TC dishes plated on Friday (Figure 1) so that you have enough cells for 48 flasks at 11×10⁶ cells/flask.

    If desired, AAV production can be done using T-225 or T-182 cell culture flasks. Table 2 lists the pertinent adjustments when using different sized flasks.

21. To one 150mm dish, add 8–10×10⁶ cells (there should be enough cells left over after Step 8), and bring up to 15ml with culturing media. Incubate in a 37°C water-jacketed incubator with 5% CO₂ 16–24 hours.

22. On the next day, perform a 1:8 split of the single 150mm TC dish from Step 21, keeping 4 dishes, as described in Steps 8 to 12. This restarts the protocol from Step 8 to Step 20, for a subsequent full AAV preparation.

    According to Strategic Planning (Figure 1), you should be performing this 1:8 split on Friday, so that four 150mm TC dishes are confluent by Monday.

23. On the next day, begin the third major step of this protocol: Transfection of AAVPro-293T cells on T-175 flasks. Prepare six 50ml conical tubes. To three of the 50ml conicals, dilute the transfection reagent, polyethylenemine (PEI), with the volumes listed in Table 3. To the remaining three 50ml conicals, dilute the helper plasmids and recombinant AAV plasmid according to the volumes listed in Table 3. Allow to sit at room temperature for 5 minutes.

    The helper plasmid, pAdDelta6, encodes the functional proteins that facilitate AAV production. pAAV2/9 specifically encodes capsids of AAV serotype 9, though vendors like Addgene have plasmids encoding capsid proteins for various AAV serotypes. We generate AAV serotype 9, as that has proven to be the most cardiotropic, our organ of interest (Bish, 2008; Inagaki, 2006). The recombinant AAV plasmid is your cloned or purchased recombinant AAV plasmid that will become the recombinant AAV genome.

24. Combine the diluted PEI from one conical with the diluted plasmids from one conical. Repeat for the remaining 2 pairs of conical. Vortex vigorously for 1 minute. Incubate mixture at room temperature for 30 minutes, then combine the contents of one 50ml conical (roughly ~18ml of PEI and plasmid mixture) with 270ml of transfection media (See reagents and solutions; Table 3). The contents of each 50ml conical are calculated for 16 T-175 flasks.

    Critical Point! Keep to the times stated to ensure PEI and DNA complex formation.

    If using different sized flasks, use the volumes listed in Table 3.

25. Aspirate culturing media from the 16 T-175 flasks and replace with 18ml of the transfection media and PEI/plasmid mixture from Step 24. Rock 4–5 times before placing in a water-jacketed 37°C 5% CO₂ incubator.

26. Repeat Steps 24 and 25 for the remaining two 50ml conicals and 32 T-175 flasks. Incubate all 48 T-175 flasks in a water-jacketed incubator 37°C 5% CO₂ incubator for 3 to 4 days. This completes the third major step of Basic Protocol 1: Transfection of AAVPro-293T cells.

    Incubation at 3 or 4 days has resulted in the same AAV yield (Figure 4).

27. Three or 4 days later (Monday or Tuesday, if following the Strategic Planning outline in Figure 1), begin the fourth major step of Basic Protocol 1: Harvesting of AAV from the cells. First, set up twenty-four 50ml conical tubes in a laminar flow hood.

28. Then, move 12 of the 48 T-175 flasks from the 37°C 5% CO₂ incubator into the laminar flow hood. Collect all of the media from 2 T-175 flasks with a serological pipet into one 50ml conical.

29. Repeat for the remaining 10 T-175 flasks. You should end with six 50ml conicals with ~36ml of collected media. To each bare T-175 flask, add 3ml of TrypLE, rock 3–4 times to evenly coat the surface, and place back in the 37°C 5% CO₂ incubator for about 10 minutes.

30. During the 10-minute incubation time, repeat Steps 28 and 29 with another 12 T-175 flasks,. By the time these flasks have TrypLE and are ready to incubate in the 37°C 5% CO₂ incubator, the first set of flasks from Steps 28 and 29 will be ready for cell harvest. Move the first set of flasks back into the laminar flow hood, and rock 3–4 times to ensure the cells have been efficiently trypsinized from the surface of the flask. To 2 T-175 flasks, with a 10ml pipet, add ~10ml of the collected media from one 50ml conical to each flask, and wash the surface of the flask to collect the trypsinized cells. Add the cells into the same 50ml conical. Repeat for the remaining 10 flasks.

31. By the end of Step 30, the second set of 12 T-175 flasks should be ready for cell harvest. Repeat Step 30 for the second set of T-175 flasks. By the end of this step, you should have 24 empty T-175 flasks, and twelve 50ml conicals full of media and cells (~39ml/conical).

    Optional: if intending to perform a dual AAV preparation of the same virus, these T-175 flasks can be re-used for the next AAV preparation but only if making the same recombinant AAV. This significantly reduces the cost associated with 2 AAV preparations. To re-use, add dPBS to wash the flasks of residual cells and TrypLE, and aspirate. Leave in the laminar flow hood without UV on. To increase efficiency, another member of the lab should be recruited for washing of T-175 flasks. We have not detected any contamination from this process that has affected our in vivo applications.

32. Centrifuge the twelve 50ml conical tubes from Step 31 gently at 300xg for 10 minutes at room temperature. After centrifugation, there should be a pellet of cells. Remove the media supernatant with a 25ml serological pipet into a biohazard waste collection container. Add 3ml of AAV lysis buffer (See reagents and solutions) to each 50ml conical tube, resuspend with a 10ml pipet, and combine cell pellets from 6 conicals into one. By the end of this step, you should have two 50ml conical tubes with about ~20ml of cell lysate. Place these conical tubes on ice.

    AAV particles in waste media can be subjected to ammonium sulfate extraction to harvest any virus remaining in the media; however, in our hands generates a negligible increase in the yield of AAV (Figure 4). For this reason, media disposal in a biohazard waste collection container is recommended.

    The biohazard waste collection container can be bleached for an hour before disposal or disposed of according to your institution’s recommendations.

33. Repeat Steps 28 to 32 for the remaining 24 T-175 flasks. By the end of this step, there should now be a total of four 50ml conical tubes with ~20ml of cell lysate.

    The set up for Steps 28 to 33 are designed according to our optimized workflow, but can be adjusted, as long as a total of ~80ml of cell lysate is obtained at the end, as this volume is crucial for the success of Basic Protocol 2: AAV Purification.

    Note that Steps 28 to 33 take about 2 hours for one individual. It is recommended that 1–2 more personnel be recruited if wanting to reduce the time to 1 hour.

34. Next, perform 3 rounds of freeze-thaw on the four 50ml conicals of cell lysate. Freeze lysates by placing in a bath of dry ice and methanol for 20–30 minutes, and thaw in a 37°C water bath for 20–30 minutes. After 3 rounds of freeze-thaw, store at −80°C until ready for Basic Protocol 2 – AAV Purification.

    You may freeze cell lysates at −80°C, but expect 1 hour for complete freeze.

Figure 2. Representative image of AAVPro-293T cell density 24 hours after thaw.

A frozen vial of approximately 10×10⁶ AAVPro-293T cells were seeded onto one 150mm TC dish. The image shows the confluency after 24 hours of incubation. The image shown is the maximal confluency needed for continued passaging. Ideally, cells should appear as shown in the image or 20–30% less confluent at minimum. Line bar indicates 380 µm; Magnification: 10X.

Figure 3. Optimal AAVPro-293T cell density on T-175 flasks for AAV production.

A. Western blot and Coomassie blue stain of a Bis-Tris gel loaded with a standard of known viral titer and two AAV preparations, one produced from AAVPro-293T cells seeded at 14×10⁶ cells/T-175 flask, and the other at 11×10⁶ cells/T-175 flask. The AAV yields listed are from 48 T-175 flasks incubated for 3 days post-transfection; B. Representative images of AAVPro-293T cells seeded on T-175 flasks at 14 or 11×10⁶ cells/flask. vp: viral particles, M: million; note the # Units at 1×10¹¹ vp is indicative of how many mice can be injected at 1×10¹¹ vp dose, our typical dose injected to animals by tail-vein. Line bar indicates 380 µm; Magnification: 10X.

Table 2.

Adjustments for different flask sizes.

Flask Size:	Culturing/Transfection Media (ml)	AAVPro-293T Cell Density (Million/ml):	#Flasks for a single AAV preparation
T-175	18	11	48
T-182	19	12	46
T-225	23	14	38

Table 3.

AAVPro-293T Transfection.

Prepare the transfection reagent by diluting with DMEM as follows:
		Per 16 T-175 flasks (Add the listed volumes to 3 50ml conicals each)	Per 12 T-182 flasks (Add the listed volumes to 3 50ml conicals each)		Per 19 T-225 flasks (Add the listed volumes to 2 50ml conicals each)
1X Polyethylenimine (PEI) *Dilute the 10X PEI stock (0.517mg/ml) to 1X with molecular biology grade water		2.56ml	2.56ml		3.84ml
1:1 DMEM:F12 no antibiotic		16ml	16ml		24ml
Prepare the helper and recombinant AAV plasmids by diluting with DMEM as follows:
		Per 16 T-175 flasks (Add the listed volumes to 3 50ml conicals each)	Per 12 T-182 flasks (Add the listed volumes to 3 50ml conicals each)			Per 19 T-225 flasks (Add the listed volumes to 2 50ml conicals each)
AAV plasmid		80µg	80µg			120µg
pAdDeltaF6		160µg	160µg			240µg
pAAV2/9n		80µg	80µg			120µg
1:1 DMEM:F12 no antibiotic		16ml	16ml			24ml
Allow the diluted PEI and plasmids to sit separately for ~5 minutes at room temperature. Then, combine the diluted PEI to the diluted plasmids and vortex vigorously for 1 minute. Add to Transfection media as follows:
Flask Size	Transfection Media (For each 50ml conical of PEI/plasmid mixture)			#Flasks (For each 50ml conical of PEI/plasmid mixture)
T-175	270			16
T-182	273			12
T-225	385			19

Figure 4. Comparison of AAV yields from cell lysate or media, and after 3 or 4 days of incubation post-transfection.

A. Western blot and Coomassie blue stain of a Bis-Tris gel loaded with a standard of known viral titer and two AAV preparations demonstrating the AAV yield from cell lysate or media, when AAVPro-293T cells are incubated for 3 or 4 days. The AAV yields are from 12 T-175 flasks; B. Representative images of AAVPro-293T cells at 3 and 4 days post-transfection. vp: viral particles, d: day; note the # Units at 1×10¹¹ vp is indicative of how many mice can be injected at 1×10¹¹ vp dose, our typical dose injected to animals by tail-vein. Line bar indicates 380 µm; Magnification: 10X.

BASIC PROTOCOL 2: adeno-associated virus (AAV) Purification

Here, we describe our Basic Protocol for AAV purification, which entails 2 major steps:

1. Iodixanol gradient centrifugation to separate the AAV particles from other cellular contents of the AAVPro-293T cell lysates.

2. Purification of AAV particles using an ultrafiltration and concentrator device, that also facilitates the replacement of Iodixanol with Lactated Ringer’s solution.

By the end of Basic Protocol 2, high titer AAV in a safe, injectable solution will be ready for administration in vivo.

CAUTION: AAV is a Biosafety Level 2 pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms.

Materials:

Benzonase 250U/µl (Millipore Sigma, cat. no. 71205–3)

MgCl₂ (1M) (Sigma M1208)

Optiprep (Sigma-Aldrich, cat. no. 1556)

15%, 25%, 40%, and 60% Iodixanol solutions (See reagents and solutions)

TD buffer (5X) (See reagents and solutions)

NaCl (3M) (See reagents and solutions)

Molecular biology grade water

Phenol Red (0.1%) (Sigma-Aldrich P4758)

Bis-Tris protein gels (4–12%) (Bio-Rad, cat. no. 3450124)

MES Running buffer (20X) (Bio-Rad, cat. no. 1610789)

Laemmli sample buffer (4X) (Bio-Rad, cat. no. 1610747)

β-Mercaptoethanol (Sigma-Aldrich, cat. No. M6250)

Precision Plus Kaleidescope prestained protein ladder (Bio-Rad, cat. no. 1610375)

Lactated Ringer’s solution (NDC 0990–7953-09)

Coomassie blue stain (InstantBlue, Abcam, cat. no. ab119211)

Centrifuge equipped with a swinging bucket rotor (Eppendorf, or equivalent)

Swinging bucket rotor with 50ml conical inserts (Eppendorf, or equivalent)

OptiSeal ultracentrifuge 32.4ml tube kit (Beckman, cat. no. 361662)

Laminar flow hood (Labconco Purifier BSC Class II, or equivalent)

Motorized pipette controller (Pipet aid, VWR, or equivalent)

Serological pipets, sterile (25ml, 10ml, 5ml, VWR, or equivalent)

Spinal needle: 18G x 3.5 inches (Med Vet International, cat. no. 29660)

Weighing Scale (Ohaus, cat. no. 1450-SD)

Optima XE-90 ultracentrifuge (Beckman, cat. no. A9983)

Type 70 Ti fixed-angle titanium rotor (Beckman, cat. no. 337922)

18G hypodermic needles (BD 305195, or equivalent)

10ml sterile syringes (Fisher 14–955-459, or equivalent)

2.0ml sterile tubes (Fisher-Scientific, cat. no. 02–681-375)

Single channel pipet (1000µl, 200µl, 20µl, 10 µl)

1.5ml microcentrifuge tubes (VWR, or equivalent)

Heat block

Criterion cell and power supply (Bio-Rad, cat. no. 1656019)

Container for coomassie blue staining

Microcentrifuge for 1.5ml/2ml tubes (Thermo Scientific, or equivalent)

Vivaspin 20 100kDa MWCO (Sartorius, cat. no. 28–9323-63)

Protocol steps with step annotations:

1. Thaw the four 50ml conical tubes of cell lysate containing AAV particles from Basic Protocol 1 in a 37°C water bath for 20–30 minutes. Once thawed, to each of the four 50ml conical tubes, add 11µL Benzonase Nuclease and 20µL of 1M MgCl₂. Incubate in a 37°C water bath for 30 minutes.

2. Centrifuge at 3200xg for 20 minutes at room temperature to collect insoluble cellular contents. The supernatant will contain soluble proteins, as well as AAV that can now be subjected to purification by Iodixanol gradient centrifugation.

3. Set up 8 OptiSeal ultracentrifuge tubes for iodixanol gradient centrifugation in a laminar flow hood. To each ultracentrifuge tube, 15%, 25%, 40%, and 60% iodixanol phases will be added, according to the volumes listed in Table 4. Begin by adding the 15% iodixanol phase with a 5ml serological pipet to the bottom of each ultracentrifuge tube. Then, use a spinal needle and 10ml syringe to set up each subsequent phase, placing the needle all the way at the bottom of the tube, so that each phase is layered below the other (Figure 5A and 5B). Then, layer the AAV9-containing cell lysate (~10ml/ultracentrifuge tube) carefully on top of the 15% phase using an 18G needle and 10ml syringe. Bring the cell lysate layer up to the base of the tube’s neck with AAV lysis buffer.

   Tip: Bend the 18G needle so that it touches the inner plastic tube so that transferred contents falls gently on top of the 15% phase (Figure 5C and D).

   8 ultracentrifuge tubes are sufficient for ~80ml of cell lysate generated from 48 T175 flasks in Basic Protocol 1.

   The spinal needle and syringe used to set up the 25%, 40%, and 60% iodixanol phases can be re-used. Between the setup of each phase, wash the needle and syringe with molecular biology grade water. Do not use the syringe or needle for any purpose other than iodixanol gradient setup.

4. Balance the ultracentrifuge tubes using a weighing scale (Figure 5E). When balancing tubes, place a pair on the balance together, and remove/add lysate from one to the other tube until they are balanced. This pair of tubes will be placed directly across from each other on the 70 Ti rotor to ensure perfect balance.

   Critical: Ensure ultracentrifuge tubes are balanced before centrifugation!

5. Seal the tubes with the plastic corks provided in the OptiSeal ultracentrifuge tube kit. Press down firmly on caps to ensure closure.

6. Centrifuge in a 70 Ti rotor in an ultracentrifuge at 69,000 RPM at 16°C for 1 hour with maximum acceleration and coast (no brake) on deceleration. On an Optima XE ultracentrifuge, the setting for acceleration and deceleration is 0 and 10, respectively. At the end of ultracentrifugation, the genome containing AAV will settle into the 40–60% interface and the clear 40% iodixanol phase (Figure 5F), while all other proteins and empty AAV capsids will settle in the upper 25% phase and 25–40% interface, respectively (Kimura, 2019; Lopez-Gordo, 2019; Zolotukhin, 1999).

   At coast, expect about 30 minutes for the rotor to stop. The coast setting is to ensure that there is no disturbance of the gradient. However, we have set the deceleration to the next lowest setting from, i.e. 9 on an Optima XE, with no disturbance of the layers. At this setting, it takes the rotor about 15 minutes to stop.

7. After the centrifugation is complete, the iodixanol gradients should look as depicted in Figure 5F. Prepare a metal support stand with a burette clamp, as depicted in Figure 5G, in a laminar flow hood. Wipe each ultracentrifuge tube with 70% ethanol. Remove the cap on top of the tube, being mindful not to apply too much force that would disturb the layers. Gently secure one iodixanol gradient tube onto the burette clamp so that it is held in place. For collection of the 40% phase and the 40–60% interface in subsequent steps, set up five 2.0ml tubes with caps off in a rack underneath the iodixanol gradient tube, as shown in Figure 5G.

8. Attach a 5 or 10ml syringe to an 18G needle, and use the syringe to guide the insertion of the needle about ~2 mm below the 40%−60% interface. While applying pressure as you insert the needle, twist the needle clockwise and counterclockwise until a drop appears from the tube, and the needle breaks through the ultracentrifuge tube into the 60% iodixanol phase. Ensure the needle is positioned bevel side up inside the tube.

   No further drops should emanate once the needle is fully inserted through the ultracentrifuge casing. However, twisting of the needle to let out an initial drop has proven crucial for steady flow of solution through the needle at the next step. If no drop occurs, we have found the flow of the solution to be slow, but at no detriment to AAV retrieval. Proceed as usual.

9. Once the needle is fully inserted into the tube, unfasten the syringe from the needle. As soon as the syringe is unfastened, the iodixanol solution will quickly begin to flow through the needle hub.

   Ensure that the first 2.0ml tube is positioned directly beneath the lip of the needle hub to catch the solution as it drips. Once the first 2.0ml tube is about 1ml full, move to the next tube, collecting about 0.5 to 1ml, then the next, collecting 0.25 to 0.5ml, and so on until most of the clear 40% phase has been collected. In total, about 3–4ml is collected per iodixanol gradient tube, divided into fractions among the five 2.0ml tubes. Do not collect any of the 40–25% interface or any of the 25% phase, since these regions contain cellular proteins and empty AAV particles (Kimura, 2019).

   When the 25% phase gets near to the needle, you should be at the last or second to last tube. This ensures that any contamination from the 25–40% interface and 25% phase lies only in the last two tubes, which can be discarded if needed with minor loss to total AAV retrieval.

   Optional: Rather than removing the syringe and allowing the iodixanol phase to drip through the needle hub, you can leave the syringe fastened and pull the 40–60% interface and 40% phase into the syringe directly. However, this method increases the risk of contamination from the 25% phase.

10. Determine the purity of the collected virus by loading 18µl from each 2.0ml tube (supplemented with 6µl of 4X laemmli buffer, 0.3µl of β-mercaptoethanol, and boiled for 5 minutes at 95°C) on a 4–12% Bis-Tris gel with 1X MES buffer. Include a lane with 4µl of Kaleidescope ladder. Run the gel for 200V for 40 minutes. Then, stain with InstantBlue for at least 15 minutes to visualize the viral capsid proteins, VP1, VP2 and VP3, whose molecular weights are 87, 73, and 62 kDa, respectively (Kimura, 2019). If any of the 25% phase was accidentally collected, a smear of proteins running at various sizes will be clear (Figure 6).

    Samples from the last 2–3 tubes need only be run, since they should be the only ones at risk of having been contaminated by the 25% phase. The intensity of the viral capsid protein bands can also provide a subjective estimate of the titer of virus produced.

    Optional: 4–12% Bis-Tris gels are not necessary. Any percent gel can be used.

11. Discard any samples with obvious contamination of the 25% phase (Figure 6). Combine all pure samples into two 50ml conical tubes, with about 10–15ml of sample in each tube. Bring the volume of each conical to at least 40ml with Lactated Ringer’s solution.

12. Wash 4 Vivaspin 20 ultrafiltration devices by adding 10ml of Lactated Ringer’s solution. Centrifuge for 5 minutes at 200xg in a swinging bucket rotor at room temperature.

    Fewer Vivaspin 20 ultrafiltration devices can be used. Four are used in this protocol for the sake of time. If using less than 4 Vivaspin devices, consider diluting the sample by adding Lactated Ringer’s for up to a volume of 50ml in Step 11. This is to safeguard the Vivaspin device from being overloaded with iodixanol. It will take longer to pack ~100ml of 40–60% iodixanol onto fewer columns but diluting the iodixanol ensures optimal flow through the Vivaspin device.

13. Add 20ml of the diluted sample from Step 11 to each of the 4 Vivaspin 20 devices, and centrifuge at 3000xg for 10–15 minutes at room temperature, until the volume of sample has reached between 1 and 5ml, as indicated on the Vivaspin 20 device. After centrifugation, the virus will remain inside the column of the Vivaspin 20 device. The attached collection tube holds the effluent that is to be discarded.

    If the volume reaches between 0.5 to 1ml, decrease the centrifugation time. Do not allow the column to dry. Work quickly to add more sample during this process. If volume continues to reach 0.5 to 1ml despite decreasing centrifugation time, consider lowering the speed to 2000xg.

14. Repeat Step 13 if there is any remaining sample from Step 11. Bring the volume to 20ml with Lactated Ringer’s solution.

15. Then, fill the Vivaspin 20 device up to 20ml with Lactated Ringer’s solution, and proceed to centrifuge at 3000xg for 10–15 minutes at room temperature, until the volume is between 1 and 5ml. Discard the effluent.

    If the volume reaches between 0.5 to 1ml, decrease the centrifugation time or speed.

16. Repeat Step 15 two more times. This repeated centrifugation is necessary for thorough replacement of iodixanol with Lactated Ringer’s solution.

17. Perform a final centrifugation, as in Step 15 but continue to centrifuge the Vivaspin 20 devices until the volume of each sample reaches the 0.2ml mark on the device. This ensures highly concentrated virus. The timing of this step is variable but can take up to 30 minutes.

18. Unscrew the cap of the Vivaspin 20 device, and with a 200µl pipet, collect all traces of sample from inside the column and combine into one 1.5ml tube. Since each device should have at least 0.2ml of sample, there should be at least ~800µl total. Separate 16µl of the virus into 2 microcentrifuge tubes each (8µl/tube). For the remaining sample, measure and take note of the total volume, and aliquot the virus into four 1.5ml tubes. Store at −80°C.

19. To each tube with 8µl of virus, add 4µl of dPBS, then 4µl of 4X Laemmli buffer and 0.3µl of β-mercaptoethanol. Set aside.

20. Prepare a dilution series of an AAV of known titer, i.e. the standard, by adding 16µl of the standard to 4µl of Lactated Ringer’s solution in a tube. From this tube, take 10µl and add to 10µl of dPBS in a new tube, and so on until you have 5 tubes in total. Prepare a technical replicate of two.

    Our standard was provided as a gift from Dr. Roger Hajjar’s lab (Lopez-Gordo, 2019), but an AAV of known titer from Addgene or an alternate source can be used.

21. Boil the samples from Step 19 and 20 at 95°C for 5 minutes. Then, let cool at room temperature for 1 minute, then centrifuge at >12,000xg for 1 minute. Load all samples on a 4–12% Bis-Tris gel with 1X MES running buffer, including 4µl of Kaleidescope ladder. Run for 200V for 40 minutes. Stain with InstantBlue for 1 hour, or overnight to obtain the most sensitive detection of the viral capsid proteins. Wash the gel several times with water to wash off the background stain. This will lead to a cleaner gel for quantification.

22. Perform densitometry analysis of VP3 of the standard AAV and your new AAV sample preparation, using ImageJ. Densitometry of VP3 is performed due to its greater intensity relative to the other viral capsids. Densitometry of the standard virus will yield a standard curve with which you use to determine the titer of the new AAV sample preparation. Specifically, the results of this analysis yields a standard curve plotted as measured densitometry units (arbitrary units on the Y-axis) vs. known concentration (viral particles on the X-axis), with an R-squared value between 0.97 to 1.0. A high R-squared value will ensure the most accurate quantification of your sample. An R-squared value of 0.96 or lower likely indicates pipetting error, and Steps 19 to 21 should be repeated with more careful consideration for sample set-up and gel loading. At the end of Basic Protocol 2, purified recombinant AAV in a safe injectable solution is generated for use to genetically manipulate transgenes in vivo.

Table 4.

Iodixanol Gradient Set-up.

Iodixanol (OptiPrep)	Volume
15% iodixanol	7.3ml
25% iodixanol	4.9ml
40% iodixanol	4ml
60% iodixanol	4ml

Figure 5. Iodixanol gradient ultracentrifugation workflow.

Set-up of iodixanol gradient in Optiseal ultracentrifuge tubes, starting with an illustration of A. A spinal needle and 10ml syringe adding 25% iodixanol phase under the 15% phase to an ultracentrifuge tube. This method is to be repeated for all subsequent phases, resulting in the iodixanol gradient shown in B. Subsequently, C. illustrates cell lysate being layered on top of the 15% iodixanol phase with an 18G needle and 10ml syringe, resulting in the complete iodixanol gradient shown in D. Note the color of the iodixanol phases have changed to a slightly pinker hue over time, but have no impact on subsequent steps. Weigh the ultracentrifuge tubes using weighing scales as in E, followed by ultracentrifugation. After ultracentrifugation, the iodixanol gradient tubes appear as shown in F. Set up and method for 40% iodixanol phase extraction is shown in G and H, respectively. L=AAVPro-293T cell lysate.

Figure 6. Analysis of 40% iodixanol fractions collected after iodixanol gradient ultracentrifugation.

A. Western blot and Coomassie Blue stain of a Bis-Tris gel loaded with 18µl from the last three 2.0ml tubes used to collect the 40% phase after ultracentrifugation. Lanes marked 3, 4, 5 indicate samples from the last three 2.0ml tubes collected from the first iodixanol ultracentrifuge tube. Lanes marked 8, 9, 10 indicate samples from the last three 2.0ml tubes collected from the second iodixanol ultracentrifuge tube, and so on, for a total of four iodixanol ultracentrifuge tubes represented. B. and C. show the same set-up as A., but with samples representing 25% phase contamination, indicated by X. These samples are to be discarded to preserve AAV purity.

REAGENTS AND SOLUTIONS:

Culturing Media

• 10% FBS – 110ml of Fetal Bovine Serum (Omega, or equivalent)

• 1X PSG – 10ml of 100x PSG

• 80µl of 10mg/ml Amphoterecine B

• Sterile filter with a Nalgene filter and receiver

• Store at 4°C for up to 6 months

• This recipe is for 1L. Two liters will be needed for one AAV preparation

• Preparation should be conducted in a laminar flow hood to maintain sterility

Transfection Media

• 2% FBS – 20ml of Fetal Bovine Serum (Omega, or equivalent)

• 1x PSG – 10ml of 100x PSG

• 80µl of 10mg/ml Amphoterecine B

• Sterile filter with a Nalgene filter and receiver

• Store at 4°C for up to 6 months

• This recipe is for 1L. Two liters will be needed for one AAV preparation

• Preparation should be conducted in a laminar flow hood to maintain sterility

AAV Lysis Buffer

• 150mM NaCl – 8.77g of NaCl

• 50mM Tris-HCl – 6.06g of Tris Base

• Bring up to 1L with molecular biology grade water

• pH to 8.5

• Sterile filtration is not necessary here but recommended

• Store at room temperature for up to 6 months

5X TD Buffer

• 5x PBS – 500ml of 10x PBS

• 5mM MgCl₂ – 5.0ml of 1M MgCl₂

• 12.5 mM KCl – 0.93g of KCl

• Bring up to 1L with molecular biology grade water

• Sterile filtration is not necessary here but recommended

• Store at room temperature for up to 6 months

3M NaCl

• 43.83g of NaCl

• Bring up to 250ml with molecular biology grade water

• Sterile filtration is not necessary here but recommended

• Store at room temperature for up to 6 months

15% Iodixanol

• 45ml Optiprep

• 60ml of 3M NaCl

• 36ml of 5x TD buffer

• 39ml of molecular biology grade water

• Do not sterile filter, but preparation should be conducted in a laminar flow hood

• Store at room temperature for up to 3 months

• This recipe is for 180ml. This will be sufficient for 3 sets of 8 ultracentrifuge tubes

25% iodixanol

• 50ml Optiprep

• 24ml of 5x TD buffer

• 46ml of molecular biology grade water

• 300ul of 0.5% phenol red

• Do not sterile filter, but preparation should be conducted in a laminar flow hood

• Store at room temperature for up to 3 months

• This recipe is for 120ml. This will be sufficient for 3 sets of 8 ultracentrifuge tubes

40% Iodixanol

• 58ml Optiprep

• 20ml of 5x TD buffer

• 12ml of molecular biology grade water

• Do not sterile filter, but preparation should be conducted in a laminar flow hood

• Store at room temperature for up to 3 months

• This recipe is for 100ml. This will be sufficient for 3 sets of 8 ultracentrifuge tubes

60% Iodixanol

• 36ml Optiprep

• 90ul of 0.5% phenol red

• Do not sterile filter, but preparation should be conducted in a laminar flow hood

• This recipe is for 1 set of 8 ultracentrifuge tubes.

• This should be made fresh every time.

COMMENTARY:

Background Information:

AAV generation is used as a method for genetic manipulation in vivo, in addition to, or in place of traditional forms of genetic manipulation, namely transgenesis (Kumar, 2009). AAV is a popular alternative to transgenesis due to its relatively low cost of production and comparably quick production, qualities that together add to overall greater versatility of AAV as a tool. As a popular means for genetic manipulation in vivo, procedures for generating high titers of recombinant AAV have grown in popularity, with industries devoted to its production in high-throughput procedures. The method detailed here suits an academic lab/institution that seeks to generate their own AAV without specialized equipment and high costs.

Critical Parameters:

The most critical parameters to generating robust AAV yields are rooted in Basic Protocol 1, where plasmid integrity is the most common cause for low AAV yield. Ensure that helper plasmids are primarily super-coiled for efficient transfection, and that recombinant AAV plasmid has intact ITRs. A common method to check for ITR integrity is to perform a restriction digest with SmaI. An AAV plasmid should have 2 ITRs, and within each is a SmaI restriction site. Therefore, at least 2 bands should be generated from a SmaI restriction digest, more if internal SmaI sites exist. Ensure you know your sequence well to predict the result of the restriction digest and confirm intact ITRs.

In addition, cell confluency on T-175 flasks at the time of transfection is a crucial factor in determining high AAV titer (Figure 3). Lastly, the harvest of AAV at the end of Basic Protocol 1 details the use of TrypLE to harvest cells. Using TrypLE to gently detach cells from the flask is optimal for obtaining high yields of AAV. Using cell lifters/cell scrapers can result in significant loss of AAV, likely due to disruptive nature of cell lysis (Data not shown). Therefore, to most efficiently retrieve AAV produced from AAVPro-293T cells, gentle trypsinization is highly recommended.

Troubleshooting:

Table 5.

Troubleshooting Guide for Low AAV Yield Anchor this table under the header Troubleshooting

Problem	Possible Cause	Solution
Low AAV titer (<60 units worth from 48 T-175 flasks)* *Unit: 1×1011vp	Poor DNA plasmid quality	The most likely reason for low AAV yields is poor DNA plasmid quality. Ensure you check plasmid integrity for all plasmids, by gel electrophoresis. On a 1% agarose gel, run uncut helper plasmid AdF6, uncut AAV2/9n to ensure primarily supercoiled DNA. For the recombinant AAV genome plasmid, run SmaI digested plasmid to ensure integrity of ITRs.
Low AAV titer due to low volume retrieval from Vivaspin device (<400µl), normally associated with unusually quick flow through Vivaspin columns (<8 minutes/wash)	Faulty Vivaspin column, leading to uncontrollable flow through the column, and potential loss of AAV	The unusually quick flow through the column will be evident by the first wash with Lactated Ringer’s, if not earlier. Reduce speed to 2,000xg. If issue persists, immediately replace columns.
Unusually slow flow through Vivaspin columns (>25 minutes)	Iodixanol was not diluted enough Centrifugation at 4°C instead of room temperature	If the iodixanol is too concentrated, for example, you collected more of the 60% phase than intended, the flow through the Vivaspin will take significantly longer at the packing step and may damage the Vivaspin column. Immediately add more Lactated Ringer’s to dilute. If the iodixanol is sufficiently diluted, ensure the centrifuge is at room temperature. The colder the centrifuge, the slower the flow through the Vivaspin.

Understanding Results:

As a result of Basic Protocols 1 and 2, recombinant AAV will be suitable for use in animals, in vivo. Step 10 of Basic Protocol 2 involves loading a sample from the collected 40% phase of each iodixanol gradient to determine purity of the AAV particles generated. This step is also the first step that indicates successful recombinant AAV generation. Clear bands corresponding to VP1, VP2, and VP3 should be clear by 15 minutes of application of InstantBlue (Figure 6). Because genome containing AAV particles migrate to the 40–60% interface and 40% phase of the Iondixinol gradient, while empty AAV capsids sediment at the 25–40% interphase (Kimura, 2019; Zolotukhin, 1999), viral capsid protein bands are a good indicator for AAV genome-containing particle titer, assuming proper collection technique. Alternatively, qPCR of the recombinant AAV genome can be performed to ensure AAV genome-containing particles are present. However, our method has consistently produced titers that when injected by tail-vein in vivo, resulted in efficient transduction of the AAV at 1–3×10¹¹ viral particles (vp).

Using our method, each preparation of 48 T-175 flasks generates enough AAV for ~80 to 120 mice at 1×10¹¹ vp dose. It is highly recommended that a dose-response study is done in vivo to determine AAV-based gene expression at 1 and 3×10¹¹ vp, as, in our experience, different recombinant AAV express differently, depending on the promoter and the desired tissue to be transduced. However, multiple preparations of the same recombinant AAV genome have produced highly consistent transduction at the same dose; therefore, a dose-response need only be performed once for each new recombinant AAV.

Additionally, using an AAV with a fluorescent reporter is a convenient way to visualize gene transduction efficiency at histological and microscopic levels. Another approach is to apply an AAV driving Cre recombinase to a Cre-dependent fluorescent mouse model, for example the mT/mG mice provided by Jackson Laboratories (Stock No. 007676). These methods provide important information as to the AAV transduction efficiency and the strength of the promoter of the recombinant AAV generated.

Time Considerations:

For Basic Protocol 1, about 10–12 hours per week can be expected for one personnel’s time. This time can be reduced significantly with recruitment of additional personnel. The most time-consuming steps in this protocol are plating AAVPro-293T cells on 48 T-175 flasks, and AAV harvest from AAVPro-293T cell lysates. If following the Strategic Planning outlined in Figure 1, these steps fall on Thursday and Monday, respectively.

For Basic Protocol 2, about 12–13 hours per week can be expected for one personnel’s time. Consider that about 3 hours of the 12–13 consist of centrifugation time. The most time-consuming step in this protocol is setting up and running the iodixanol gradient centrifugation. If following the Strategic Planning outlined in Figure 1, this step falls on a Tuesday.

If expecting to perform 2 or more continuous AAV preps, it is recommended that at least 2 individuals be recruited for this method, with one person devoted to Basic Protocol 1 and the other for Basic Protocol 2. This ensures consistency in the protocols and manages time efficiently if the personnel are expected to perform other unrelated tasks, as is often the case in an academic lab.

DATA AVAILABILITY STATEMENT:

The data that support the findings of this study are available from the corresponding author upon request.