Assaying the Stability and Inactivation of AAV Serotype 1 Vectors (2024)

Primary cortical cultures

Rat primary cortical cultures were prepared from Sprague–Dawley embryos, as described previously¹⁹ and in accordance with approved procedures by the NIH Animal Care and Usage Committee. Cells (6.0 × 10⁴) were plated in polyethyleneimine-coated wells (96-well plate), and half-medium exchanges were performed on days in vitro (DIV) 4, 6, 8, 11, and 13. Viral transductions were performed on day 6 in culture.

AAV plasmids, packaging, purification, and titering

AAV packaging plasmids used in the study have been described and can be found on Addgene.org (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/hgtb). All AAV vectors were produced by a triple transfection method⁴ with some modification.¹⁹ Specifically, HEK293 cells are grown in media (Dulbecco's modified Eagle's medium, high glucose [DMEM-HG; Invitrogen, Carlsbad, CA] containing 5% bovine growth serum [BGS; HyClone, Logan, UT] and 1% penicillin–streptomycin) in 20 × 150 mm dishes until 80–90% confluent. Media on each plate was changed to Iscove's modified Dulbecco's medium (IMDM; Invitrogen) containing 5% BGS (transfection media). For each 150 mm dish, 25 μg of pHelper (Stratagene, La Jolla, CA), 18 μg of AAV plasmid coding for transgene, and 7 μg of rep/cap plasmid (pAAV1; aka pXX12) coding for AAV serotype 1 (generously provided by Dr. Xiao Xiao, University of North Carolina at Chapel Hill, Chapel Hill, NC) were added to 2 mL of 0.25 M CaCl₂. The solution was gently vortexed while a 2 ×  HEPES buffered saline (HBS) buffer (280 mM of NaCl, 50 mM of HEPES, and 1.5 mM of Na₂HPO₄) was slowly dripped into the solution. The DNA/CaCl₂/HBS solution was dripped onto each 150 mm dish (4 mL/plate). After 15–17 h, transfection media was replaced with 25 mL/plate 293 media. Forty hours post transfection, the cells were harvested and centrifuged at 800 g for 5 min at 4°C. The pellet was resuspended in 1 mL of 50 mM of Tris–HCl pH 8.0, 150 mM of NaCl, and 2 mM of MgCl₂ (resuspension buffer) per 150 mm dish. The resuspended cells were frozen and stored at −80°C until purification. AAV was affinity purified, as described previously.²⁰ An example of affinity-purified AAV1 is shown in Supplementary Fig. S2.

AAV freeze–thaw procedure

The AAV1-CaMKII-eNpHR3.0-iRFP aliquots were placed on dry ice, and one set of aliquots was thawed for 5 min at room temperature. The aliquots were then placed back on dry ice for 5 min, and the process was repeated until all aliquots had been frozen and thawed the appropriate number of times. At DIV 6 for the primary cortical neurons (PCNs), 95 μL of media was then removed from each well of the 96-well dish, and 5 μL of AAV was added to each well. After 2 h, 100 μL of fresh PCN media was added to each well. Cells were fed with a 50% media exchange on DIVs 8, 11, and 13.

AAV 4°C testing

AAV1-CaMKII-eNpHR3.0-iRFP was thawed, diluted 1:5 and 1:10 in dilution buffer (phosphate-buffered saline containing 0.5 mM of MgCl₂), and placed in a refrigerator at 4°C each week for 7 weeks. At DIV 6 for the PCNs, 95 μL of media was then removed from each well of the 96-well dish, and 5 μL of AAV1-CaMKII-eNpHR3.0-iRFP was added to each well. After 2 h, 100 μL of fresh PCN media was added to each well. Cells were fed with a 50% media exchange on DIVs 8, 11, and 13.

AAV drying test or “metal slug assay”

Seven days prior to transduction, one aliquot of AAV1-CMV-SP-GFP-Cre was thawed, and 5 μL of AAV was added to the center of three autoclaved slugs placed in a 24-well dish. The stainless-steel slugs were obtained from Arrow Metal Fabricators (Baltimore, MD). The slugs were 13 mm in diameter with an average thickness of 1.17 ± 0.03 mm. This was repeated each day for 1 week. At DIV 6 for the PCNs, 95 μL of media was then removed from each well of the 96-well dish, and 5 μL of AAV1-EF1α-DIO-mCherry was added to each well. AAV1-CMV-SP-GFP-Cre (5 μL) was dropped on the slug and immediately transferred to appropriate wells of the 96-well dish of PCNs. Dilution buffer (10 μL) was added to each of the remaining slugs and transferred to appropriate wells of the 96-well well plate. After 2 h, 100 μL of fresh PCN media was added to each well. Cells were fed with a 50% media exchange on DIVs 8, 11, and 13.

AAV autoclaving test

AAV1-CMV-GFP-Cre (5 μL) was added to the center of nine slugs placed in a 24-well dish. Dilution buffer (5 μL) was also added to the center of nine slugs and allowed to dry for 90 min. The slugs were separated into groups of three. Two sets of AAV and two sets of dilution buffer were autoclaved in two different autoclaves (121°C exposure for 30 min with 30 min drying time), while the other set of AAV and dilution buffer were kept at room temperature. The media (95 μL) was then removed from each well of the 96-well dish of PCNs (DIV 6), and 5 μL of AAV1-EF1α-DIO-mCherry was added to each well. Dilution buffer (10 μL) was added to each of the slugs and transferred to appropriate wells of the 96-well well plate. After 2 h, 100 μL fresh PCN media was added to each well. Cells were fed with a 50% media exchange on DIVs 8, 11, and 13.

AAV decontamination testing

AAV1-CMV-SP-GFP-Cre (2 μL) was added to 18 μL of 10% bleach solution, Gladiator (Cello, Havre de Grace, MD), 3.9% peracetic acid (PAA; Sigma–Aldrich, St. Louis, MO), povidone iodine solution (Thermo Fisher Scientific, Waltham, MA), cavicide (Metrex, Orange, CA), 70% isopropanol (Sigma–Aldrich), or dilution buffer. For pre-made PAA solutions are 5–6% PAA in ViorOXLS&D (PeroxyChem, Philadelphia, PA), 0.23% PAA Accel 5 RTU (Virox Technologies, Oakville, ON), and 0.05% PAA VIRASEPT (Ecolab, St Paul, MN). After 5 min and 30 min, 480 μL was added to each mixture to bring the volume up to 0.5 mL. The mixtures were placed in 20,000 MWCO dialysis cassettes (Pierce Chemical, Dallas, TX) and dialyzed in dilution buffer, changing the dialysis buffer three times, with each dialysis lasting at least 2 h. Solutions were removed from the dialysis cassettes, placed in an Amicon spin column (10 KDa; Merck Millipore, Billerica, MA), and spun at 4,000 g for 40 min at 4°C. The concentrated solution was placed in a 4°C refrigerator overnight. At DIV 6 for the PCNs, 95 μL of media was then removed from each well of the 96-well dish, and 5 μL of AAV1-EF1α-DIO-mCherry was added to each well. AAV/decon solution (10 μL) was added to each appropriate well of the 96-well plate (n = 3). After 2 h, 100 μL of fresh PCN media was added to each well. Cells were fed with a 50% media exchange on DIVs 8, 11, and 13.

Immunocytochemistry and image analysis

The media was removed from each well, and 100 μL of fresh 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA) was added to each well for 1 h at room temperature. The PFA was removed, and 200 μL of PBS was added to each well. The plates were then sealed with parafilm to prevent evaporation and aluminum foil to prevent photobleaching, and stored in a 4°C cabinet. For DAPI staining, 100 μL of PBS containing 0.1% Triton X-100 (Sigma–Aldrich) and DAPI (Invitrogen) diluted to 10 μg/mL were added to each well, and the cells were incubated for 10 min at room temperature. The cells were then washed twice in PBS, sealed with parafilm and aluminum foil, and placed in the 4°C cabinet until imaging.

For the AAV drying, decontamination, and autoclaving tests, images were taken with a Nikon Eclipse TE2000-E inverted microscope (Nikon, Melville, NY) equipped with a iXon3 (Andor, Belfast, United Kingdom) and NIS elements software (Nikon). Cells were imaged using three different filters: UV filter for DAPI nuclear staining, FITC green for GFP, and TRITC for mCherry. Exposure times were kept constant for each filter.

For the 4°C and freeze–thaw testing, plates were scanned on the Odyssey infrared scanner (Licor, Lincoln, NE) with the following settings: resolution 42 μm, quality high, focus offset 3.54 cm, and intensity 5.5. The LiCor Odyssey software v3.0 was used to calculate integrate intensities of iRFP expression for each well.

4°C storage

As part of routine use of viral vectors, there are practical considerations such as the storage conditions and how these conditions affect the activity or transduction efficiency. Using an AAV1 vector expressing an infrared fluorescent protein under the control of a neuronal promoter (AAV1-CaMKII-eNpHR3.0-iRFP), this study examined the transduction of primary cortical neurons after storage at 4°C for up to 7 weeks. For undiluted virus (2.1 −5.9 × 10¹² vg/mL), there was up to a 20% loss in transgene expression over 7 weeks at 4°C (Fig. 1a) with a trend of improved but more variable transduction over time. Virus diluted 1:5 or 1:10 had a slightly higher loss (up to 40%) over 7 weeks, with the biggest decrease in activity during the first week.

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Figure 1.

Adeno-associated virus (AAV) stability at 4°C and after freeze–thaw cycles. (a) AAV1-CaMKII-eNpHR3.0-iRFP was diluted and stored at 4°C for up to 7 weeks. Rat primary cortical neurons were transduced on DIV6 with AAV and imaged on day in vitro (DIV) 15. Graphs show each sample normalized to control with mean and standard deviation (SD). # indicates significantly different from the freshly thawed group (0 weeks at 4°C), p < 0.05; three independent experiments, three samples per experiment). * indicates significantly different from all others in group, p < 0.05). (b) AAV1-CaMKII-eNpHR3.0-iRFP was frozen and thawed up to 10 times. Rat primary cortical neurons were transduced on DIV 6 and imaged on DIV 15. Graphs show each sample normalized to control with mean and SD. No significant difference among groups (three independent experiments, two to three samples per experiment).

Freeze–thaw testing

The effect of multiple freeze–thaw cycles (10 maximal) on virus transduction was tested, using the same AAV1 vector as the 4°C stability testing described above. Compared with the first thaw of an aliquot stored at −80°C, the transduction efficiency based on transgene expression was reduced by 10% after the second freeze–thaw cycle (Fig. 1B). The efficiency of transgene expression was further decreased by another 10% on average after the third freeze–thaw cycle. Additional freeze–thaws showed increased variability in transduction ranging from 60% to 120% of the initial thaw (Fig. 1B).

Dehydration testing

AAV viral vectors are produced in biosafety cabinets with stainless-steel surfaces. For neuroscience applications, such as optogenetic experiments in rodents, steel syringes or cannula are often used to infuse AAV vectors into the brain. The “metal slug assay” was developed to examine the stability of AAV on stainless-steel surfaces. Concave metal slugs generated as “hole punches” in stainless-steel metal sheets were obtained, and these were used to hold small volumes of concentrated virus inside 24-well plates (Fig. 2). To create a robust assay for active vector (i.e., capable of conveying transgene expression to target cells), an AAV1 vector expressing a Cre-recombinase translationally fused to green fluorescent protein (AAV-GFP-Cre) was used. In the presence of Cre-dependent reporters that use a strong promoter such as EF1a, any Cre-dependent recombination will lead to production of fluorescent reporter. AAV-GFP-Cre was applied to metal punches and allowed to dry on the slug for up to 6 days at room temperature. Reconstituted virus and a Cre-dependent AAV expressing mCherry fluorescent protein (AAV-EF1a-DIO-mCherry) were used to co-transduce rat primary cortical neurons. Although a time-dependent decrease in the amount of mCherry expression was observed, active AAV-GFP-Cre virus was detected after 6 days on the slug at room temperature (Fig. 2). Nuclear staining with DAPI demonstrated equivalent cells were present at all time points (Supplementary Fig. S3).

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Figure 2.

Metal slug assay. (a) Sterile stainless-steel slugs were placed in a 24-well plate. (b) Ten microliters of AAV1-SP-GFP-Cre was placed in the center of each slug and allowed to dry at room temperature for up to 6 days. AAV was reconstituted and added to rat primary cortical neuron cultures (DIV 6), along with AAV1-EF1α-DIO-mCherry. (c) Cells were imaged on DIV 15 for GFP fluorescence (green) or mCherry fluorescence (red). Images are representative of two independent experiments with two to three samples per experiment. Scale bar = 100 μm.

Ethylene oxide and autoclave testing

Based on the above data, the integrity of the AAV capsid is stable through temperature fluctuations and dehydration. First, ethylene oxide (EtO) was tested for its ability to inactive AAV on a metal surface. EtO is commonly used to decontaminate delicate instruments that cannot be autoclaved. EtO did not completely inactivate AAV-GFP-Cre, but steam sterilization autoclaving resulted in no detectable AAV-GFP-Cre activity (Fig. 3). Independent testing of EtO sterilizer using a Verify^® disposable biological EO test pack (Steris, Mentor, OH) confirmed the EtO was functioning properly (data not shown).

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See Also

Human Immune Responses to Adeno-Associated Virus (AAV) Vectors

Figure 3.

Autoclave and ethylene oxide (EtO) inactivation of AAV. AAV1-SP-GFP-Cre was placed in the center of stainless-steel slugs and allowed to dry. Slugs were left at room temperature and sterilized by autoclave or ETO sterilization as indicated. AAV was reconstituted and added to rat primary cortical neuron cultures (DIV 6), along with AAV1-EF1α-DIO-mCherry. Cells were imaged on DIV 15 for DAPI-stained nuclei (blue), GFP fluorescence (green), and mCherry fluorescence (red). Images are representative of two (ETO) and three (autoclave) independent experiments with three samples per experiment. Scale bar = 100 μm.

Decontamination solution testing

Next, the ability of a variety of commonly used laboratory disinfectants to inactivate an AAV1 vector was examined. Undiluted AAV-GFP-Cre virus was incubated in decontamination solutions for 5 or 30 min and then was diluted, dialyzed, concentrated, and used to co-transduce rat primary neurons with AAV-EF1a-DIO-mCherry (Fig. 4). Of the reagents tested, only 0.5% peracetic acid, 10% bleach, and iodine (1% available) eliminated AAV-GFP-Cre-mediated transduction (Fig. 4). The integrity of the dialysis cassettes after exposure to various decontamination solutions was tested, and any observed loss of transduction was not due to loss of virus from the cassette (Supplementary Fig. S4). Although the use of bleach and iodine was effective, they are not optimal for cleaning of stainless-steel surfaces or instruments due to their corrosive nature. PAA-based solutions are commonly used for decontamination of medical, agricultural, and food production facilities. Three ready-made, commercially available PAA-based solutions were evaluated at the working stock and 1:10 for their ability to inactivate AAV-Cre-GFP. As before, virus was incubated in various decontamination solutions for 5 or 30 min and then was diluted, dialyzed, concentrated, and used to co-transduce rat primary neurons with AAV-EF1a-DIO-mCherry (Fig. 5). Vendor A's stock solution (5–6% PAA) was 100% effective at 5 min using stock or 1:10 dilution. Vendor B's stock solution (0.23% PAA) was 100% effective at the stock solution at 5 min but required 30 min at 1:10 to inactivate AAV-GFP-Cre. Vendor C's stock solution (0.05%) required 30 min and did not completely inactivate AAV. Based on these data, decontamination solutions with 0.25% PAA for 5 min are recommended for inactivating AAV1 vectors.

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Figure 4.

Inactivation of AAV using decontamination solutions. AAV1-SP-GFP-Cre was incubated at room temperature for 5 or 30 min in decontamination solutions (1:10 ratio of AAV to indicated decontamination solution). Solutions were diluted, dialyzed overnight, and concentrated. The AAV was added to rat primary cortical neuron cultures (DIV 6), along with AAV1-EF1α-DIO-mCherry. Cells were imaged on DIV 15 for GFP fluorescence (green) or mCherry fluorescence (red). Images are representative of one (iodine), four (peracetic acid [PAA]), and five (70% IPA, Gladiator, Cavicide, 10% bleach, control) independent experiments with three samples per experiment. Scale bar = 100 μm.

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Figure 5.

Comparison of PAA solutions for AAV inactivation. PAA solutions were obtained from three different vendors (Vendor A = 5–6% PAA, Vendor B = 0.23% PAA, Vendor C = 0.05% PAA). AAV1-SP-GFP-Cre was incubated at room temperature for 5 or 30 min in decontamination solutions (1:10 ratio of AAV to indicated dilution of decontamination solution). Solutions were diluted, dialyzed overnight, and concentrated. The AAV was added to rat primary cortical neuron cultures (DIV 6) along with AAV1-EF1α-DIO-mCherry. Cells were imaged (three independent samples) on DIV 15 for mCherry fluorescence (red). Scale bar = 100 μm.

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Supplemental data:

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