Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.
This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.
Recombinant adeno-associated viral (rAAV) vectors are widely used tools for the treatment of genetic diseases, including spinal muscular atrophy, retinal dystrophy, and hemophilia A1,2,3. rAAV vectors are engineered to lack viral genes present in wild-type AAV4, a small, non-envelope icosahedral virus with a linear single-stranded 4.7 kb DNA genome. AAV was first discovered in the 1960s as a contaminant of adenovirus preparations5. Despite its small capsid size, which limits the size of the transgene that can be packaged to a maximum of 4.9 kb excluding ITRs6, AAV is useful for transgene delivery because it is non-pathogenic in humans, allows expression of transgene in many dividing and nondividing cell types, and has limited immunogenic effects7.
As members of the genus dependoparvovirus, the production of rAAVs relies on the expression of helper genes present in adenovirus or herpes simplex virus8. Several strategies to produce rAAV have been developed, but production in HEK293 cells transformed with the adenoviral E1A/E1B helper genes is the most established method used today9. The general approach of rAAV production begins with the transfection of HEK293 cells with three plasmids containing the transgene within inverted terminal repeats (ITRs), AAV rep and cap genes, and additional adenoviral helper genes, respectively. Seventy-two hours after transfection, cells are harvested and processed to purify rAAV containing the transgene.
In the development of new rAAV vectors for therapeutic purposes, a major goal is producing vectors with increased transduction efficiency. An increase in the transduction efficiency of target cells would mean a decrease in the necessary clinical dose of rAAV, thus decreasing the likelihood of adverse immunogenic effects ranging from antibody-mediated neutralization to acute toxicities10,11. To improve the transduction efficacy of rAAV vectors, alterations can be made to the packaged genome or to the capsid. Viable methods to tune transduction efficacy via packaged genome design include the incorporation of strong and tissue-specific promoters, thoughtful selection of mRNA processing elements, and coding sequence optimization to improve translation efficiency12. Alterations to the capsid are made with the goal of increasing tropism for target human cell types. Efforts towards developing new rAAV transgene delivery vector capsids are generally characterized by a focus on either rational design of AAV capsids with specific mutations targeting specific cell receptors or directed evolution to identify capsids with tropism for specific cell types from high-complexity combinatorial capsid libraries without targeting one specific receptor (although some groups combine these approaches)13,14,15. In the directed evolution approach, combinatorial capsid libraries are constructed using a particular serotype backbone with mutated variable regions on the capsid exterior16. Combinatorial capsid libraries are often constructed from AAV serotypes not originating in humans, decreasing the risk of preexisting immunity during clinical use10. Therefore, purification methods that can be applied to any serotype are ideal to eliminate the need for serotype-specific optimization for the less commonly used serotypes serving as backbones for these libraries.
Iodixanol density gradient centrifugation is utilized to purify high titers of rAAV with high infectivity17. In this protocol, rAAV is produced in suspension cell culture to decrease the amount of labor needed to produce large titers of AAV. A centrifugation step is also included to clear cell lysate to reduce the presence of contaminating proteins and decrease the risk of virus precipitation. This protocol is a cost-effective method to produce preparations of high-purity rAAV suitable for pre-clinical use.
The composition of the solutions and buffers used in this protocol are provided in Table 1.
Solution | Composition | |
AAV lysis buffer | 1.2 mL of 5 M NaCl solution | |
2 mL of 1 M Tris-HCl pH 8.5 solution | ||
80 uL of 1 M MgCl2 solution | ||
mQ water to 40 mL | ||
AAV precipitation solution | 40 g PEG 8000 | |
50 mL of 5 M NaCl solution | ||
mQ water to 100 mL | ||
15% iodixanol fraction | 7.5 mL OptiPrep | |
3 mL of 10X DPBS | ||
6 mL of 5 M NaCl solution | ||
30 uL of 1 M MgCl2 solution | ||
mQ to 30 mL | ||
25% iodixanol fraction | 12. 5 mL OptiPrep | |
3 mL of 10X DPBS | ||
30 uL of 1 M MgCl2 solution | ||
60 uL phenol red solution | ||
mQ to 30 mL | ||
40% iodixanol fraction | 33.3 mL OptiPrep | |
5 mL of 10X DPBS | ||
50 uL of 1 M MgCl2 solution | ||
mQ to 50 mL | ||
60% iodixanol fraction | 50 mL OptiPrep | |
100 uL phenol red solution | ||
AAV buffer solution | 8 mL of 5 M NaCl | |
20 uL of 10% Pluronic F-68 | ||
PBS to 200 mL |
Table 1: Solution compositions for solutions used in this protocol.
1. Triple transfection of Expi293 cells
Figure 1: Expi293 cells expressing GFP two days after transfection. After transfection with a plasmid containing a gene for GFP, the Expi293 cells transiently express eGFP. The cell morphology is round. The image was captured with a 15 ms exposure time. Microscope images are acquired using an inverted microscope equipped with epi-fluorescence illumination and a 10x/0.30 objective. Scale bar = 100 µm. Please click here to view a larger version of this figure.
2. rAAV vector purification
Figure 2: Iodixanol gradient with 40%-60% iodixanol interface labeled. (A) First iodixanol gradient. Phenol red is used in the 40% iodixanol and 60% iodixanol fractions. It appears as a different color because of the difference in pH between the two fractions. The arrow indicates where the syringe should be inserted to harvest the rAAV fraction, just below the 40%-60% iodixanol interface. (B) Second iodixanol gradient. Only the 40% and 60% iodixanol fractions are used in this step. The arrow indicates where the syringe should be inserted to harvest the rAAV fraction. Please click here to view a larger version of this figure.
3. Buffer exchange and virus concentration
This method can be used to obtain titers of at least 1012 viral particles per mL. A titer can be obtained (Figure 3) by qPCR using the ITR primers provided in Supplementary Table 1, by ddPCR, or by any other titering method. Suboptimal titers could result from using a cap gene encoding a capsid with poor packaging efficiency.
Another possible source of suboptimal results is the poor transduction efficiency of Expi293 cells. It is recommended to have cells at a density of 3-4 x 106 vc/mL on the day of transfection and that the cell viability is close to 98%. In the present study, the authors obtained a titer of 1.07 x 1012 vg following this protocol. This yield aligns with previous yields obtained from packaging AAVrh7418.
Figure 3: Titer determination by qPCR. Curve 1 was generated with a standard concentration of 3.66 x 1011 vg/mL, curve 2 was generated with a standard concentration of 3.66 x 1010 vg/mL, curve 3 was the final concentrated AAV sample, curve 4 was generated with a standard concentration of 3.66 x 109 vg/mL, and curve 5 was generated with a standard concentration of 3.66 x 108 vg/mL. Each qPCR reaction was performed in duplicate. (A) qPCR amplification curve. Standards were generated by serial dilution of an AAV standard tittered by ddPCR. The concentration of the undiluted standard was 3.66 x 1012 vg/mL. (B) Standard curve generated by qPCR. The produced AAV was determined to have a concentration of 1.85 x 1011 vg/mL in 5.75 mL, for a total yield of 1.07 x 1012 vg. Please click here to view a larger version of this figure.
Supplementary Table 1: Forward and reverse primer sequences for AAV inverted terminal repeats (ITRs). Please click here to download this File.
The double iodixanol density gradient purification protocol is the universal method because it is applicable to any AAV mutant variants, regardless of their receptor specificity. Early methods of AAV purification relied on particle density and included isopycnic centrifugation in CsCl and continuous sucrose density gradient centrifugation19. Later, serotype-specific approaches were developed, which made use of monoclonal antibodies bound to Sepharose columns20. A novel density-based purification method using a discontinuous iodixanol gradient was developed in 1999 and yielded AAV isolates with higher infectivity than those isolated from CsCl gradients21. Methods to purify AAV continued to be developed into the early 2000s, as some groups used high-performance liquid chromatography to purify rAAV with recovery from crude lysate exceeding 70%22,23,24. These methods are still used in large-scale manufacturing processes25. Despite the development of chromatography methods for isolating AAV, purification by iodixanol density gradient centrifugation is still widely used due to its lower cost and serotype agnosticism26,27.
While this method is well suited for the time-efficient pre-clinical production of rAAV, it is very limited in its scalability and ability to be adapted for cGMP manufacturing25. Thus, as described above, other purification methods are preferred for large-scale production. Additionally, the transgene capacity of rAAV vectors is limited. The size limit for an AAV genome that can be packaged intact has been found to be about 5.2 kb, for a maximum transgene size of 4.9 kb, accounting for ITRs6. This complicates the expression of proteins whose cDNA exceeds this capacity. These proteins cannot be expressed by the system described here without modification of the transgene itself or the use of a split vector system. Chamberlain et al. describe several methods to express transgenes larger than 4.9 kb28.
This method is particularly useful for the production of high-complexity combinatorial capsid libraries containing AAV capsids with varied biochemical properties due to mutations present on the capsid surface. If the rAAV preparation is destined for injection in animals, especially in non-human primates, it is critical to limit endotoxin contamination. Great care must be taken to separate areas used to grow bacterial cultures from rAAV production to prevent bacterial contamination during all steps. Perform all work with human cells and crude AAV in a biosafety cabinet using disposable plasticware. This prevents bacterial contamination and is a necessity for working with AAV, as it is a BSL1 agent.
If a lab does not have access to a CO2 incubator with a shaker, adherent cell culture with HEK293 cells can be used in place of Expi293 suspension cell culture. Follow manufacturer instructions for subculturing and use the transfection protocol described in Crosson et al., 201817.
Critical steps in this protocol are Expi293 transfection, preparation of the iodixanol gradient, and aspiration of the iodixanol fraction containing AAV. Errors during any of these steps can result in suboptimal rAAV yields or failure to produce rAAV.
Transfection of the Expi293 cells is a critical step in the production of rAAV using this protocol. On the day of transfection, cells are resuspended in half a volume of fresh media to provide them with the nutrients necessary to recover from transfection29; transfection is a very taxing process for the cells. However, the conditioned media is not entirely discarded because it contains growth factors and other metabolites important for cell maintenance and growth. It is critical that PEI and DNA incubate for a full ten minutes before being applied to cells in order to form a charged complex30. Without forming a complex with PEI, DNA will not be efficiently uptaken by cells.
When producing rAAV, the proportion of virus found in the media depends on the particular serotype's interactions with Expi293 cell receptors. Some serotypes are released into cell media at higher rates than others31, necessitating that viral particles be precipitated from cell media to capture the highest yield possible and prevent the loss of any variants based on a lack of affinity for Expi293 cell receptors. In contrast, the serotype AAV2 has strong receptor interactions with heparan sulfate proteoglycans present in 293 cells, so very little packaged virus is found in the media32. When producing a derivative of AAV2, omit the PEG precipitation step. For the production of combinatorial capsid libraries or rAAV of alternate serotypes, precipitation of viral particles with PEG is appropriate.
It is critical that 1 M sodium chloride is present in the 15% iodixanol fraction to disassociate aggregated AAV particles during ultracentrifugation. The omission of NaCl in this fraction can lead to undesired outcomes, such as decreases in transduction efficiency and immunogenicity33. Finally, take great care in aspirating AAV from the iodixanol gradient. Aspirating too much of the iodixanol fraction can result in the presence of empty capsids in the final product17.
This method can be used to package rAAV combinatorial capsid libraries for use in downstream-directed evolution experiments to identify capsids with higher tropism for certain human cell types. rAAV is stable at 4 °C for short-term use and at -70 °C for long-term storage34. Currently, the recombinant AAV is in use in five FDA-approved medications treating conditions including Duchenne muscular dystrophy35,36,37. There are several additional rAAV therapies in clinical and pre-clinical trials38,39,40. Further research on this vector is thus critical to the development of additional gene therapies.
The authors have nothing to disclose.
None.
5810 R benchtop centrifuge | Eppendorf | 22625501 | |
8-channel peristaltic pump | Watson-Marlow | 020.3708.00A | |
Automated cell counter | NanoEntek | EVE-MC | |
Avanti J-E high-speed centrifuge | Beckman Coulter | 369001 | |
Benzonase | Thermo Scientific | 88701 | |
Biological safety cabinet | Labconco | 322491101 | |
CO2 incubator with shaker | Set at 8% CO2 and 37 °C | ||
Conical centrifuge tubes | Thermo Scientific | 339652 | 50 mL |
Conical centrifuge tubes | Thermo Scientific | 339650 | 15 mL |
Disposable micro-pipets | Fisherbrand | 21-164-2G | Capillaries |
Dulbecco's phosphate buffered saline without CaCl2 and MgCl2 (DPBS) (10x) | Sigma-Aldrich | D1408 | |
ECLIPSE Ts2R-FL inverted microscope | Nikon | ||
Expi293 Expression Medium | Gibco | A1435101 | |
Expi293F cells | Gibco | A14527 | |
Filter tips | USA Scientific | 1126-7810 | 1000 µL |
Filter tips | USA Scientific | 1120-8810 | 200 µL |
Filter tips | USA Scientific | 1120-1810 | 20 µL |
Filter tips | USA Scientific | 1121-3810 | 10 µL |
Hypodermic needles | Tyco Healthcare | 820112 | 20 GA x 1-1/2 A |
Ice bucket with lid | VWR | 10146-184 | |
JS-5.3 rotor | Beckman Coulter | 368690 | |
Magnesium chloride solution (1 M) | Millipore Sigma | M1028-100ML | |
Metal stand and clamp | Fisherbrand | 05-769-6Q | |
Microcentrifuge tubes | Eppendorf | 22600028 | 1.5 mL |
Needle nose pliers | |||
Optima XE-90 ultracentrifuge | Beckman Coulter | A94471 | |
Opti-MEM I Reduced-Serum Medium | Gibco | 31985062 | |
OptiPrep density gradient media (iodixanol) | Serumwerk | AXS-1114542 | 60% iodixanol solution |
P1000 Pipet | Gilson | F144059M | |
P2 Pipet | Gilson | F144054M | |
P20 Pipet | Gilson | F144056M | |
P200 Pipet | Gilson | F144058M | |
Phenol red solution | Sigma-Aldrich | P0290 | |
Phosphate buffered saline (PBS) | Sigma-Aldrich | P4474 | |
Pipet-Aid XP pipette controller | Drummond Scientific | 4-000-101 | |
Plasmid pCapsid | De novo or Addgene, etc. | N/A | We used pACGrh74. |
Plasmid pHelper | Addgene | 112867 | |
Plasmid pTransgene | De novo or Addgene, etc. | N/A | We used pdsAAV-GFP. |
Pluronic F-68 polyol solution (10%) | Mp Biomedicals | 92750049 | |
Polyethylene glycol 8000 | Research Products International | P48080-500.0 | |
Polyethylenimine HCl Max (PEI-Max) | Polysciences | NC1038561 | Dilute in water to 40 μM |
Polypropylene centrifuge tubes, sterile | Corning | 431123 | 500 mL |
Polypropylene centrifuge tubes, sterile | Corning | 430776 | 250 mL |
Polypropylene Optiseal tubes | Beckman Coulter | 361625 | |
Serological pipettes | Alkali Scientific | SP250-B | 50 mL |
Serological pipettes | Alkali Scientific | SP225-B | 25 mL |
Serological pipettes | Alkali Scientific | SP210-B | 10 mL |
Serological pipettes | Alkali Scientific | SP205-B | 5 mL |
Shaker flasks | Fisherbrand | PBV1000 | 1 L |
Shaker flasks | Fisherbrand | PBV50-0 | 500 mL |
Shaker flasks | Fisherbrand | PBV250 | 250 mL |
Shaker flasks | Fisherbrand | PBV12-5 | 125 mL |
Sodium chloride solution (5 M) | Fisher Scientific | NC1752640 | |
Sterile syringes | Fisherbrand | 14-955-458 | 5 mL |
Syringe filter | Millipore | SLGV013SL | 0.22 micron |
Tris-HCl pH 8.5 (1 M) | Kd Medical | RGE3363 | |
Trypan blue solution | Gibco | 15250061 | |
Tube rack assembly | Beckman Coulter | 361646 | |
Tube spacers (x4) | Beckman Coulter | 361669 | |
Tubing for peristaltic pump | Fisher Scientific | 14190516 | |
Type 70 Ti fixed-angle titanium rotor | Beckman Coulter | 337922 | |
Ultra low temperature freezer | Set at -70 °C | ||
Vivaspin 20 centrifugal concentrator | Sartorius | VS2041 | |
Water bath | Set at 37 °C |