* These authors contributed equally
This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.
Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.
Adeno-associated virus (AAV) vector is conquering its way as one of the most promising delivery systems in current gene therapy studies. Initially identified in 19651, AAV is a small, non-enveloped virus, with an icosahedral protein capsid of about 25 nm in diameter, harboring a single-stranded DNA genome. AAVs belong to the Parvoviridae family and to the Dependoparvovirus genus owing to their unique dependence on co-infection with a helper virus, such as herpes simplex virus or, more frequently, adenovirus, to complete their lytic cycle2,3.
The 4.7 kilobase genome of AAVs consists of two open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs) that form characteristic T-shaped hairpin ends4. ITRs are the only cis-acting elements critical for AAV packaging, replication, and integration, therefore being the only AAV sequences retained in recombinant AAV (rAAV) vectors. In this system, the genes necessary for vector production are supplied separately, in trans, allowing the gene of interest to be packaged inside the viral capsid5,6.
Each viral gene codifies different proteins through alternative splicing and start codons. Within the Rep ORF, four non-structural proteins (Rep40, Rep52, Rep68, and Rep78) are encoded, playing crucial roles in replication, site-specific integration, and encapsidation of viral DNA7,8. The Cap ORF serves as a template for the expression of three structural proteins differing from each other at their N-terminus, (VP1, VP2, and VP3), that assemble to form a 60-mer viral capsid at a ratio of 1:1:104,9. Additionally, an alternative ORF nested in the Cap gene with a nonconventional CUG start codon encodes an assembly-activating protein (AAP). This nuclear protein has been shown to interact with the newly synthesized capsid proteins VP1-3 and promote capsid assembly10,11.
Differences in the amino acid sequence of the capsid account for the 11 naturally occurring AAV serotypes and over 100 variants isolated from humans and non-human primate tissues7,12,13. Variations in the conformation of structurally variable regions govern the distinct antigenic properties and receptor-binding specificities of capsids from different strains. This results in distinct tissue tropisms and transduction efficiencies across different mammalian organs14.
Early production methods of rAAVs relied on adenovirus infection for helper purposes15,16,17,18,19. Despite being efficient and usually easy to produce on a large scale, several problems arise from this infection. Even after the purification and a heat-denaturing step for inactivation, adenoviral particles may still be present in AAV preparations, constituting an unwanted safety issue20. Moreover, the presence of denatured adenoviral proteins is unacceptable for clinical use. Other production strategies take advantage of recombinant herpes simplex virus strains engineered to bring the Rep/Cap and transgene into the target cells21 or of the baculovirus-insect cell system22. Although these systems offer advantages in terms of scalability and GMP compatibility, they still face similar problems.
The triple transfection method for rAAV production has been commonly adopted to easily overcome these issues. Briefly, the rAAV assembly relies on the transient transfection of cells with three plasmids encoding for: 1) the transgene expression cassette packed between the ITRs from the wild-type AAV2 genome (pITR); 2) the Rep/Cap sequences necessary for replication and virion assembly (pAAV-RC); and 3) the minimal adenoviral proteins (E1A, E1B, E4, and E2A) along with the adenovirus virus-associated RNAs required for the helper effect (pHelper)6,20,23. While plasmid transfection methods provide simplicity and flexibility for rAAV production in preclinical studies, these procedures have limitations in terms of scalability and reproducibility when applied to large-scale production. As an alternative approach, rAAV production can be achieved through the use of AAV producer cell lines (of both adherent and suspension growth), stably expressing either AAV Rep/Cap genes or Rep/Cap in combination with vector constructs. In these systems, the adenoviral helper genes are introduced through plasmid transfection. Even though this strategy improves the scalability of the cell culture process, it is technically complex and time-consuming21,24,25.
In either case, the producer cells are then lysed and subjected to one or multiple purification steps. Currently, the principal methods for purifying rAAVs include the use of cesium chloride (CsCl) or iodixanol ultra-high speed density gradient centrifugation followed, or not, by chromatography techniques26. The original purification scheme for viral precipitation used ammonium sulfate, followed by two or three rounds of ultracentrifugation through a CsCl gradient. The main advantages of this process include the possibility to purify all serotypes and the ability to physically separate full particles from empty capsids based on their different densities. This method, however, is elaborate, time-consuming, and has limited scalability, often resulting in poor yield and low sample quality27,28,29,30. Moreover, dialysis against a physiological buffer is often necessary before in vivo studies due to the toxic effects that CsCl can exert on mammals.
Iodixanol has also been used as an alternative iso-osmotic gradient medium to purify rAAV vectors, with advantages over CsCl from both safety and vector potency points of view. However, like CsCl, the iodixanol method presents some drawbacks related to the loading capacity of cell culture lysate (and thus the scalability of rAAV purification) and it remains a time-consuming and expensive method30,31.
To overcome these constraints, researchers turned their attention to chromatography techniques. In this regard, several purification approaches were developed that either incorporate affinity, hydrophobic, or ion-exchange chromatography methods. These methods rely on the biochemical properties of a particular serotype, including their natural receptors, or the charge characteristics of the viral particle32. For instance, the discovery that AAV2, AAV3, AAV6, and AAV13 preferably bind to heparan sulfate proteoglycans (HSPG), opened the possibility of using the closely related heparin in affinity chromatography purification. However, the binding sites to HSPG can differ among serotypes, mediating AAV attachment and infection of target cells in different ways2,33,34,35,36. On the other hand, AAV1, AAV5, and AAV6 bind to N-linked sialic acid (SA), while AAV4 uses O-linked SA2,14,34. Following the same rationale, a single-step affinity chromatography protocol for the purification of rAAV5 has also been developed based on the use of mucin, a mammalian protein highly enriched in SA37. Like heparin-based techniques, this purification is also dependent on the specific serotype being generated. Apart from heparin and mucin, other ligands have been explored for affinity chromatography, such as the A20 monoclonal antibody and camelid single-domain antibodies (AVB Sepharose and POROS CaptureSelect)22,23,38,39,40,41. Other innovative strategies to improve the previously existing purification methods involve the introduction of small modifications in the rAAV capsid to present specific binding epitopes. For instance, hexa-histidine-tagged or biotinylated rAAVs can be purified using ligands that target those epitopes (nickel nitrilotriacetic acid and avidin resins, respectively)42,43,44.
In an effort to broaden the desired characteristics of rAAVs, investigators have cross-dressed the virions by mixing their capsids. This is accomplished by supplying the capsid gene from two distinct AAV serotypes in equimolar or different ratios during production, giving rise to a capsid structure composed of a mixture of capsid subunits from different serotypes34,45,46,47,48,49,50. Previous studies provide physical evidence that co-expressing capsid proteins from AAV2 with AAV1 (1:1 ratio) and AAV2 with AAV9 (1:1 ratio) results in the generation of mosaic rAAV1/2 and rAAV2/9 vectors, respectively45,46,48. A major benefit of the generation of mosaic rAAVs is the capacity to integrate advantageous traits from different AAV serotypes, resulting in synergistic improvements in transgene expression and tropism, while maintaining other properties useful during rAAV production. Interestingly, certain mosaic variants even exhibit novel properties different from either parental virus46,47,49. By taking advantage of the heparin-binding ability of AAV2, mosaic rAAV vectors could potentially be generated and purified by mixing AAV2 with other natural or new AAV capsids generated by directed evolution and/or rational design. Nonetheless, previous studies have highlighted the importance of capsid subunit compatibility when attempting to assemble mosaic vectors. For instance, Rabinowitz and colleagues demonstrated that although the transcapsidation of AAV1, AAV2, and AAV3 led to an efficient co-assembly of mosaic capsids, the cross-dressing of these serotypes with AAV4 hindered the generation of stable virions34,45,47. Additionally, AAV1, AAV2, and AAV3 showed low compatibility with AAV5, given the reduced viral titers obtained when mixing these capsids at different ratios. Interestingly, mosaic rAAV2/5 showed decreased heparin-binding properties, while maintaining mucin-binding ability like parental AAV5. However, rAAV3/5 at a 3:1 ratio preserved the dual binding to heparin and mucin. Overall, the generation of new mosaic rAAVs with enhanced transduction, specific tropism, or low immunogenicity could greatly benefit from our understanding of capsid assembly and receptor interactions, with specific combinations still requiring thorough investigation and optimization.
In the present work, we describe a step-by-step protocol for the production and purification of rAAVs using an optimized heparin affinity chromatography method. rAAVs are produced by transient transfection and are purified using a fast protein liquid chromatography (FPLC) system. After the concentration of selected purified fractions, the resulting viral stocks are characterized in terms of titer, purity, intrinsic physical properties, and biological activity in vitro and in vivo. As a proof of concept, we demonstrate the improvements and applicability of this protocol for the generation of mosaic rAAV1/2 and rAAV2/9 vectors. The choice of each serotype was based on their strikingly different tropisms, potentially conferring their unique characteristics to the mosaic versions as well. AAV serotype 1, with an overall moderate tropism for the central nervous system (CNS), has the ability to transduce neurons and glia (to a lesser extent) and undergoes axonal transport in the anterograde and retrograde directions in vivo2,7,8. Additionally, AAV serotype 9 was chosen for its natural ability to cross the blood-brain barrier and target the CNS in both neonatal and adult mice51,52. Finally, AAV serotype 2 was selected given its ability to bind to heparin, allowing affinity chromatography33. The purified rAAV1/2 and rAAV2/9 particles combine the properties of both parental AAV serotypes and, therefore, constitute suitable vectors for the transduction of the CNS45,46,48,49.
NOTE: See Figure 1 for an illustration summarizing the protocol. See the Table of Materials for details about all materials, instruments, and reagents used in this protocol. All work involving cells and viruses should be performed in dedicated biosafety cabinets and incubators, separated from those regularly used for the maintenance of cell lines. The equipment and reagents coming in contact with cultured cells and viruses should be sterile. It is essential that the disposal of hazardous reagents and materials contaminated with viruses is performed in accordance with the material safety data sheets and in compliance with the national laws and guidelines provided by each institution's environmental health and safety office. As of April 2019, the NIH guidelines for research involving recombinant or synthetic nucleic acid molecules categorize as Risk Group 1 agents (not associated with disease in healthy adult humans) all AAV serotypes, as well as recombinant or synthetic AAV constructs. This classification applies when the transgene does not encode a potentially tumorigenic gene product or a toxin, and the constructs are produced without an helper virus.
All experiments involving animals were carried out in compliance with the European Union Community directive (2010/63/EU) for the care and use of laboratory animals, transposed into the Portuguese law in 2013 (Decree Law 113/2013). Additionally, animal procedures were approved by the Responsible Organization for the Animal Welfare of the Faculty of Medicine and Center for Neuroscience and Cell Biology of the University of Coimbra licensed animal facility. The researchers received adequate training (FELASA-certified course) and certification from the Portuguese authorities (Direcção Geral de Alimentação e Veterinária, Lisbon, Portugal) to perform the experiments.
1. Plasmid constructs
2. Cell culture
3. rAAV production by transient transfection
4. rAAV extraction and FPLC purification
5. Concentration of purified rAAVs
6. Quantification of purified rAAVs
7. SDS-PAGE, Coomassie blue staining, and western blot
8. Transmission electron microscopy (TEM)
9. Consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis
10. In vitro transduction assays
11. In vivo experiments
NOTE: The animals were housed in a temperature-controlled room, maintained on a 12 h light/dark cycle. Food and water were provided ad libitum. All efforts were made to minimize animal suffering.
In this work, we present a detailed protocol for the production, purification, and characterization of mosaic rAAVs (summarized in Figure 1), which have the potential to target and transduce the CNS (e.g. AAV1 and AAV9), being simultaneously suitable for heparin affinity chromatography purification (AAV2). To achieve that, capsids from natural AAV serotypes 1, 2, and 9 were used to develop mosaic rAAV1/2 and rAAV2/9 vectors.
Before starting, plasmid preparations were screened for structural integrity. In addition to the digestions necessary to validate the correct insertion of cloning fragments, it is essential to consistently screen pITR plasmids to detect potential ITR deletions/insertions. As an example, the integrity of ITRs in different clones of a pITR plasmid was monitored after the plasmid digestion with the restriction enzyme SmaI (Supplementary Figure S1).
Both types of mosaic vectors were generated by the co-transfection of the respective AAV capsid plasmids in a 1:1 ratio, according to standard transfection methods6. Briefly, HEK293T cells were transfected with i) a plasmid containing the transgene of interest packed between the ITR (pITR) sequences, ii) a plasmid containing the wild-type AAV genome Rep and Cap ORFs of AAV2 and AAV1 or AAV9 (pAAV-RC plasmids) and iii) a plasmid codifying the adenoviral proteins (E1A, E1B, E4, and E2A) as well as the adenovirus virus-associated RNAs essential for helper functions (pHelper). Forty-eight hours later, the cells were harvested6,36, and rAAVs were purified from the cell homogenate by affinity chromatography using an FPLC system. As depicted in Figure 2A, after column equilibration (equilibration step), the cell lysate containing the rAAVs was applied to the column (sample loading). Due to the natural affinity of rAAV2 for heparin33, rAAVs bound to the column's resin, while other components were carried out in the running buffer and detected by the UV monitor (flowthrough), resulting in an increase in the absorbance. The column was subsequently washed (washing step) and rAAVs were finally eluted by an increase of NaCl concentration (elution step). The eluted viruses were detected by the UV monitor and collected in 1 mL fractions.
A representative elution peak profile of rAAV1/2 and rAAV2/9 is shown in Figure 2B and Supplementary Figure S2A, respectively, with different viral batches consistently presenting a single peak starting at fraction F7 up to F16. Peak height is variable among rAAV productions, with higher peaks usually leading to higher rAAV yields. Each fraction of the produced rAAV1/2 and rAAV2/9 was subsequently characterized by RT-qPCR to assess viral titers (Figure 2C and Supplementary Figure S2B).
To characterize the purity of the eluted material, 40 µL of each fraction and of the respective flowthrough were examined by 10% SDS-polyacrylamide gel electrophoresis (Figure 2D for rAAV1/2 and Supplementary Figure S2C for rAAV2/9). Coomassie blue staining revealed three major bands in fractions F7-F16, with molecular weights corresponding to the VP1 (87 kDa), VP2 (72 kDa), and VP3 (62 kDa) capsid proteins of AAVs at the appropriate ratios 1:1:10, as previously described by Van Vliet and colleagues14. In both cases, and based on UV absorbance, RT-qPCR, and gel band intensity, it is clear that the majority of mosaic rAAVs is present in fractions F7 and F8 and starts to gradually decrease in fractions F9-F16. In addition to the three viral capsid proteins, another protein (or proteins) of approximately 17 kDa in size was/were detected in fractions F8-F16.
To eliminate this co-purifying protein(s), fractions F7-16 were subsequently filtered and concentrated using 100 KDa centrifugal filter units and the final rAAV titer was determined by RT-qPCR (as shown in Figure 3A,B for rAAV1/2). The final yield of an rAAV production is dependent on the length and complexity of the pITR, the integrity of ITR sequences, cell culture conditions (e.g., number of cell passages), and transfection efficiency24,58,59,60,61. Nonetheless, the final titer can be adjusted by performing multiple centrifugations of the rAAV preparation using 0.5 mL centrifugal filter units (concentration step 2). Following this protocol, for a final volume in the range of 50 to 100 µL, concentrations are usually comprised between 2 × 109 and 5 × 1010 vg/µL (quantification performed using the referenced titration kit).
The purity of the final rAAV preps was then evaluated on a 10% SDS-polyacrylamide gel. As depicted in Figure 3C, only three bands representing the rAAVs capsid proteins were observed for the rAAV1/2 preparation and no detectable co-purifying proteins were identified. These results were consistent with those obtained for rAAV2/9 (Supplementary Figure S2C). To confirm the identity and further characterize the purity of rAAV1/2 and rAAV2/9 vectors, viral fractions and concentrated stocks were analyzed by western blot, with the specific antibodies B1 (Supplementary Figure S3A and Supplementary Figure S4A) and A69 (Supplementary Figure S3B and Supplementary Figure S4B). While the antibody B1 recognizes a C-terminal epitope common to all VP proteins of most AAV serotypes62, the clone A69 only recognizes epitopes of VP1 and VP263. Nonetheless, some faint bands with molecular weights lower than VP3 (<62 kDa) can also be detected upon B1 and A69 labeling.
To characterize the structural morphology and further evaluate the purity of rAAVs, the viral particles were directly visualized by TEM. This technique has been the standard procedure to assess sample integrity and purity in viral samples, as it allows the quantification of empty and full rAAV particles, as well as the assessment of contamination in a sample29,64,65,66,67. As shown in Figure 3D, large amounts of rAAV particles, ~25 nm in diameter, could be observed on a clean background. Empty particles (black arrow) with an electron-dense center, as well as full vectors (white arrow) could also be observed throughout the sample field.
We also performed quality control of the purified rAAVs using Stunner, a platform that combines ultraviolet-visible (UV-Vis) spectroscopy, static light scattering (SLS), and dynamic light scattering (DLS)68. For each sample, the total amount of protein, ssDNA, as well as absorbing impurities and background turbidity, were measured by UV-Vis spectroscopy (Figure 3E and Supplementary Figure S5A). SLS and DLS were then applied to assess the light-scattering behavior of rAAV capsids. Given that AAVs have a mean diameter of 25 nm, particles within a 15-45 nm diameter range are considered intact. Larger particles typically represent viral aggregates, and everything smaller comprises most likely small particles, including unassembled capsid proteins68. For rAAV1/2, a single peak corresponding to intact capsid particles was observed at 30 nm (Figure 3F), with 0% of aggregate intensity and 0% of small particle intensity. For the rAAV2/9 preparation, a peak at 30 nm was also detected representing a 78% capsid intensity (Supplementary Figure S5B). Even though the small particle intensity was 0%, for this sample, an aggregate intensity of 22% was measured (depicted in grey), with the major contribution (19.9%) from large aggregates with a mean diameter of 620 nm (Supplementary Figure S5B). Through the combination of UV-Vis spectroscopy with SLS and DLS information, Stunner revealed the overall total capsid titer, full capsid titer, free and aggregated protein, as well as free and aggregated ssDNA for the two viral preparations, shown in Figure 3G and Supplementary Figure S5C (specific values indicated in each figure legend).
In parallel, to evaluate the biological activity of the developed mosaic AAV vectors, HEK293T cells were infected with 50 µL of each FPLC-obtained fraction (F2-F16) of either the rAAV1/2 or rAAV2/9 preparation. Since the rAAV1/2 vector encodes a single-strand green fluorescent protein (GFP), under the control of a CMV promoter (pAAV-CMV-ssGFP), and the rAAV2/9 vector encodes a self-complementary GFP, under the control of CMV promoter (pAAV-CMV-scGFP53), direct GFP fluorescence was examined in these cells 48 hours post-infection (Supplementary Figure S6 and Supplementary Figure S7). Consistently with the previous observations for RT-qPCR, Coomassie blue, and western blot, the highest infectivity level was achieved for viral fractions F7 and F8, gradually decreasing in fractions F9 to F16.
To confirm whether the biological activity of rAAVs was maintained after ultrafiltration and concentration steps, Neuro2A cells, plated in both 24-well plates and an 8-well chamber slide, were infected with the concentrated rAAV1/2 vector, encoding scGFP under the control of CMV promoter (pAAV-CMV-scGFP53). Brightfield and fluorescence images were acquired 48 h post-infection (Figure 4A,B for higher resolution images).
Aiming to explore the infectious capacity of the produced rAAVs in a more relevant and reflective cell model, semi-dense primary neuronal cultures from the cortex were seeded on a 12-well plate and infected with the previously used rAAV1/2 - CMV-scGFP. Forty-eight hours after infection, cells were fixed and labeled with DAPI and WGA conjugated with Alexa Fluor 633, a widely used lectin to label fixed cells. The images shown in Figure 4C,D were acquired with a Zeiss Axio Observer Z1 and on a Zeiss confocal LSM 710. As depicted in these figures by direct GFP fluorescence, concentrated mosaic viruses preserve their gene transfer properties for neuronal cells.
Having characterized mosaic rAAVs in terms of purity, physical properties, and functionality in vitro, we next evaluated the possibility of using the purified rAAV1/2 mosaic vectors to transduce the cerebellum of C57BL/6 mice. For that, a stereotaxic injection was performed in 9-week-old mice and the GFP expression was evaluated 12 weeks later. As anticipated, animals injected with PBS exhibited no fluorescence upon GFP immunolabeling. Epifluorescence images from mice injected with rAAV1/2 vectors encoding GFP under the control of synapsin 1 promoter (rAAV1/2 - Syn-ssGFP) revealed that rAAV1/2 vectors successfully transduced several regions of the cerebellum, namely the deep cerebellar nuclei (DCN) region, as well as the different lobules of the cerebellum (Figure 5). These results demonstrate the prolonged expression of the transgene in the mammalian brain (12 weeks).
Figure 1: Schematic representation of the rAAV production and purification protocol. rAAVs are produced by transient transfection of HEK293T cells using polyethylenimine (PEI). Subsequently, cells are harvested and lysed, and rAAVs are purified from the cell homogenate via affinity chromatography. The collected fractions containing rAAVs are then concentrated, and the final viral stocks are characterized in terms of titer, purity, morphological features, and biological activity. Abbreviations: rAAV = recombinant adeno-associated virus; PEI = polyethylenimine; RT-qPCR = real-time quantitative polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Please click here to view a larger version of this figure.
Figure 2: FPLC purification protocol and representative elution profile of rAAV1/2. (A) Schematic representation of a complete chromatogram profile, showing the different stages of the rAAV purification process. After a column equilibration step, the sample is applied. The column is then washed, and the elution is performed with increasing concentrations of NaCl. The unbound material (flowthrough) and 1 mL fractions of the eluted viruses are collected for analysis. The absorbance at 280 nm is expressed in mAU and the x-axis indicates the volume in mL. (B) Enlarged partial chromatogram showing an rAAV1/2 elution peak (in black), with the corresponding fraction numbers (F2-F16) and waste (indicated in red). The issued concentration of buffer B and conductivity (expressed in mS/cm) are also shown in green and purple, respectively. (C) RT-qPCR of each fraction collected during affinity purification (F2-F16) and flowthrough. The titer in vg/µL is represented on a logarithmic scale. (D) SDS-PAGE analysis of the collected viral fractions. Equal volumes (40 µL) of each fraction from the elution step (F2-F16), and respective flowthrough were loaded and resolved on a 10% SDS-polyacrylamide gel. Protein bands were visualized by Coomassie blue staining. Bands corresponding to AAV capsid proteins VP1, VP2, and VP3 are indicated. Standard protein size ladder is designated as (L) and the corresponding molecular weights are also indicated. Abbreviations: rAAV = recombinant adeno-associated virus; RT-qPCR = real-time quantitative polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Please click here to view a larger version of this figure.
Figure 3: Characterization of the concentrated rAAV1/2 vectors. (A) Amplification curves of a concentrated rAAV1/2 sample (in blue), serially diluted standards from 2 × 107 vg/µL to 2 × 102 vg/µL (in black) and a no-template control (in green), obtained during RT-qPCR. (B) Standard curve (linear regression) for the determination of the titer of an rAAV sample in vg/µL. (C) SDS-PAGE analysis of the concentrated viral particles. A total of 2.3 × 1010 vg of the concentrated stock were pooled on the gel. (D) Transmission electron microscopy image of rAAV1/2 particles with ~25-30 nm in diameter. Empty particles with an electron-dense center (evidenced by black arrows) can be distinguished from full capsids (evidenced by white arrows). Scale bar = 100 nm. (E) Absorbance spectrum of an rAAV1/2 preparation measured by Stunner (in black). The contribution of proteins (in blue), ssDNA (in green), other UV-absorbing compounds or impurities (in purple), and background turbidity (in grey) are also shown. (F) DLS intensity distribution of rAAV1/2 with a single peak at 30 nm, measured by Stunner. A capsid scattering intensity of 100% was determined by measuring the area under the curve from 15 to 45 nm (shaded green). (G) Stunner analysis of an rAAV1/2 vector preparation exhibiting a total capsid titer of 1.19 × 1014 cp/mL (dark blue) and a full capsid titer of 1.73 × 1013 vg/mL (dark green). A free and aggregated protein of 7.16 × 1012 cp/mL equivalents (light blue), as well as a free and aggregated ssDNA of 1.04 × 1012 vg/mL equivalents (light green), were also measured. Abbreviations: rAAV = recombinant adeno-associated virus; RT-qPCR = real-time quantitative polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ssDNA = single-stranded DNA; DLS = dynamic light scattering. Please click here to view a larger version of this figure.
Figure 4: In vitro infectivity assessment of a concentrated rAAV1/2 sample. (A) Neuro2A cells were infected with rAAV1/2 - CMV-scGFP or incubated with an equivalent volume of PBS, as a negative control. Brightfield and fluorescence images of cells imaged 48 h post-infection. Images were acquired in a Zeiss Axio Observer Z1 (10x objective). Scale bars = 100 µm. (B) Detailed images of Neuro2A cells 48 h post-infection with rAAV1/2 - CMV-scGFP. Images were acquired in a Zeiss LSM 710 (40x objective). Scale bars = 20 µm. (C) Semi-dense primary neuronal cultures infected with rAAV1/2 - CMV-scGFP or incubated with an equivalent volume of PBS, serving as a negative control. Cells were labeled with a nuclear stain (DAPI in blue) and a membrane stain (WGA in white). Images were acquired in a Zeiss Axio Observer Z1 (40x objective). Scale bars = 20 µm. (D) Detailed images of semi-dense primary neuronal cultures 48 h post-infection with rAAV1/2 - CMV-scGFP. Images were acquired in a Zeiss LSM 710 (40x objective). Scale bars = 20 µm. Abbreviations: rAAV = recombinant adeno-associated virus; CMV = cytomegalovirus; scGFP = self-complementary green fluorescent protein; PBS = phosphate-buffered saline; DAPI = 4',6-diamidino-2-phenylindole; WGA = wheat germ agglutinin. Please click here to view a larger version of this figure.
Figure 5: In vivo transduction efficiency of rAAV1/2 following an intraparenchymal injection. Representative immunofluorescence images showing the widespread GFP expression (in green) throughout the cerebellum upon a central injection of rAAV1/2 - Syn-ssGFP in the cerebellum. Nuclei were stained with DAPI (in blue). Scale bars = 500 µm. Abbreviations: rAAV = recombinant adeno-associated virus; Syn = Synapsin 1; ssGFP = single-strand green fluorescent protein; DAPI = 4',6-diamidino-2-phenylindole; PBS = phosphate buffered saline. Please click here to view a larger version of this figure.
Supplementary Figure S1: Agarose gel analysis of an rAAV vector plasmid digested with SmaI. Six clones (C1-C6) of a pITR were digested with SmaI restriction enzyme (lanes 2, 4, 6, 8, 10, and 12), which cuts twice within each inverted terminal repeat. In this case, a complete digestion of this pITR would be expected to generate two bands (3,796 bp and 3,013 bp). In successful preparations (C1, C3, C4, and C5) a band of 6809 bp, resulting from partial digestion is still visible (~5% of the total). In preparations with ITR recombination, the proportions are reversed (C2), or the digestion did not occur (C6). The respective non-digested clones are also presented (lanes 3, 5, 7, 9, 11, 13). Abbreviations: rAAV = recombinant adeno-associated virus; ITR = inverted terminal repeat. Please click here to download this File.
Supplementary Figure S2: rAAV2/9 purification by heparin-based affinity chromatography. (A) Elution profile of rAAV2/9 exhibiting a single peak (in black), following an increase in the concentration of NaCl. The collected fractions are indicated by numbers (2-16) in red at the bottom of the graph, the absorbance at 280 nm is expressed in mAU, conductivity is expressed in mS/cm, and the x-axis indicates the volume in mL. (B) rAAV titers quantified by RT-qPCR for each pooled fraction (F2-F16) and flowthrough. Values are represented on a logarithmic scale. (C) Purity assay by SDS-PAGE and Coomassie blue staining. Equal volumes (40 µL) of each fraction (F2-F16) and the respective flowthrough were loaded and resolved on a 10% SDS-PAGE. Concentrated stock was quantified by RT-qPCR and 2.3 × 1010 vg were diluted in 40 µL of PBS and pooled on the gel. Protein bands were visualized by Coomassie blue staining. The AAV capsid proteins (VP1, VP2, and VP3) are indicated. Standard protein size ladder is designated with (L) and the corresponding molecular weights are also indicated. Abbreviations: rAAV = recombinant adeno-associated virus; RT-qPCR = real-time quantitative polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Please click here to download this File.
Supplementary Figure S3: Western blot analysis of rAAV1/2 vectors purified by FPLC. (A) The collected fractions and concentrated rAAV1/2 vectors were resolved on an SDS-PAGE gel and probed with mouse monoclonal anti-AAV antibody (B1) that recognizes VP1, VP2, and VP3 capsid proteins. (B) The collected fractions and concentrated rAAV1/2 vectors were resolved on an SDS-PAGE gel and probed with mouse monoclonal anti-AAV antibody (A69) that recognizes VP1 and VP2 capsid proteins. Abbreviations: rAAV = recombinant adeno-associated virus; FPLC = fast protein liquid chromatography; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; L = standard protein size ladder. Please click here to download this File.
Supplementary Figure S4: Western blot analysis of rAAV2/9 vectors purified by FPLC. (A) The collected fractions and concentrated rAAV2/9 vectors were resolved on an SDS-PAGE gel and probed with mouse monoclonal anti-AAV antibody (B1) that recognizes VP1, VP2, and VP3 capsid proteins. (B) The collected fractions and concentrated rAAV2/9 vectors were resolved on an SDS-PAGE gel and probed with mouse monoclonal anti-AAV antibody (A69) that recognizes VP1 and VP2 capsid proteins. Abbreviations: rAAV = recombinant adeno-associated virus; FPLC = fast protein liquid chromatography; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; L = standard protein size ladder. Please click here to download this File.
Supplementary Figure S5: rAAV2/9 vector quantification and characterization via Stunner. (A) Absorbance spectrum (black) of an rAAV2/9 vector measured by Stunner. The contribution of proteins (blue), ssDNA (green), other UV-absorbing compounds or impurities (purple), and background turbidity (grey) are also depicted. (B) DLS intensity distribution of rAAV2/9 with a major peak at 30 nm corresponding to a capsid scattering intensity of 78%, as determined by measuring the area under the curve from 15 to 45 nm (shaded green). A total aggregate intensity of 22% (shaded in grey) was also measured with a main contribution from large aggregates (19.9%) with a mean diameter of 620 nm. (C) Stunner analysis of an rAAV2/9 vector preparation exhibiting a total capsid titer of 2.18 × 1014 cp/mL (dark blue) and a full capsid titer of 2.35 × 1013 vg/mL (dark green). A free and aggregated protein of 2.92 × 1013 cp/mL equivalents (light blue), as well as a free and aggregated ssDNA of 3.14 × 1012 vg/mL equivalents (light green), were also measured in this preparation. Abbreviations: rAAV = recombinant adeno-associated virus; ssDNA = single-stranded DNA; DLS = dynamic light scattering. Please click here to download this File.
Supplementary Figure S6: In vitro transduction efficiency and viability of the purified fractions of rAAV1/2. HEK293T cells expressing GFP (direct fluorescence) 48 h after the transduction with 50 µL of FPLC fractions of an rAAV1/2 vector encoding ssGFP (rAAV1/2 - CMV-ssGFP). Scale bars = 100 µm. Abbreviations: rAAV = recombinant adeno-associated virus; FPLC = fast protein liquid chromatography; ssGFP = single-strand green fluorescent protein. Please click here to download this File.
Supplementary Figure S7: In vitro transduction efficiency and viability of the purified fractions of rAAV2/9. HEK293T cells were infected with 50 µL of each FPLC fraction (F2-F16) or flowthrough of an rAAV2/9 vector encoding scGFP under the control of the CMV promoter. The GFP-expressing cells were visualized 48 h post-infection. Scale bars = 100 µm. Abbreviations: rAAV = recombinant adeno-associated virus; FPLC = fast protein liquid chromatography; scGFP = self-complementary green fluorescent protein; CMV = cytomegalovirus. Please click here to download this File.
The rapidly expanding AAV vector toolkit has become one of the most promising gene delivery systems for a wide range of cell types through different routes of administration. In this work, we aimed to develop an improved protocol for the production, purification, and characterization of mosaic rAAV vectors that could prove their worth in preclinical studies. For that purpose, the generation of rAAV1/2 and rAAV2/9 mosaic vectors is described here, but the procedure can also be applied to purify standard rAAV2 vectors (data not shown).
Mosaic rAAVs were produced following an optimized transfection method using PEI as a transfection reagent. A transient transfection method was selected due to its greater flexibility and speed, considerable advantages in early-stage preclinical studies. Once a particular transgene and serotype have been validated, the production system can be fine-tuned to achieve better scalability and cost-effectiveness by establishing a stable transfected cell line that expresses a subset of the specific Rep/Cap genes, with additional genes provided by an infection process24. Compared to calcium-phosphate transfection, PEI presents several advantages. It is a stable and cost-effective transfection reagent that operates effectively within a broader pH range. Additionally, it eliminates the requirement to change the cell medium after transfection, resulting in a significant reduction in both cost and workload69.
In an attempt to circumvent some of the limitations imposed by CsCl or iodixanol gradients, the produced rAAVs were harvested and purified by affinity chromatography. This strategy offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers. Indeed, chromatography techniques using an FPLC system can be automated and scaled up by packing more resin volume in a column with a higher bed height. The protocol described herein can be easily adapted to incorporate 5 mL HiTrap Heparin HP columns (data not shown). Additionally, heparin columns may be reused several times, thus contributing to the cost-effectiveness of this method.
The purified rAAVs were then characterized in terms of titer, purity, morphological features, and biological activity. Interestingly, in Coomassie blue staining, a band with approximately 17 kDa was detected in fractions F8-F16 in addition to the three typical viral capsid proteins. However, this band is no longer present after the concentration step of rAAVs. Moreover, some faint bands with molecular weights lower than VP3 (<62 kDa) can also be detected upon B1 and A69 labeling, suggesting that these could be fragments of the VP1, VP2, and VP3 capsid proteins70. Another possibility is that these are in fact other co-purifying proteins such as ferritin or other cellular proteins with polypeptides that share similar protein fingerprints with the AAV capsid proteins and could be involved in the AAV biology, as previously suggested26,71,72.
TEM and stunner analysis also revealed the presence of empty particles at variable levels across different productions. Similarly, other studies previously reported the generation of variable and high levels (>65%) of empty capsids for rAAVs prepared by transfection or infection methods24,73. The mechanism behind rAAV generation starts with the rapid formation of empty capsids from newly synthesized VP proteins, followed by a slow rate-limiting step of genome packaging into the preformed capsids mediated by Rep proteins74,75. Therefore, empty capsids are generated in rAAV productions, although the proportion of empty and full capsids can vary depending on the size and sequence of the transgene of interest and cell culture conditions58,73. Empty capsids raise some concerns since, by lacking the genome of interest, they are unable to provide a therapeutic effect and may also potentially increase an innate or adaptive immune response. However, some reports have also shown that, by adjusting their ratio, empty AAV capsids may serve as highly effective decoys for AAV-specific neutralizing antibodies and therefore, increase transduction efficiencies60,76,77. If the presence of empty capsids is critically detrimental and given the slightly less anionic character of empty particles compared to full vectors, a potential solution could involve conducting a second polishing purification step using anion exchange chromatography techniques64.
This study also provides compelling evidence that the generated mosaic rAAVs are able to efficiently transduce not only in vitro neuronal cultures but also the CNS upon intracranial injection of rAAV1/2. Overall, these results suggest that the described production and purification protocol renders highly pure and biologically active rAAVs ready to use in 6 days, presenting itself as a versatile and cost-effective method for the generation of rAAVs in preclinical studies.
We are grateful for the collaboration, insights, and technical assistance provided by Dr. Mónica Zuzarte, at the Coimbra Institute for Clinical and Biomedical Research (iCBR) and Center for Innovative Biomedicine and Biotechnology (CIBB), regarding the TEM analysis of rAAVs. We extend our appreciation to Dr. Dominique Fernandes, at the Center for Neuroscience and Cell Biology of the University of Coimbra (CNC-UC) and the Institute for Interdisciplinary Research of the University of Coimbra (IIIUC), for her invaluable technical assistance and insights regarding the primary neuronal culture experiments. The pRV1, pH21, and pFdelta6 plasmids, essential for this study, were generously provided by Dr. Christina McClure, at the School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, for which we are grateful. This work was funded by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Program; through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation, and Portuguese national funds via FCT - Fundação para a Ciência e a Tecnologia, under the projects: UIDB/04539/2020, UIDP/04539/2020, LA/P/0058/2020, ViraVector (CENTRO-01-0145-FEDER-022095), Imagene (PTDC/BBB-NAN/0932/2014 | POCI-01-0145-FEDER-016807), ReSet - IDT-COP (CENTRO-01-0247-FEDER-070162), Fighting Sars-CoV-2 (CENTRO-01-01D2-FEDER-000002), BDforMJD (CENTRO-01-0145-FEDER-181240), ModelPolyQ2.0 (CENTRO-01-0145-FEDER-181258), MJDEDIT (CENTRO-01-0145-FEDER-181266); by the American Portuguese Biomedical Research Fund (APBRF) and the Richard Chin and Lily Lock Machado-Joseph Disease Research Fund, ARDAT under the IMI2 JU Grant agreement No 945473 supported by EU and EFPIA; GeneT- Teaming Project 101059981 supported by the European Union's Horizon Europe program. M.M.L. was supported by 2021.05776.BD; C.H. was supported by 2021.06939.BD; A.C.S. was supported by 2020.07721.BD; and D.D.L. was supported by 2020.09668.BD. Figure 1 was created using BioRender.com.
Name | Company | Catalog Number | Comments |
10% povidone-iodine | Medline | MDS093943 | |
12-well plates | Thermo Scientific | 11889684 | |
24-well plates | VWR | 734-2325 | |
4’,6-diamidino-2-phenylindole (DAPI) | Invitrogen | D1306 | |
96-well Stunner plate | Unchained Labs | 701-2025 | 96-well quantification plate for the consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis of rAAV samples |
AAVpro Titration Kit (for Real-Time PCR) Ver.2 | Takara | 6233 | For determining the titer of AAV using RT-qPCR. This kit contains DNase I, Lysis Buffer, Dilution Buffer, positive control, Taq II mix, primer forward, primer reverse, water |
Acetic acid glacial | Fisher Chemical | A/0360/PB17 | |
ÄKTA pure 25 | Cytiva | 29018224 | FPLC system controlled by UNICORN software, version 6.3 |
Alkaline phosphatase-linked goat anti-mouse | Invitrogen | 31328 | |
Amicon ultra-0.5 centrifugal filter unit | Merck Millipore | UFC5100 | |
Amicon ultra-15 centrifugal filter unit | Merck Millipore | UFC9100 | |
Benzonase Nuclease | Merck Millipore | E1014 | |
Bromophenol blue | Sigma-Aldrich | B0126 | |
CFX96 Real-Time PCR detection system | Biorad | 184-5096 | |
ChemiDoc Touch Imaging System | Bio-Rad Laboratories | 1708370 | |
Chicken polyclonal anti-GFP primary antibody | Abcam | ab13970 | |
Coomassie Blue G250 | Fisher Chemical | C/P541/46 | |
Dithiothreitol (DTT) | Fisher Bioreagents | BP17225 | |
DMEM | Sigma-Aldrich | D5796 | |
ECF Substrate for Western Blotting | Cytiva | RPN5785 | |
FastDigest SmaI | Thermo Scientific | FD0663 | |
FEI-Tecnai G2 Spirit Biotwin | FEI | Biotwin | Transmission electron microscope |
Fetal bovine serum | Biowest | S1810 | |
Fluorescence mounting medium | Dako | S3023 | |
Formvar-carbon coated 200 mesh grid | TAAB Laboratories Equipment | F077/025 | |
Gas evacuation apparatus | RWD | R546W | |
Glycerol | Fisher BioReagents | 10021083 | |
Goat polyclonal anti-chicken antibody, Alexa Fluor 488 | Invitrogen | A-11039 | |
Hamilton needle 30G, Small Hub RN Needle, 25 mm, PST3 | Hamilton | 7803-07 | |
Hamilton syringe (10 µL) | Hamilton | 7653-01 | |
HEK293T | American Type Culture Collection | CRL-11268 | |
HiTrap Heparin HP 1 x 5 mL | Cytiva | 17040701 | Pre-packed heparin column |
HiTrap Heparin HP 5 x 1 mL | Cytiva | 17040601 | Pre-packed heparin column |
Immobilon-P PVDF Membrane | Merck Millipore | IPVH00010 | |
Isoflurane | Braun | 469860 | |
Ketamine | Dechra Pharmaceuticals | N/A | Nimatek |
Low-retention microcentrifuge tubes (2 mL) | Fisher Scientific | 11906965 | |
Lunatic & Stunner Client software | Unchained Labs | N/A | Client analysis software version 8.0.1.235. Software for the consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis of rAAV samples |
Methanol | Fisher Chemical | M/4000/FP21 | |
Mouse monoclonal anti-AAV, VP1, VP2 antibody (A69) | American Research Products | 03-61057 | |
Mouse monoclonal anti-AAV, VP1, VP2, VP3 antibody (B1) | American Research Products | 03-61058 | |
Neuro2a | American Type Culture Collection | CCL-131 | |
Normal goat serum | Gibco | 16210064 | |
NucleoBond Xtra Maxi EF | Macherey-Nagel | 12738422 | |
NZYColour Protein Marker II | NZYtech | MB09002 | |
pAAV-CMV-scGFP | Addgene | 32396 | Addgene plasmid # 32396; http://n2t.net/addgene:32396; RRID:Addgene_32396 |
pAAV-CMV-ssGFP | Addgene | 105530 | Addgene plasmid # 105530; http://n2t.net/addgene:105530; RRID:Addgene_105530 |
pAAV2/9n | Addgene | 112865 | Addgene plasmid # 112865; http://n2t.net/addgene:112865; RRID:Addgene_112865 |
Paraformaldehyde | Acros Organics | 10342243 | |
PBS | Fisher BioReagents | BP2438 | |
Penicillin-streptomycin | Gibco | 15140-122 | |
Pluronic F-68 Non-ionic Surfactant (100x) | Gibco | 24040032 | |
Polyethylenimine MAX, MW 40,000 | Polysciences Europe | 24765 | |
R500 Series Compact Small Animal Anesthesia Machine - Isoflurane | RWD | N/A | |
Sample Inlet Valve V9-IS | Cytiva | 29027746 | |
Sample pump P9-S | Cytiva | 29027745 | |
Sodium azide | Sigma-Aldrich | S2002 | |
Sodium chloride | Fisher Scientific | 10428420 | |
Sodium deoxycholate | Sigma-Aldrich | D6750 | |
Sodium dodecyl sulfate (SDS) | Fisher Bioreagents | BP166 | |
Sterile PVDF syringe filter | Fisher Scientific | 15191499 | |
Stunner Platform | Unchained Labs | 700-2002 | Equipment for the consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis of rAAV samples |
Superloop 150 mL | Cytiva | 18-1023-85 | |
Superloop 50 mL | Cytiva | 18-1113-82 | |
SURE 2 supercompetent cells | Stratagene, Agilent Technologies | HPA200152 | |
Treated culture dishes | Corning | 734-1711 | |
Tris base | Fisher BioReagents | BP152 | |
Tris hydrochloride | Fisher BioReagents | BP153 | |
Triton X-100 | Sigma-Aldrich | T8787 | |
Trypsin-EDTA | Gibco | 25200-072 | |
Wheat Germ Agglutinin (WGA) conjugated with Alexa Fluor 633 | Invitrogen | W21404 | |
Xylazine | Dechra Pharmaceuticals | N/A | Sedaxylan |
Zeiss Axio Observer Z1 | Carl Zeiss Microscopy GmbH | N/A | Inverted fluorescence microscope equiped with an EC Plan-Neofluar 10x/0.30 objective and a Plan-Apochromat 40x/0.95 objective |
Zeiss Axio Scan.Z1 | Carl Zeiss Microscopy GmbH | N/A | Slide scanner fluorescence microscope equipped with a Plan-Apochromat 20x/0.8 objective |
Zeiss LSM 710 | Carl Zeiss Microscopy GmbH | N/A | Inverted confocal microscope equipped with a Plan-Apochromat 40x/1.4 Oil DIC objective |
µ-Slide 8 well Ibidi | Ibidi | 80826 | 8-well chamber slide |
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