We are grateful for the collaboration, insights, and technical assistance provided by Dr. M&#243;nica 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 - Funda&#231;&#227;o para a Ci&#234;ncia 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&#39;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.

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

The authors declare no conflicts of interest.

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 (&#60;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 (&#62;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.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% povidone-iodine</td>
			<td>Medline</td>
			<td>MDS093943</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Thermo Scientific</td>
			<td>11889684</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>VWR</td>
			<td>734-2325</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Stunner plate</td>
			<td>Unchained Labs</td>
			<td>701-2025</td>
			<td>96-well quantification plate for the consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis of rAAV samples</td>
		</tr>
		<tr>
			<td>AAVpro Titration Kit (for Real-Time PCR) Ver.2</td>
			<td>Takara</td>
			<td>6233</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Acetic acid glacial</td>
			<td>Fisher Chemical</td>
			<td>A/0360/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>&#196;KTA pure 25</td>
			<td>Cytiva</td>
			<td>29018224</td>
			<td>FPLC system controlled by UNICORN software, version 6.3&#160;</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase-linked goat anti-mouse</td>
			<td>Invitrogen</td>
			<td>31328</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon ultra-0.5 centrifugal filter unit</td>
			<td>Merck Millipore</td>
			<td>UFC5100</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon ultra-15 centrifugal filter unit</td>
			<td>Merck Millipore</td>
			<td>UFC9100</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Merck Millipore</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR detection system</td>
			<td>Biorad</td>
			<td>184-5096</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touch Imaging System</td>
			<td>Bio-Rad Laboratories</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken polyclonal anti-GFP primary antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Blue G250</td>
			<td>Fisher Chemical</td>
			<td>C/P541/46</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Fisher Bioreagents</td>
			<td>BP17225</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>ECF Substrate for Western Blotting</td>
			<td>Cytiva</td>
			<td>RPN5785</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest SmaI</td>
			<td>Thermo Scientific</td>
			<td>FD0663</td>
			<td></td>
		</tr>
		<tr>
			<td>FEI-Tecnai G2 Spirit Biotwin</td>
			<td>FEI</td>
			<td>Biotwin</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence mounting medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Formvar-carbon coated 200 mesh grid</td>
			<td>&#160;TAAB Laboratories Equipment</td>
			<td>F077/025</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas evacuation apparatus</td>
			<td>RWD</td>
			<td>R546W</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>10021083</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat polyclonal anti-chicken antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11039</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton needle 30G, Small Hub RN Needle, 25 mm, PST3</td>
			<td>Hamilton</td>
			<td>7803-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (10 &#181;L)</td>
			<td>Hamilton</td>
			<td>7653-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td>American Type Culture Collection</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap Heparin HP 1 x 5 mL</td>
			<td>Cytiva</td>
			<td>17040701</td>
			<td>Pre-packed heparin column</td>
		</tr>
		<tr>
			<td>HiTrap Heparin HP 5 x 1 mL</td>
			<td>Cytiva</td>
			<td>17040601</td>
			<td>Pre-packed heparin column</td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF Membrane</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Braun</td>
			<td>469860</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Dechra Pharmaceuticals</td>
			<td>N/A</td>
			<td>Nimatek</td>
		</tr>
		<tr>
			<td>Low-retention microcentrifuge tubes (2 mL)</td>
			<td>Fisher Scientific</td>
			<td>11906965</td>
			<td></td>
		</tr>
		<tr>
			<td>Lunatic &#38; Stunner Client software</td>
			<td>Unchained Labs</td>
			<td>N/A</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Chemical</td>
			<td>M/4000/FP21</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-AAV, VP1, VP2 antibody (A69)</td>
			<td>American Research Products</td>
			<td>03-61057</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-AAV, VP1, VP2, VP3 antibody (B1)</td>
			<td>American Research Products</td>
			<td>03-61058</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuro2a</td>
			<td>American Type Culture Collection</td>
			<td>CCL-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Gibco</td>
			<td>16210064</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi EF</td>
			<td>Macherey-Nagel</td>
			<td>12738422</td>
			<td></td>
		</tr>
		<tr>
			<td>NZYColour Protein Marker II</td>
			<td>NZYtech</td>
			<td>MB09002</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-CMV-scGFP</td>
			<td>Addgene</td>
			<td>32396</td>
			<td>Addgene plasmid # 32396; http://n2t.net/addgene:32396; RRID:Addgene_32396</td>
		</tr>
		<tr>
			<td>pAAV-CMV-ssGFP</td>
			<td>Addgene</td>
			<td>105530</td>
			<td>Addgene plasmid # 105530; http://n2t.net/addgene:105530; RRID:Addgene_105530</td>
		</tr>
		<tr>
			<td>pAAV2/9n</td>
			<td>Addgene</td>
			<td>112865</td>
			<td>Addgene plasmid # 112865; http://n2t.net/addgene:112865; RRID:Addgene_112865</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>10342243</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher BioReagents</td>
			<td>BP2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-68 Non-ionic Surfactant (100x)</td>
			<td>Gibco</td>
			<td>24040032</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine MAX, MW 40,000</td>
			<td>Polysciences Europe</td>
			<td>24765</td>
			<td></td>
		</tr>
		<tr>
			<td>R500 Series Compact Small Animal Anesthesia Machine - Isoflurane</td>
			<td>RWD</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Inlet Valve V9-IS</td>
			<td>Cytiva</td>
			<td>29027746</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample pump P9-S</td>
			<td>Cytiva</td>
			<td>29027745</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>10428420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Bioreagents</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PVDF syringe filter</td>
			<td>Fisher Scientific</td>
			<td>15191499</td>
			<td></td>
		</tr>
		<tr>
			<td>Stunner Platform</td>
			<td>Unchained Labs</td>
			<td>700-2002</td>
			<td>Equipment for the consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis of rAAV samples</td>
		</tr>
		<tr>
			<td>Superloop 150 mL</td>
			<td>Cytiva</td>
			<td>18-1023-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Superloop 50 mL</td>
			<td>Cytiva</td>
			<td>18-1113-82</td>
			<td></td>
		</tr>
		<tr>
			<td>SURE 2 supercompetent cells</td>
			<td>Stratagene, Agilent Technologies</td>
			<td>HPA200152</td>
			<td></td>
		</tr>
		<tr>
			<td>Treated culture dishes</td>
			<td>Corning</td>
			<td>734-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher BioReagents</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Fisher BioReagents</td>
			<td>BP153</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin (WGA) conjugated with Alexa Fluor 633</td>
			<td>Invitrogen</td>
			<td>W21404</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Dechra Pharmaceuticals</td>
			<td>N/A</td>
			<td>Sedaxylan</td>
		</tr>
		<tr>
			<td>Zeiss Axio Observer Z1</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>N/A</td>
			<td>Inverted fluorescence microscope equiped with an EC Plan-Neofluar 10x/0.30 objective and a Plan-Apochromat 40x/0.95 objective</td>
		</tr>
		<tr>
			<td>Zeiss Axio Scan.Z1</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>N/A</td>
			<td>Slide scanner fluorescence microscope equipped with a Plan-Apochromat 20x/0.8 objective</td>
		</tr>
		<tr>
			<td>Zeiss LSM 710</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>N/A</td>
			<td>Inverted confocal microscope equipped with a Plan-Apochromat 40x/1.4 Oil DIC objective</td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 well Ibidi</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>8-well chamber slide</td>
		</tr>
	</tbody>
</table>

isolation of adeno-associated viral vectors through a single-step and semi-automated heparin affinity chromatography protocol

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.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

In this work, we intend to develop an improved protocol for the production, purification, and characterization of mosaic adeno-associated viral vectors that could prove their worth in preclinical studies. Despite efforts to establish stable producer cell lines, transient transfection in mammalian cells is still the predominant workflow for AAV production. Then the AAVs are purified by ultra-high speed density gradient centrifugation, or by chromatography techniques that rely on the biochemical properties of viral particles.The commonly used methods to purify AAVs make use of cesium chloride, or iodixanol density gradient ultracentrifugation. Despite their advantages, they have some limitations. They are time-consuming, they have limited scalability, and they are often giving results to some vectors with low purity.Now, to overcome these constraints, several researchers have turned their attention to chromatography techniques. We describe a step-by-step protocol for the generation of mosaic rAAVs based on the heparin-binding ability of AAV2. This method renders highly pure and biologically active rAAVs ready to use in six days, presenting itself as a semi-automated, scalable, and cost-effective strategy to generate rAAVs for preclinical studies.Having demonstrated the applicability of this method for the generation of mosaic rAAVs, the production system can now be fine-tuned to achieve a better scalability and cost effectiveness by establishing a producer cell line. Additionally, we aim to remove empty particles by including a second polishing purification step.

Read this Scientific Article Protocol about Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors at JoVE.com

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. ...

isolation-adeno-associated-viral-vectors-through-single-step-semi

Research

JoVE Journal

Bioengineering

Production of Recombinant AAV by Transient Transfection

FPLC Purification and Characterization of Recombinant AAV

1.4K Views.  University of Coimbra.  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.

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Summary