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><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&amp;rsquo;,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>&amp;Auml;KTA pure 25</td><td>Cytiva</td><td>29018224</td><td>FPLC system controlled by UNICORN software, version 6.3&amp;nbsp;</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>&amp;nbsp;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 &amp;micro;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&amp;nbsp;</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 &amp;amp; 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>&amp;micro;-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.

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

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

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.

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. ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.8K Views.  University of Coimbra. In dit werk zijn we van plan een verbeterd protocol te ontwikkelen voor de productie, zuivering en karakterisering van mozaïekadeno-geassocieerde virale vectoren die hun waarde zouden kunnen bewijzen in preklinische studies. Ondanks inspanningen om stabiele productiecellijnen op te zetten, is voorbijgaande transfectie in zoogdiercellen nog steeds de overheersende workflow voor AAV-productie. Vervolgens worden de AAV's gezuiverd door centrifugatie met ultrahoge snelheid van dichtheidsgradiën...

Video: Auteur in de schijnwerpers: verbeterde methode voor productie en zuivering van adeno-geassocieerde virale vectoren

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Immunology and Infection

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Neuroscience

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields &#62;1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. ...

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

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.

Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications

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.

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

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