M.R. is supported by a Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO) postdoctoral fellowship (133722/1204517N) and acknowledges the continuous support of the Fundaci&#243;n Cardiovascular de Colombia and the Administrative Department of Science, Technology and Innovation (Grant CT-FP44842-307-2016, project code 656671250485). M.M. is supported by an FWO doctoral fellowship (1S48018N). M.G.H. was supported by a VIB institutional grant and external support from the Thierry Latran Foundation (SOD-VIP), The Foundation for Alzheimer Research (SAO-FRA) (P#14006), the FWO (Grant 1513616N), and the European Research Council (ERC) (Starting Grant 281961 - AstroFunc; Proof of Concept Grant 713755 - AD-VIP). The authors acknowledge Jeason Haughton for his help with mouse husbandry, Stephanie Castaldo for her help performing the tail vein injections, and Caroline Eykens for providing the images of transfected HEK293T cells. M.G.H. acknowledges Michael Dunlop, Peter Hickman, and Dean Harrison.

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<li>Bachman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunohistochemistry+on+Freely+Floating+Fixed+Tissue+Sections%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Immunohistochemistry on Freely Floating Fixed Tissue Sections.</a> Methods in Enzymology. 533, 207-215 (2013).</li>
<li>Hordeaux, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Neurotropic+Properties+of+AAV-PHP.B+Are+Limited+to+C57BL%2F6J+Mice%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice.</a> Molecular Therapy. 26 (3), 664-669 (2018).</li>
<li>Rincon, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Widespread+transduction+of+astrocytes+and+neurons+in+the+mouse+central+nervous+system+after+systemic+delivery+of+a+self-complementary+AAV-PHP.B+vector%5BTitle%5D)+AND+%22Gene+Therapy%22%5BJournal%5D)">Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector.</a> Gene Therapy. 25 (2), 83-92 (2018).</li>
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</ol>

The authors have nothing to disclose.

The production of recombinant AAV vectors described here uses materials and equipment common to most molecular biology labs and cell culture facilities. It allows the user to obtain pure, preclinical grade AAV vectors that can be used to target multiple cell and tissue types across a range of in vitro and in vivo applications. One of the greatest advantages of this protocol, compared to others (i.e., CsCl-based purification), is the shorter working time needed. Ready-to-use AAV vectors are obtainable in a maximum of 6 working days after the initial transfection of HEK293T cells.
Several factors can negatively influence the final yield or the quality of the AAV vector. Poor transfection efficiency is one of the main reasons for a low viral yield33. A major recommendation is the use of HEK293T cells that have not been passaged&#160;more than 20 times and do not have a cell confluence greater than 90% at the time of transfection21. In addition, the transfection method selected has a major impact on the results. This protocol is based on the use of PEI. PEI is a cationic polymer with the ability to deliver exogenous DNA to the cell nucleus through the generation of complexes of polymer and nucleic acid, known as polyplexes, which are taken up&#160;by the cell and trafficked via endosomes34. PEI-based transfection is easy and fast to perform, in contrast to other widely used methods, such as the co-precipitation of DNA with calcium phosphate35. Also, PEI-based transfection is much cheaper when compared to other newly introduced methods, such as the usage of cationic lipids and magnet-mediated transfection36.
The purification strategy plays a key role in the protocol. Compared to other methods, iodixanol-based purifications tend to contain a higher percentage of empty viral particles (20%)20. This is offset, to a degree, by the fact that iodixanol-based purification routinely results in AAV vector preparations with a particle-to-infectivity ratio of less than 100. This represents a significant improvement in comparison to conventional CsCl-based procedures, for which substantial loss of particle infectivity is reported37. Another common alternative method to purify AAV vectors is chromatography-based purification. However, this method has the major drawback that a specific column is required for each vector capsid used: for example, while AAV2 is classically isolated using heparin columns, this methodology does not work with AAV4 and AAV5, which do not possess heparin-binding sites on their capsids38. Considering that chromatography purification is also expensive, iodixanol-based purification is generally more suitable for laboratories that wish to produce high-quality batches of AAV vectors on a small scale33,39,40. However, to maximize the final yield and purity of the vector, extreme care is needed when making the iodixanol gradients. The various iodixanol fractions should be transferred to the ultracentrifugation tube using a sterile Pasteur pipette whose tip is touching the wall of the tube: iodixanol should be expelled from the pipette slowly and continuously. As the vector particles accumulate in the 40% iodixanol layer, care needs to be taken to ensure that the gradient interfaces do not mix20. Finally, the fraction containing the vector should be recovered by the insertion of a stainless-steel blunt needle with a gauge not larger than 20 G. To maximize vector recovery, the clear fraction should be retrieved in its entirety. During this step, timing is critical. To avoid compromising the purity of the preparation, it is essential to stop the collection before other (contaminating) phases of the gradient are collected.
Differences in the obtained vector&#160;titer can be also attributed to the intrinsic ability of the vector&#160;to produce packaged&#160;particles. A comparison between different AAV serotypes showed that some AAV vectors are more difficult to produce at a higher titer than others (e.g., AAV2)41. Precipitation of the vector, during the desalting step, can be a possible reason for a lower titer and easily prevented by avoiding overconcentration33. Moreover, it is also possible that the efficiency of iodixanol gradient-based purification slightly differs between serotypes and, therefore, discrepancies in the titer obtained with different serotypes can be observed41.
Finally, it needs to be pointed out that&#160;even though qPCR is a very accurate method for DNA quantification&#160;some inherent variability in the technique can be observed. Accuracy in the titration primarily depends on the precise pipetting and proper vortexing of all the solutions. To guarantee the most accurate titer reading, the qPCR can be independently repeated, and the obtained values averaged. The choice of primers presented in this protocol is based on the sequence of the CBA promoter located in the pTransgene plasmid used in our laboratory. The CBA promoter is a strong synthetic promoter that is widely used in the vector field to drive expression across multiple cell types. It incorporates multiple elements, including the cytomegalovirus (CMV) early enhancer element; the promoter, first exon, and the first intron of the CBA gene; and the splice acceptor of the rabbit &#946;-globin gene. However, primers can be designed for virtually any element located within the expression cassette (including the promoter, transgene, and regulatory elements). The comparison of titer across batches is also possible, providing primers are used against regions common to the vectors in question.
In conclusion, this protocol can be used to produce AAV vectors with a variety of capsids, genome configurations, promoter types&#160;and transgene cargos. This will allow users to easily adapt the final characteristics of their vectors to best suit experimental needs. In the example presented in the representative results, the use of the PHP.B capsid, which efficiently crosses the BBB, gave&#160;highly efficient gene expression in the CNS, following tail vein injection32. The systemic administration of CNS penetrant vectors has considerable advantages in terms of possible side-effects2,17,32. A possible alternative to peripheral injection, while avoiding the caveats of invasive techniques, is intrathecal delivery, which consists of&#160;delivery of the AAV vector into the cerebrospinal fluid. This delivery route is proven to be effective, showing&#160;widespread expression of transgene across the CNS, less off-target effects in peripheral organs, and low levels of immune response42. However, intrathecal injections are much more challenging, as they require higher technical skills than tail vein injection.
Further capsid development to refine this technology will be driven by the opportunities for AAV vector use in gene therapy applications. Such approaches offer attractive possibilities to treat currently incurable CNS disorders, such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease,&#160;Parkinson&#8217;s and Alzheimer&#8217;s disease18.

Over the past 30 years, wild-type AAVs have been engineered to create recombinant AAV vectors, which have proven to be exceptionally useful tools for gene transfer&#160;into the CNS1,2,3,4,5,6 and gene therapy approaches to disease (including FDA- and EMA-approved therapies)4,7. Their suitability for use in the CNS largely derives from their ability to infect non-dividing, post-mitotic cells, typically found in the CNS8. However, AAV-based vectors also have the advantage of allowing a long-term expression of any given therapeutic transgene4,9 while eliciting a milder immune response compared to other viral vectors7,8,10,11,12.
The main elements of any AAV vector are the genome and the capsid. Wild-type AAVs are single-stranded (ss) DNA viruses with a genome of approximately 5 kilobases (kb)13. For the production of recombinant AAV vectors, the rep and cap genes (necessary for genome replication and the assembly of the viral capsid) are deleted from the genome of the wild-type AAV and provided in trans, leaving room for an expression cassette containing the transgene14,15. The inverted terminal repeat sequences (ITRs) of the original viral genome are the only retained elements in an AAV vector, as these are essential for replication and packaging3,10,14. AAV vectors can be engineered to enhance transgene expression; a mutation in one of the ITRs leads to the formation of a hairpin loop which effectively allows the generation of a complementary DNA strand3,7,15. The main advantage of this configuration, termed a self-complementary (sc) genome, is that it bypasses the need for&#160;second-strand synthesis typical in the conventional life cycle of AAVs, considerably increasing the speed and levels of transgene expression1. Nevertheless, using an scAAV genome reduces the cargo capacity of the vector to approximately 2.4 kb. This includes transgene sequences, as well as any regulatory sequences such as promoters or microRNA-binding sites, to limit expression to specific cell types16.
The AAV capsid determines the vector-host cell interaction and confers a degree of cell type or tissue tropism for an AAV serotype, which can also be exploited to limit transgene expression to specific locations. Several AAV serotypes are found in nature, whereas others have been produced in laboratories through recombinant approaches (i.e., PHP.B). In addition, some capsids also bestow other useful characteristics, such as the ability to cross the BBB, resulting in the delivery of transgenes throughout the CNS after systemic administration. This has been shown for AAV9, as well as for the recently described PHP.B capsid17. As a consequence, these serotypes are proving to be particularly relevant for new gene therapy approaches for&#160;neurodegenerative disorders1,17,18.
The aim of this protocol is to describe a cost-effective method for the small-scale production of AAV vectors with high titer and purity. Although the results presented here use the PHP.B capsid and a scAAV expression cassette, the protocol is suitable for the production of several AAV vector serotypes and genome configurations, allowing maximum experimental flexibility. However, the vector yield and final purity can vary depending on the chosen serotype.
The protocol itself is a variant of the classical tri-transfection method for viral vector production and incorporates the use of an iodixanol gradient for vector clean-up, which, in comparison to the classical use of cesium chloride (CsCl) gradients, has been reported to produce AAV vectors of higher purity in a more time-efficient manner19,20,21.
The transfection, purification&#160;and concentration steps are intended to be performed according to&#160;good laboratory practice (GLP) in a tissue culture laboratory rated for viral vector work. Each task needs to be performed in compliance with the relevant local and national legislation concerning viral vector production and use. Work must be carried out under a laminar flow hood and in sterile conditions. Inside the vector facility, it is recommended to wear a lab apron, in addition to the regular tissue culture lab coat. Moreover, a double pair of gloves, as well as plastic overshoes, should be worn at all times.
Before starting the vector production, ensure all necessary equipment and plasmids are available. 1) pCapsid plasmid contains the rep gene that encodes four non-structural proteins necessary for replication, namely Rep78, Rep68, Rep52, and Rep40, and the cap gene that encodes three structural capsid proteins, namely VP1, VP2, and VP3. 2) pHelper plasmid contains the genes E4, E2A, and VA from adenovirus, which facilitate AAV production in HEK293T cells. 3) pTransgene plasmid contains the transgene expression cassette, flanked by two ITRs. These plasmids can be synthesized de novo in the laboratory using sequences available online22. For plasmids made de novo, especially those containing novel transgenes, sequencing is required, to make sure that the transgene and ITRs are correct. Alternatively, premade plasmids can be directly acquired through online plasmid repositories. When necessary, plasmids can be amplified and purified using standard kits, according to the manufacturer&#39;s instructions23.
Vector titer and purity can adversely affect the transduction ability of the vector. Additional protocols are supplied to evaluate the quality of the produced vector. The final vectors will be useful for studies of the CNS cell function in both in vitro and in vivo applications.

<table><tbody><tr><td>&lt;strong&gt;Plasmid production&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pTransgene plasmid</td><td>&lt;em&gt;De novo &lt;/em&gt;design or obtained from a plasmid repository</td><td>N/A</td><td>See step 1 of main protocol for further details</td></tr><tr><td>pCapsid</td><td>&lt;em&gt;De novo&lt;/em&gt; design or obtained from a plasmid repository</td><td>N/A</td><td>See step 1 of main protocol for further details</td></tr><tr><td>pHelper</td><td>Agilent</td><td>240071</td><td></td></tr><tr><td>Plasmid &lt;em&gt;Plus &lt;/em&gt;maxi kit</td><td>Qiagen</td><td>12963</td><td></td></tr><tr><td>QIAquick PCR purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>AAV Helper-Free System</td><td>Agilent</td><td>240071</td><td></td></tr><tr><td>&lt;strong&gt;Cell culture and transfection &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM), high glucose, no glutamine</td><td>Life technologies</td><td>11960-044</td><td>Supplement DMEM with FBS (1% or 10% v/v) and GlutaMAX 200 mM (1% v/v) then filter sterilize the medium using a 0.22 &amp;micro;m filter</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GIBCO</td><td>10500-064</td><td></td></tr><tr><td>GlutaMAX supplement</td><td>GIBCO</td><td>35050038</td><td></td></tr><tr><td>(200 mM)</td><td></td><td></td><td></td></tr><tr><td>Corning bottle-top vacuum filter system</td><td>Sigma-Aldrich</td><td>CLS430769</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS) with no calcium and no magnesium</td><td>GIBCO</td><td>14190094</td><td></td></tr><tr><td>HEK293T cells</td><td>American Tissue Culture Collection</td><td>CRL3216</td><td>Upon receipt, thaw the cells and culture as described in the protocol. After a minimal number of passages, freeze a subfraction for future in aliquots.&amp;nbsp;Always use cells below passage number 20. Once cultured cells have been passaged more than 20 times, restart a culture from the stored aliquots</td></tr><tr><td>Cell culture dishes</td><td>Greiner Cellstar</td><td>639160</td><td>15 cm diameter culture dishes</td></tr><tr><td>Cell scrapers</td><td>VWR</td><td>10062-904</td><td></td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polyscience</td><td>23966-2</td><td>PEI in powder form is dissolved at 1 &amp;micro;g/&amp;micro;L in deionized water (&lt;sub&gt;dd&lt;/sub&gt;water) at pH=2 (use HCl). Prepare in a beaker and stir for 2-3 h. When dissolved, bring the pH back to 7 with NaOH. Filter sterilize and store the resuspended stock solution in 1 ml aliquots at -20 &amp;deg;C. PEI aliquots can 
 be freeze/thawed multiple times.</td></tr><tr><td>Virkon solution</td><td>Fisher Scientific</td><td>NC9821357</td><td>Disinfect any material that has been in contact with assembled vector particles with Virkon solution</td></tr><tr><td>Mutexi long-sleeve aprons</td><td>Fisher Scientific</td><td>11735423</td><td>Wear an apron over the top of a regular lab coat</td></tr><tr><td>Fisherbrand maximum protection disposable overshoes</td><td>Fisher Scientific</td><td>15401952</td><td></td></tr><tr><td>&lt;strong&gt;AAV Purification and desalting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optiprep density gradient medium</td><td>Sigma-Aldrich</td><td>D1556</td><td>Optiprep is a 60% (w/v) solution of iodixanol in water (sterile). CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Phenol red</td><td>Sigma-Aldrich</td><td>P0290</td><td>CAUTION. Use under a laminar flow hood</td></tr><tr><td>Pasteur pipette</td><td>Sigma-Aldrich</td><td>Z627992</td><td>Sterilize before use</td></tr><tr><td>OptiSeal Polypropylene tubes</td><td>Beckman</td><td>361625</td><td></td></tr><tr><td>Benzonase (250 U/&amp;micro;L)</td><td>Sigma-Aldrich</td><td>E1014</td><td>Supplied as a ready-to-use solution</td></tr><tr><td>Acrodisc syringe filter</td><td>Pall corporation</td><td>4614</td><td></td></tr><tr><td>Omnifix syringe (5 mL)</td><td>Braun</td><td>4617053V</td><td></td></tr><tr><td>Blunt syringe needle</td><td>Sigma Aldrich</td><td>Z261378</td><td>Stainless steel 316 syringe needle, pipetting blunt 90&amp;deg; tip gauge 16, L 4 in. Referred to in the text as a blunt-end needle</td></tr><tr><td>Aqua Ecotainer</td><td>B. Braun</td><td>0082479E</td><td>Sterile endotoxin-free water. Referred to as &#039;Ultrapure water&#039;</td></tr><tr><td>Amicon ultra-15 centrifugal filter unit</td><td>Millipore</td><td>UFC910024</td><td>These filters concentrate the final product by collecting the vector particles in consecutive centrifugation steps</td></tr><tr><td>Pluronic F68 (100x)</td><td>Thermo Fisher</td><td>24040032</td><td>Non-ionic surfactant. Dilute in sterile PBS to use at 0.01% (v/v)</td></tr><tr><td>Fisherbrand Sterile Microcentrifuge Tubes with Screw Caps (2 mL)</td><td>Fisher Scientific</td><td>02-681-374</td><td>Use skirted tubes for easy handling</td></tr><tr><td>&lt;strong&gt;AAV Titration &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Restriction enzyme: StuI (10 U/&amp;micro;L)</td><td>Promega</td><td>R6421</td><td></td></tr><tr><td>DNAse I (1 U/&amp;micro;L)</td><td>Fisher Scientific</td><td>EN0521</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>3115852001</td><td>Reconstitute in ultrapure water and use at a final concentration of 10 mg/mL. Solution can be stored at -20 &amp;deg;C</td></tr><tr><td>EasyStrip Plus Tube Strips (with attached caps)</td><td>Fisher Scientific</td><td>AB2000</td><td></td></tr><tr><td>Eppendorf microtube 3810x</td><td>Sigma-Aldrich</td><td>Z606340-1000EA</td><td></td></tr><tr><td>LightCycler 480 SYBR Green I Master Mix</td><td>Roche</td><td>4707516001</td><td></td></tr><tr><td>LightCycler Multiwell Plates, 96 wells</td><td>Roche</td><td>4729692001</td><td>White polypropylene plate (with unique identifying barcode)</td></tr><tr><td>Microseal &#039;A&#039; PCR plate and PCR Plate Sealing Film</td><td>Bio-Rad</td><td>msa5001</td><td></td></tr><tr><td>&lt;strong&gt;AAV Purity control &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Sigma-Aldrich</td><td>A3678</td><td>Reconstitute in ultrapure water to 10% (v/v). CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Sigma-Aldrich</td><td>T9281</td><td>CAUTION. Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Tris Base ULTROL Grade</td><td>Merck</td><td>648311</td><td>CAUTION: Use under a laminar flow hood. Wear gloves</td></tr><tr><td>UltraPure Agarose</td><td>Thermo Fisher</td><td>16500-500</td><td></td></tr><tr><td>Rotiphorese&amp;reg; Gel 30 (37,5:1)</td><td>Carl Roth</td><td>3029.3</td><td>Aqueous 30% acrylamide and bisacrylamide stock solution at a ratio of 37.5:1. CAUTION: Use under a laminar flow hood. Wear gloves</td></tr><tr><td>Serva Blue G</td><td>Sigma-Aldrich</td><td>6104-58-1</td><td></td></tr><tr><td>Precision Plus prestained marker</td><td>Bio-Rad</td><td>1610374edu</td><td></td></tr><tr><td>1-Butanol</td><td>Sigma-Aldrich</td><td>B7906</td><td>CAUTION: Use under a laminar flow hood. Wear gloves</td></tr><tr><td>&lt;strong&gt;Immunohistochemistry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rabbit anti-GFP</td><td>Synaptic Systems</td><td>132002</td><td>1:300 dilution</td></tr><tr><td>Anti-rabbit Alexa Fluor 488</td><td>Invitrogen</td><td>A21206</td><td>1:1,000 dilution</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Vector production lab&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rotina 380 bench-top centrifuge *</td><td>Hettich</td><td>1701</td><td></td></tr><tr><td>Optima XPN 80 ultracentrifuge *</td><td>Beckmann Coulter</td><td>A95765</td><td></td></tr><tr><td>Type 50.2 Ti fixed-angle titanium rotor *</td><td>Beckmann Coulter</td><td>337901</td><td></td></tr><tr><td>Entris digital scale *</td><td>Sartorius</td><td>2202-1S</td><td></td></tr><tr><td>Warm water bath *</td><td></td><td></td><td>Set at 37 &amp;deg;C</td></tr><tr><td>Ice bucket *</td><td>VWR</td><td>10146-290</td><td>Keep material used in the vector production lab separate from that used in standard lab areas</td></tr><tr><td>Pipetboy pro *</td><td>Integra</td><td>156,400</td><td></td></tr><tr><td>Graduated pipettes: Cell star *</td><td>Greiner bio-one</td><td>606180</td><td>Capacity of 5 mL</td></tr><tr><td>Graduated pipettes: Cell star *</td><td>Greiner bio-one</td><td>607180</td><td>Capacity of 10 mL</td></tr><tr><td>Graduated pipettes: Cell star *</td><td>Greiner bio-one</td><td>760180</td><td>Capacity of 25 mL</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator CB150 *</td><td>Binder</td><td>9040-0038</td><td>Set at 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; and 95% humidity</td></tr><tr><td>Nuaire safety cabinet NU 437-400E *</td><td>Labexchange</td><td>31324</td><td>Clean all the surfaces with 70% ethanol and Virkon before and after use</td></tr><tr><td>&lt;strong&gt;Conventional lab&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>T100 thermal cycler *</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>LightCycler 480 Instrument II *</td><td>Roche</td><td>5015278001</td><td></td></tr><tr><td>ThermoMixer *</td><td>Eppendorf</td><td>C 5382000015</td><td></td></tr><tr><td>Nanodrop *</td><td>ThermoFisher Scientific</td><td>ND 2000</td><td></td></tr><tr><td>Mini-Protean Tetra Cell*</td><td>Bio-Rad</td><td>1658001FC</td><td>For use with handmade or precast gels</td></tr><tr><td>ProteoSilver silver stain kit</td><td>Sigma-Aldrich</td><td>PROTSIL1</td><td>High sensitivity protein detection with low background</td></tr><tr><td>Centrifuge 5804 R *</td><td>Eppendorf</td><td>B1_022628045</td><td>High speed centrifuge for medium capacity needs (up to 250 mL)</td></tr><tr><td>Graduated pipettes Cell star *</td><td>Greiner bio-one</td><td>606180</td><td>5 mL</td></tr><tr><td>Graduated pipettes Cell star *</td><td>Greiner bio-one</td><td>607180</td><td>25 mL</td></tr><tr><td>Graduated pipettes Cell star *</td><td>Greiner bio-one</td><td>760180</td><td>25 mL</td></tr><tr><td>Filter tips *</td><td>Greiner bio-one</td><td>750257</td><td>1250 &amp;micro;L</td></tr><tr><td>Filter tips *</td><td>Greiner bio-one</td><td>738257</td><td>300 &amp;micro;L</td></tr><tr><td>Filter tips *</td><td>Greiner bio-one</td><td>771257</td><td>10 &amp;micro;L</td></tr><tr><td>Ice bucket with lid *</td><td>VWR</td><td>10146-290</td><td></td></tr><tr><td>Mini diaphragm vacuum pump, VP 86 *</td><td>VWR</td><td>181-0065</td><td></td></tr><tr><td>Pipetman P2</td><td>Gilson</td><td>F144801</td><td></td></tr><tr><td>Pipetman P20</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>Pipetman P100</td><td>Gilson</td><td>F123615</td><td></td></tr><tr><td>Pipetman P200</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>Pipetman P1000</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>* Materials marked with an asterisk are expensive pieces of equipment and are usually central infrastructure items shared between multiple labs. These items can also be replaced by equivalents if available. Note, when a different ultracentrifuge is used, care must be taken to select the correct rotor and centrifuge tubes.</td><td></td><td></td><td></td></tr></tbody></table>

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.

AAV9 was considered, until recently, to be the most effective AAV vector serotype in crossing the BBB and transducing cells of the CNS, following peripheral administration. A significant advance in capsid design was achieved when Deverman et al. reported the use of a capsid selection method called Cre recombination-based AAV-targeted evolution (CREATE)17. Using this method, they identified a new capsid, named PHP.B, which they reported as able to transduce the majority of astrocytes and neurons in multiple CNS regions, following systemic injection17. At this point, it should be noted that even though PHP.B provides positive results in C57/Bl6 mice (which was the strain used in the initial isolation experiments), preliminary reports suggest its efficiency may vary in a strain-dependent manner. Further experiments will, no doubt, shed more light on this issue31.
However, despite these issues, PHP.B offers exciting possibilities for noninvasive gene manipulation in the CNS of mice, including proof-of-concept gene therapy experiments in disease models. As such, we chose to evaluate the efficiency of transgene expression using PHP.B versus AAV9, which has been the &#39;gold-standard&#39; vector for CNS transduction following peripheral administration since 20092. To perform a direct comparison of both serotypes, under optimal conditions for transgene expression, we used an sc genome configuration32. Both vectors carried the transgene for green fluorescent protein (GFP) under the control of the ubiquitous chicken &#946;-actin (CBA) promoter. Female C57/Bl6 mice at postnatal day 42 (approximately 20 g in weight) received a dose of 1 x 1012 vg per mouse of either scAAV2/PHP.B-CBA-GFP or scAAV2/9-CBA-GFP. Vector administration was performed via tail vein injection. The experiments were approved by the Ethical Committee of the KU Leuven.
Three weeks postinjection, the mice underwent transcardial perfusion with ice-cold PBS, followed by 4% (w/v) ice-cold paraformaldehyde (PFA). Their brains were harvested and underwent further postfixation by an overnight incubation in the same fixative, before transferring to 0.01% (w/v) Na-azide/PBS for storage until further analysis. Afterward, the brains were sectioned using a vibrating microtome, and immunohistochemistry was performed on 50 &#181;m thick sections.
To evaluate the levels of transgene expression, sections were stained with primary antibodies against GFP (rabbit anti-GFP), with detection using secondary antibodies conjugated to a fluorescent dye (anti-rabbit Alexa Fluor 488) (Figure 4A, B). Fluorescence intensity measurements (in arbitrary units [au]) confirmed a significant increase in GFP expression when an sc genome and the PHP.B capsid were used relative to AAV9. Increases in GFP were observed in the cerebrum (2105 &#177; 161 vs. 1441 &#177; 99 au; p = 0.0032), the cerebellum (2601 &#177; 196 vs. 1737 &#177; 135 au; p = 0.0032), and the brainstem (3082 &#177; 319 vs. 2485 &#177; 88 au; p = 0.0038) (Figure 4C).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58960/58960fig4.jpg" /> 
Figure 4: Systemic delivery of scAAV2/PHP.B-CBA-GFP leads to a high GFP expression in the CNS. scAAV2/PHP.B-CBA-GFP or scAAV2/9-CBA-GFP (1 x 1012 vg/mouse) was administered to 6-weeks-old C57/Bl6 mice via tail vein injection. GFP was detected using immunohistochemistry on coronal brain sections 3 weeks postinjection. (A) The cerebrum and (B) the cerebellum and brainstem are shown. Scale bars = 1 mm. (C) The quantification of relative fluorescence intensities was performed to determine the levels of GFP signal achieved with each vector (10 sections per mouse; three mice per vector group). A one-way ANOVA test was performed, followed by a two-tailed Student&#39;s t-test; the data are expressed as mean &#177; standard deviation; **p &#60; 0.01; au. arbitrary units. pCapsid, used for AAV vector production, contains the gene rep from serotype 2 and the gene cap from serotype PHP.B or AAV9, accordingly. This figure has been modified from Rincon et al.32. <a href="https://www.jove.com/files/ftp_upload/58960/58960fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors at JoVE.com

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

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), ...

production-purification-quality-control-for-adeno-associated-virus

Research

JoVE Journal

Neuroscience

35.9K Views.  VIB-KU Leuven Center for Brain and Disease Research. 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 (≥1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

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

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

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

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

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. ...

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

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

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. ...

Bioengineering

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. ...

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...

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.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a ...

Medicine

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.