Name: production, purification, and quality control for adeno-associated virus-based vectors
Uploaded: 2019-01-29T14:00:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors at JoVE.com

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.

<ol>
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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 colspan="4">Plasmid production</td>
			<td></td>
		</tr>
		<tr>
			<td>pTransgene plasmid</td>
			<td>De novo design or obtained from a plasmid repository</td>
			<td>N/A</td>
			<td>See step 1 of main protocol for further details</td>
			<td></td>
		</tr>
		<tr>
			<td>pCapsid</td>
			<td>De novo design or obtained from a plasmid repository</td>
			<td>N/A</td>
			<td>See step 1 of main protocol for further details</td>
			<td></td>
		</tr>
		<tr>
			<td>pHelper</td>
			<td>Agilent</td>
			<td>240071</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Plus maxi kit</td>
			<td>Qiagen</td>
			<td>12963</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV Helper-Free System</td>
			<td>Agilent</td>
			<td>240071</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell culture and transfection </td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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 mm filter</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GIBCO</td>
			<td>10500-064</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td rowspan="2">GIBCO</td>
			<td rowspan="2">35050038</td>
			<td rowspan="2"></td>
			<td></td>
		</tr>
		<tr>
			<td>(200 mM)</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filter system</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430769</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS) with no calcium and no magnesium</td>
			<td>GIBCO</td>
			<td>14190094</td>
			<td></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.&#160;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>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes</td>
			<td>Greiner Cellstar</td>
			<td>639160</td>
			<td>15 cm diameter culture dishes</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>VWR</td>
			<td>10062-904</td>
			<td></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 &#181;g/&#181;L in deionized water (ddwater) 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 &#176;C. PEI aliquots can freeze/thawed multiple times.</td>
			<td></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 viral particles with Virkon solution</td>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand maximum protection disposable overshoes</td>
			<td>Fisher Scientific</td>
			<td>15401952</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">AAV Purification and desalting</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>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td>CAUTION. Use under a laminar flow hood</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Sigma-Aldrich</td>
			<td>Z627992</td>
			<td>Sterilize before use</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiSeal Polypropylene tubes</td>
			<td>Beckman</td>
			<td>361625</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase (250 U/&#181;L)</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>Supplied as a ready-to-use solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrodisc syringe filter</td>
			<td>Pall corporation</td>
			<td>4614</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnifix syringe (5mL)</td>
			<td>Braun</td>
			<td>4617053V</td>
			<td></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&#176; tip gauge 16, L 4 in. Referred to in the text as a blunt-end needle</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua Ecotainer</td>
			<td>B. Braun</td>
			<td>0082479E</td>
			<td>Sterile endotoxin-free water. Referred to as &#39;Ultrapure water&#39;</td>
			<td></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 viral particles in consecutive centrifugation steps</td>
			<td></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>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">AAV Titration </td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Restriction enzyme: StuI (10 U/&#181;L)</td>
			<td rowspan="2">Promega</td>
			<td rowspan="2">R6421</td>
			<td rowspan="2"></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I (1 U/&#181;L)</td>
			<td>Fisher scientific</td>
			<td>EN0521</td>
			<td></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&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyStrip Plus Tube Strips (with attached caps)</td>
			<td>Fisher scientific</td>
			<td>AB2000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microtube 3810x</td>
			<td>Sigma-Aldrich</td>
			<td>Z606340-1000EA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 SYBR Green I Master Mix</td>
			<td>Roche</td>
			<td>4707516001</td>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td>Microseal &#39;A&#39; PCR Plate and PCR Tube Sealing Film</td>
			<td>Bio-Rad</td>
			<td>msa5001</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">AAV Purity control </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 laminar flow hood. Wear gloves</td>
			<td></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>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Thermo Fisher</td>
			<td>16500-500</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotiphorese&#174; 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 laminar flow hood. Wear gloves</td>
			<td></td>
		</tr>
		<tr>
			<td>Serva Blue G</td>
			<td>Sigma-Aldrich</td>
			<td>6104-58-1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus prestained marker</td>
			<td>Bio-Rad</td>
			<td>1610374edu</td>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Immunohistochemistry</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFP</td>
			<td>Synaptic System</td>
			<td>132002</td>
			<td>1:300 dilution</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>1:1000 dilution</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Vector production lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotina 380 bench-top centrifuge *</td>
			<td>Hettich</td>
			<td>1701</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XPN 80 ultracentrifuge *</td>
			<td>Beckmann Coulter</td>
			<td>A95765</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Type 50.2 Ti fixed-angle titanium rotor *</td>
			<td>Beckmann Coulter</td>
			<td>337901</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Entris digital scale *</td>
			<td>Sartorius</td>
			<td>2202-1S</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water bath *</td>
			<td></td>
			<td></td>
			<td>Set at 37&#176;C</td>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td>Pipetboy pro *</td>
			<td>Integra</td>
			<td>156,400</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated pipettes: Cell star *</td>
			<td>Greiner bio-one</td>
			<td>606180</td>
			<td>Capacity of 5 ml, 10 ml and 25 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated pipettes: Cell star *</td>
			<td>Greiner bio-one</td>
			<td>607180</td>
			<td>Capacity of 5 ml, 10 ml and 25 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated pipettes: Cell star *</td>
			<td>Greiner bio-one</td>
			<td>760180</td>
			<td>Capacity of 5 ml, 10 ml and 25 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Co2 incubator CB150 *</td>
			<td>Binder</td>
			<td>9040-0038</td>
			<td>Set at 37&#176;C, 5% CO2 and 95% humidity</td>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Conventional lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T100 thermal cycler *</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 Instrument II *</td>
			<td>Roche</td>
			<td>5015278001</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer *</td>
			<td>Eppendorf</td>
			<td>C 5382000015</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop *</td>
			<td>ThermoFisher Scientific</td>
			<td>ND 2000</td>
			<td></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>
			<td></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>
			<td></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>
			<td></td>
		</tr>
		<tr>
			<td>Graduated pipettes Cell star *</td>
			<td>Greiner bio-one</td>
			<td>606180</td>
			<td>5 ml, 10 ml and 25 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated pipettes Cell star *</td>
			<td>Greiner bio-one</td>
			<td>607180</td>
			<td>5 ml, 10 ml and 25 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated pipettes Cell star *</td>
			<td>Greiner bio-one</td>
			<td>760180</td>
			<td>5 ml, 10 ml and 25 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips *</td>
			<td>Greiner bio-one</td>
			<td>750257</td>
			<td>2 &#181;l, 20 &#181;l, 200 &#181;l</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips *</td>
			<td>Greiner bio-one</td>
			<td>738257</td>
			<td>2 &#181;l, 20 &#181;l, 200 &#181;l</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips *</td>
			<td>Greiner bio-one</td>
			<td>771257</td>
			<td>2 &#181;l, 20 &#181;l, 200 &#181;l</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket with lid *</td>
			<td>VWR</td>
			<td>10146-290</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini diaphragm vacuum pump, VP 86 *</td>
			<td>VWR</td>
			<td>181-0065</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P2, P20, P100, P200, P1000</td>
			<td>Gilson</td>
			<td>F144801</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P2, P20, P100, P200, P1000</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P2, P20, P100, P200, P1000</td>
			<td>Gilson</td>
			<td>F123615</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P2, P20, P100, P200, P1000</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P2, P20, P100, P200, P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">* Materials marked with an asterisk are expensive vpieces 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>
		</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), ...

Research

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Neuroscience

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Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

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An In Ovo Model for Testing Insulin-mimetic Compounds

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Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

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Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

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Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

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