This work was supported by a grant from the Royal Netherlands Academy of Arts and Sciences (KNAW) research fund and a grant from Start2Cure (0-TI-01). We thank Leisha Kopp for her input and advice in the setup of the protocol. Figures were created using Biorender.

All experimental procedures were approved by the institutional animal care and use committee of the Royal Netherlands Academy of Sciences (KNAW) and were in accordance with the Dutch Law on Animal Experimentation under project number AVD8010020199126. In Figure 1, a schematic overview of the complete protocol is provided. From seeding cells to AAV purification, the protocol takes 6 days to complete.
1. Reagent preparation
<ol>
	<li>Plasmid purification
	<ol>
		<li>Perform plasmid extraction as described by the manufacturer with a few adjustments to ensure sterility of the plasmids.</li>
		<li>Prepare 70% ethanol using sterile distilled water and absolute ethanol.</li>
		<li>After the isopropanol centrifugation step, dry plasmids in the flow cabinet hood and add sterile 70% ethanol to the tubes. After ethanol precipitation, dry the plasmids in the flow cabinet hood and resuspend the plasmid in sterile distilled water. Take care to use sterilized microcentrifuge tubes.</li>
		<li>Take an aliquot for quantification, restriction analysis, and, if needed, sequencing. It is advised to check the integrity of the inverted terminal repeats (ITRs) as mutations can have a negative impact on yield. Adjust the sample concentration to 1 &#181;g/&#181;L.</li>
	</ol>
	</li>
	<li>Preparation of 10x Tween lysis buffer
	<ol>
		<li>Dissolve 30.3 g Tris buffer and 2.033 g MgCl2.6H2O in 400 mL of sterile distilled water, set pH to 8.0, and increase total volume to 450 mL.</li>
		<li>Keep Tween 20 sterile, add 50 mL of Tween 20 to buffer solution in the hood, and filter sterilize using a 0.2&#160;&#181;M vacuum filter.</li>
	</ol>
	</li>
	<li>Iodixanol dilutions
	<ol>
		<li>Iodixanol solution is provided at 60% concentration. Use the starting solution to make 15%, 25%, and 40% solutions.</li>
		<li>For 15% solution, dilute 60 mL of 60% solution with 48 mL of 5 M NaCl and 48 mL of PBS-MK (5x PBS with 5 mM MgCl2 and 12.5 mM KCl), and add distilled water up to 240 mL.</li>
		<li>For 25% solution, dilute 67 mL of 60% solution and 32 mL of PBS-MK. Add distilled water up to 160 mL. Mix well and add 1.6 mL of phenol red solution.</li>
		<li>For 40% solution, dilute 160 mL of 60% solution with 48 mL of PBS-MK and add distilled water up to 240 mL. For 60% solution, add 1 mL of phenol red to 100 mL of 60% solution.</li>
	</ol>
	</li>
	<li>PBS 5% sucrose solution
	<ol>
		<li>Add 25 g of sucrose to a 500 mL bottle of DPBS without calcium or magnesium. Shake well and filter using a 0.2 &#181;M bottle vacuum filter.</li>
	</ol>
	</li>
	<li>Polyethylene glycol (PEG) 8000 solution
	<ol>
		<li>Dissolve 400 g of PEG 8000 and 24 g of NaCl into sterile distilled water and adjust to a final volume of 1000 mL. Stir with heating until fully dissolved. Adjust pH to 7.4 using pH paper.</li>
		<li>Filter sterilize using a 0.2&#160;&#181;M bottle vacuum filter to obtain a 40% PEG 8000 solution. Note that filtration will take a while due to the viscosity of the solution-store at 4 &#730;C.</li>
	</ol>
	</li>
</ol>
2. Culture of HEK293 suspension cells
<ol>
	<li>Use wipes soaked in 70% ethanol for cleaning. Clean biosafety hood, prepare and clean all materials needed for culturing cells.</li>
	<li>Prewarm 30 mL of suspension cell medium to 37 &#176;C in a 50 mL conical culturing tube. Retrieve viral production cells from liquid nitrogen. Quickly thaw cells in a 37 &#176;C water bath. Just before the vial is completely thawed, clean it with a wipe soaked in 70% ethanol and transfer the vial to the biosafety hood. 
	NOTE: The details of the viral production cells used here are provided in the Table of Materials.</li>
	<li>Quickly transfer cells to the prewarmed 50 mL conical culturing tube. Culture cells in a shaking incubator at 37 &#176;C, 80% humidity, 8% CO2,&#160;200 revolutions per min (RPM), and a shaking diameter of 50 mm. Passage cells, once viability is above 90% and cell density is above 1-3 x 106 cells/mL. 
	NOTE: The incubator used has a shaking diameter of 50 mm; culture conditions should be adjusted for a different shaking diameter.</li>
	<li>Passage cells 3-4 days after thawing. Check culture tube(s) to make sure there are no signs of contamination; for example, fungus will appear as a discolored (black or green) ring.</li>
	<li>Prepare 400 &#181;L of 0.4% trypan blue solution per sample using a 1 mL stripette.</li>
	<li>Quickly retrieve suspension cells from the incubator. Immediately extract 500 &#181;L from the tube using a 1 mL stripette. Gently pipet up and down.</li>
	<li>Add 100 &#181;L of cell suspension to the prepared trypan blue tube. Gently invert the tube to mix; do not pipet to mix, as this will lead to cell death. Return cells to incubator.</li>
	<li>Take 50 &#181;L of trypan blue-treated cell suspension and apply it to the hemocytometer slide. Count total viable cells and calculate the amount of cell suspension and medium needed for passage or transfection the following day.</li>
	<li>Warm medium in the incubator for 10-15 min. Add cell suspension to warmed medium and return to incubator. For passaging cells for 3 days (i.e., Monday-Thursday), set to 0.5 x 106 cells/mL; for passaging cells for 4 days (i.e., Thursday-Monday), set to 0.3 x 106 cells/mL. 
	NOTE: According to the manufacturer, viral production cells double every 26 h and should be between 3.5 x 106 and 5.5 x 106 before passaging. Cells can be kept for up to passage 20.</li>
</ol>
3. Transfection
<ol>
	<li>Day 1
	<ol>
		<li>At 24 h before transfection, culture 1 x 106 cells per/mL in 300 mL of media.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Warm medium, plasmids, and transfection reagent to room temperature. For the amounts to prepare, see Table 1.</li>
		<li>Briefly vortex plasmids and reagents. Add plasmids to the prepared medium, vortex. Add transfection reagent and vortex briefly. Do not agitate the mixture after this step. Incubate at room temperature for 30 min.</li>
		<li>In the meantime, count cells prepared on day 1. Cells should be between 2 and 2.5 x 106 cells per mL and &#62; 95% viable.</li>
		<li>After incubation, dropwise, add the transfection mixture to cells while gently swirling the tube. Incubate for 72-96 h.</li>
	</ol>
	</li>
</ol>
4. Harvesting the cells
<ol>
	<li>Day 5
	<ol>
		<li>Add 33 mL of 10x cell lysis buffer (500 mM Tris pH 8, 10% Tween 20, 20 mM MgCl2), mix by gently shaking, and incubate at 37 &#176;C for 1.5 h with shaking.</li>
		<li>Centrifuge at 3428 x g at 4 &#176;C for 60 min. Filter through a 0.45 &#181;M PES vacuum filter to clarify the cell lysate, leaving the clarified lysate in the container of the filter. 
		NOTE: Optional after filtration: take 50 &#181;L of sample for quantitative-PCR (Q-PCR).</li>
		<li>Add 90 mL of 40% PEG 8000 solution to 360 mL of filtered cell lysate.</li>
		<li>Clean the stir bar with 70% ethanol and add to the sample. Stir on ice or in the cold room at 300 RPM for 1 h. Incubate at 4 &#176;C without stirring overnight. 
		NOTE: Incubation times of 1 h to 72 h have been tested. Longer incubation times have a negative effect on overall protein precipitation.</li>
	</ol>
	</li>
</ol>
5. Iodixanol purification
<ol>
	<li>Day 6
	<ol>
		<li>Transfer the entire PEG precipitated culture volume sample to a clean, large conical tube and centrifuge at 2820 x g and&#160;4&#176;C for 15 min.</li>
		<li>Discard the supernatant and resuspend the PEG pellet in 15 mL of DPBS with calcium and magnesium. The pellet is difficult to resuspend; take the time to do this carefully.</li>
		<li>Transfer the resuspended part to a clean 50 mL tube. PEG will remain on the sides of the bioreactor tube. Add an extra 10 mL, taking care to gather all the PEG precipitate. Transfer all to a 50 mL container for a total volume of 25-30 mL.</li>
		<li>Add 40 &#181;L of DNaseI (10 U/&#181;L). Place in the incubator for 1 h 37&#176;C. 
		NOTE: Optional after incubation of DNase: take 50 &#181;L of sample for Q-PCR.</li>
		<li>Clean stir bars that were used for PEG precipitation well with chloride solution followed by 70% ethanol. Leave stir bars in 70% ethanol for the next experiment.</li>
		<li>Fill a 25 mm x 89 mm polyallomer tube using a glass Pasteur pipette with 15.5 mL of concentrated cell lysate in each tube. After adding cell lysate, exchange the glass Pasteur pipette for a new one.</li>
		<li>Add iodixanol solutions from 15%-60%: infuse 9 mL of the 15% iodixanol solution gently beneath the cell lysate. Next, add 5 mL of the 25% and 40% iodixanol solutions, and lastly, add 5 mL of the 60% iodixanol solution.</li>
		<li>Top off the tube using a syringe with DPBS +/+ to remove most of the air bubbles, taking care not to disturb iodixanol layers. Seal the tube using an electrical tube topper.</li>
		<li>Centrifuge in a non-swing rotor for 1 h 10 min at 490,000 x g at 16 &#176;C in an ultracentrifuge. Remove from ultracentrifuge, assemble the tubes in a metal clasp, and prepare collection tubes. Open the rotor in the hood in case of spills during spinning.</li>
		<li>Prepare one 50 mL tube (for discarding the rotor tube), one 15 mL tube (for collecting virus), a 5 mL syringe, and a 30G and 19G needle per gradient.</li>
		<li>Puncture a hole in the top of the tube with a 30G needle. Leave the needle in place. Place a 19G needle on the syringe.</li>
		<li>Carefully puncture the tube just below the 40%/60% interface, which can be seen by the phenol red indicator. Make sure the needle bevel is facing the 40% layer.</li>
		<li>Remove the 30G needle from the top of the tube using the non-dominant hand. With the needle beveled up, slowly extract the virus/iodixanol. The aim is to extract between 3 mL and 4.5 mL. About halfway and well into the 40%/clear layer, rotate the needle for the bevel to face down and continue to extract to avoid collection from the protein layer.</li>
		<li>Prepare the 50 mL tube with the non-dominant hand, carefully extract the needle while placing the tube in the 50 mL tube and discard. Dilute the AAV/iodixanol suspension 5x in DPBS by filling up to 15 mL.</li>
		<li>Transfer to a centrifugal filter tube and centrifuge at 3428 x g, 4 &#176;C for 10 min. Discard flow-through; gently remove the holder of the filter and tip the bottom container into a waste bottle. Add 15 mL of PBS-5% sucrose to filtrate and resuspend.</li>
		<li>Centrifuge at 3428 x g at 4 &#730;C for 20 min. Repeat buffer exchange at least 3x. Store virus (~150-250 &#181;L) at 4 &#176;C (PHP.B variants a maximum of 3 months) or aliquot into 50 &#181;L aliquots and store at -80 &#176;C for long term. 
		NOTE: PHP.B capsid variants are sensitive to freeze/thaw, so this should be avoided.</li>
		<li>Perform titration as described in a previous protocol10.</li>
	</ol>
	</li>
</ol>

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

<ol><li>Zhou, K., Han, J., Wang, Y., Zhang, Y., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Routes+of+administration+for+adeno-associated+viruses+carrying+gene+therapies+for+brain+diseases%5BTitle%5D)+AND+%22Front+Mol+Neurosci%22%5BJournal%5D)">Routes of administration for adeno-associated viruses carrying gene therapies for brain diseases.</a> Front Mol Neurosci. 15, 988914(2022).</li>
<li>Pietersz, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PhP.B+Enhanced+adeno-associated+virus+mediated-expression+following+systemic+delivery+or+direct+brain+administration%5BTitle%5D)+AND+%22Front+Bioeng+Biotechnol%22%5BJournal%5D)">PhP.B Enhanced adeno-associated virus mediated-expression following systemic delivery or direct brain administration.</a> Front Bioeng Biotechnol. 9, 679483(2021).</li>
<li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Several+rAAV+vectors+efficiently+cross+the+blood%E2%80%93brain+barrier+and+transduce+neurons+and+astrocytes+in+the+neonatal+mouse+central+nervous+system%5BTitle%5D)+AND+%22MolTher%22%5BJournal%5D)">Several rAAV vectors efficiently cross the blood–brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system.</a> MolTher. 19 (8), 1440-1448 (2011).</li>
<li>Deverman, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cre-dependent+selection+yields+AAV+variants+for+widespread+gene+transfer+to+the+adult+brain%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.</a> Nat Biotechnol. 34 (2), 204-209 (2016).</li>
<li>Chan, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+AAVs+for+efficient+noninvasive+gene+delivery+to+the+central+and+peripheral+nervous+systems%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.</a> Nat Neurosci. 20, 1172-1179 (2017).</li>
<li>Goertsen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAV+capsid+variants+with+brain-wide+transgene+expression+and+decreased+liver+targeting+after+intravenous+delivery+in+mouse+and+marmoset%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset.</a> Nat. Neurosci. 25 (1), 106-115 (2022).</li>
<li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat Biotechnol. 27 (1), 59-65 (2008).</li>
<li>Challis, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+AAV+vectors+for+widespread+and+targeted+gene+delivery+in+rodents%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Systemic AAV vectors for widespread and targeted gene delivery in rodents.</a> Nat Protoc. 14 (2), 379-414 (2019).</li>
<li>Fripont, S., Marneffe, C., Marino, M., Rincon, M. Y., Production Holt, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((purification%2C+and+quality+control+for+adeno-associated+virus-based+vectors%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">purification, and quality control for adeno-associated virus-based vectors.</a> J Vis Exp. (143), e58960(2019).</li>
<li>Fagoe, N. D., Eggers, R., Verhaagen, J., Mason, M. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+compact+dual+promoter+adeno-associated+viral+vector+for+efficient+delivery+of+two+genes+to+dorsal+root+ganglion+neurons%5BTitle%5D)+AND+%22Gene+Thr%22%5BJournal%5D)">A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons.</a> Gene Thr. 21 (3), 242-252 (2014).</li>
<li>Verhaagen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+gene+therapy%2C+methods+and+protocols%5BTitle%5D)+AND+%22Meth+Mol+Biol%22%5BJournal%5D)">Retinal gene therapy, methods and protocols.</a> Meth Mol Biol. 1715, 3-17 (2018).</li>
<li>Nasse, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Addgene+AAV+data+hub%3A+A+platform+for+sharing+AAV+experimental+data%5BTitle%5D)+AND+%22Nat+Meth%22%5BJournal%5D)">Addgene AAV data hub: A platform for sharing AAV experimental data.</a> Nat Meth. 20 (9), 1271-1272 (2023).</li>
<li>Grieger, J. C., Soltys, S. M., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+recombinant+adeno-associated+virus+vectors+using+suspension+HEK293+cells+and+continuous+harvest+of+vector+from+the+culture+media+for+GMP+FIX+and+FLT1+clinical+vector%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector.</a> Mol Ther. 24 (2), 287-297 (2016).</li>
<li>Blessing, D., Déglon, N., Schneider, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombinant+protein+expression+in+mammalian+cells%2C+methods+and+protocols%5BTitle%5D)+AND+%22Meth+Mol+Biol%22%5BJournal%5D)">Recombinant protein expression in mammalian cells, methods and protocols.</a> Meth Mol Biol. 1850, 259-274 (2018).</li>
</ol>

A free sample of TransIT was provided by Mirus for initial testing, after which the reagent was purchased for further use. The authors have no other competing financial interests to report.

Systemic administration of AAV is a powerful tool for&#160;gene transfer to the CNS; however, the production of AAV is an expensive and laborious process. By using suspension cells, labor and plastics are reduced compared to the adherent culture of HEK293T on 15 cm2 plates. Furthermore, the conical tubes implemented here are easy to handle and maximize the use of laboratory space. The protocol was set up by two researchers and subsequently applied by others in the lab. A series of productions by three independ...

Adeno-associated virus (AAV) was discovered in 1965 and has since been used in a myriad of applications1. AAVs have been applied in neuroscience research to study gene and neuronal function, map neurocircuits, or produce animal models for disease2. Traditionally, this is done by injecting directly at the site of interest, as most natural serotypes do not cross the blood-brain barrier or need a high dose to do so1,2,3.
With the discovery of AAV.PHP.B4 and next-generation capsids such as AAV.PHP.eB5 and AAV.CAP-B106, it is possible to target the central nervous system (CNS) using a simple systemic injection. Spatial mapping reveals the cells targeted by AAV.PHP.eB at cellular level6,7. In combination with specific promoters/enhancers, these capsids offer extensive opportunities for neuroscientists to study genes and brain function by non-invasive AAV delivery4,8.
While a lower dose is needed for AAV.PHP.eB (typically 1 to 5 x 1011 Genome Copies (GC)/mouse) compared to AAV9 (4 x 1012 GC/mouse)7, still more vector needs to be produced compared to direct injection strategies (typically 1 x 109 GC/&#181;L injection). Most natural serotypes can be produced using the classical adherent cell culture system in combination with iodixanol purification9,10,11,12. For AAV.PHP.eB this entails a labor-intensive process to culture and transfect cells to obtain sufficient vectors for one experiment8. Therefore, the production of AAV in suspension cell culture in conical tubes was developed. Conical tubes, with a capacity of up to 300 mL, are compact, saving both incubator space and plastics. Suspension cells are much easier to culture and handle in large amounts than adherent cells on 15 cm plates. The transfection components of the protocol remain the same. Therefore, plasmids previously used with the adherent system can easily be used in this protocol based on production in suspension cells. The protocol was successfully transferred to other researchers in the laboratory and successfully used for various capsids and constructs.

<table><tbody><tr><td>39 mL, Quick-Seal Round-Top Polypropylene Tube, 25 x 89 mm - 50Pk</td><td>Beckman Coulter</td><td>342414</td><td></td></tr><tr><td>Adapter 600 mL conical tubes, for rotor S-4x1000,&amp;nbsp;</td><td>eppendorf</td><td>5920701002</td><td></td></tr><tr><td>Adapter Plate fits 16 bioreactors of 600 ml</td><td>Infors HT/ TPP</td><td>587633</td><td></td></tr><tr><td>Aerosol-tight caps, for 750 mL round buckets</td><td>eppendorf</td><td>5820747005</td><td></td></tr><tr><td>Centrifuge 5920 R G, 230 V, 50-60 Hz, incl. rotor S-4x1000, round buckets and adapter 15 mL/50 mL conical tubes</td><td>eppendorf</td><td>5948000315</td><td></td></tr><tr><td>Distilled Water</td><td>Gibco</td><td>15230147</td><td></td></tr><tr><td>DNase I recombinant, RNase-free</td><td>Roche</td><td>4716728001</td><td></td></tr><tr><td>DNase I recombinant, RNase-free</td><td>Roche</td><td>4716728001</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Gibco</td><td>14040091</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Fisherbrand Disposable PES Filter Units 0,2</td><td>Fisher</td><td>FB12566504</td><td></td></tr><tr><td>Fisherbrand Disposable PES Filter Units 0,45</td><td>Fisher</td><td>FB12566505</td><td></td></tr><tr><td>Holder for 50 ml culture tubes also fits falcon tube</td><td>Infors HT/ TPP</td><td>31362</td><td></td></tr><tr><td>Holder for 600 ml cell culture tube</td><td>Infors HT/ TPP</td><td>66129</td><td></td></tr><tr><td>Incubator Minitron 50 mm</td><td>Infors HT</td><td>500043</td><td></td></tr><tr><td>LV-MAX Production Medium</td><td>Gibco</td><td>A3583401</td><td></td></tr><tr><td>N-Tray Universal</td><td>Infors HT/ TPP</td><td>31321</td><td></td></tr><tr><td>OptiPrep - Iodixanol</td><td>Serumwerk bernburg</td><td>1893</td><td></td></tr><tr><td>PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)</td><td>Poly-sciences</td><td>24765-100</td><td></td></tr><tr><td>Phenol red solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>72420100</td><td></td></tr><tr><td>Poly(ethylene glycol) 8000</td><td>Sigma-Aldrich</td><td>89510</td><td></td></tr><tr><td>TransIT-VirusGEN</td><td>Mirus</td><td>Mir 6706</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco</td><td>5250061</td><td></td></tr><tr><td>TubeSpin Bioreactors-50ml</td><td>TTP</td><td>87050</td><td></td></tr><tr><td>TubeSpin Bioreactors-600ml</td><td>TTP</td><td>87600</td><td></td></tr><tr><td>Viral Production Cells</td><td>Gibco</td><td>A35347</td><td></td></tr><tr><td>Vivaspin 20 MWCO 100 000</td><td>Cytvia</td><td>28932363</td><td></td></tr></tbody></table>

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.

Most academic labs use adherent HEK293T cells for AAV production8,9. While this works relatively well when small amounts of AAV are needed for direct injection, a 100-fold higher titer (minimally 1 x 1011 GC/mouse) is needed to achieve similar transduction with systemic capsids such as AAV.PHP.eB.
In this protocol, the production of AAV using suspension HEK293 cells cultured in conical tubes was established. Small-scale cult...

Read this Scientific Article Protocol about Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation at JoVE.com

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.

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, ...

production-high-yield-adeno-associated-vector-batches-using-hek293

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2.2K Views.  Royal Netherlands Academy of Arts and Sciences (KNAW). 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. 

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

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Bioengineering

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

Cancer Research

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher&#39;s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types&#8212;an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.

Author Spotlight: Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue ...

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

Summary

Explore More Videos

Intracranial Injection of Adeno-associated Viral Vectors

Production and Titering of Recombinant Adeno-associated Viral Vectors

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

An Efficient Method for Adenovirus Production

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

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

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

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors