The composition of the solutions and buffers used in this protocol are provided in Table 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td colspan="2">Composition</td>
		</tr>
		<tr>
			<td rowspan="4">AAV lysis buffer</td>
			<td colspan="2">1.2 mL of 5 M NaCl solution</td>
		</tr>
		<tr>
			<td colspan="2">2 mL of 1 M Tris-HCl pH 8.5 solution</td>
		</tr>
		<tr>
			<td colspan="2">80 uL of 1 M MgCl2 solution</td>
		</tr>
		<tr>
			<td colspan="2">mQ water to 40 mL</td>
		</tr>
		<tr>
			<td rowspan="3">AAV precipitation solution</td>
			<td colspan="2">40 g PEG 8000</td>
		</tr>
		<tr>
			<td colspan="2">50 mL of 5 M NaCl solution</td>
		</tr>
		<tr>
			<td colspan="2">mQ water to 100 mL</td>
		</tr>
		<tr>
			<td rowspan="5">15% iodixanol fraction</td>
			<td colspan="2">7.5 mL OptiPrep</td>
		</tr>
		<tr>
			<td colspan="2">3 mL of 10X DPBS</td>
		</tr>
		<tr>
			<td colspan="2">6 mL of 5 M NaCl solution</td>
		</tr>
		<tr>
			<td colspan="2">30 uL of 1 M MgCl2 solution</td>
		</tr>
		<tr>
			<td colspan="2">mQ to 30 mL</td>
		</tr>
		<tr>
			<td rowspan="5">25% iodixanol fraction</td>
			<td colspan="2">12. 5 mL OptiPrep</td>
		</tr>
		<tr>
			<td colspan="2">3 mL of 10X DPBS</td>
		</tr>
		<tr>
			<td colspan="2">30 uL of 1 M MgCl2 solution</td>
		</tr>
		<tr>
			<td colspan="2">60 uL phenol red solution</td>
		</tr>
		<tr>
			<td colspan="2">mQ to 30 mL</td>
		</tr>
		<tr>
			<td rowspan="4">40% iodixanol fraction</td>
			<td colspan="2">33.3 mL OptiPrep</td>
		</tr>
		<tr>
			<td colspan="2">5 mL of 10X DPBS</td>
		</tr>
		<tr>
			<td colspan="2">50 uL of 1 M MgCl2 solution</td>
		</tr>
		<tr>
			<td colspan="2">mQ to 50 mL</td>
		</tr>
		<tr>
			<td rowspan="2">60% iodixanol fraction</td>
			<td colspan="2">50 mL OptiPrep</td>
		</tr>
		<tr>
			<td colspan="2">100 uL phenol red solution</td>
		</tr>
		<tr>
			<td rowspan="3">AAV buffer solution</td>
			<td colspan="2">8 mL of 5 M NaCl</td>
		</tr>
		<tr>
			<td colspan="2">20 uL of 10% Pluronic F-68</td>
		</tr>
		<tr>
			<td colspan="2">PBS to 200 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Solution compositions for solutions used in this protocol.
1. Triple transfection of Expi293 cells
<ol>
	<li>Seed Expi293 cells (see Table of Materials) at an initial density of 5 x 105 viable cells (vc)/mL.</li>
	<li>Allow the cells to incubate at 37 &#176;C with 8% CO2 and a shaker speed of 125 rpm until they reach a density of 3-5 x 106 vc/mL. Monitor cell density and viability using trypan blue exclusion with an automated cell counter (see Table of Materials).</li>
	<li>When cells reach the desired cell density, split them 1 to 10 by adding fresh media so that their total volume is ten times their original volume. If necessary, transfer cells into a bigger flask. Return to incubation.</li>
	<li>Continue to expand cells as described in steps 1.2-1.3 until they reach a density of at least 2 x 106 vc/mL in 250 mL of media in a 1 L flask.</li>
	<li>The day before transfection, dilute cells to 2 x 106 vc/mL in 250 mL of media, discarding excess cells as necessary. Incubate the cells overnight.</li>
	<li>On the day of transfection, centrifugate half of the cells at 58 x g for 5 min at 18 &#176;C. Resuspend the cell pellet in the same volume (125 mL) of fresh medium. The cell viability should be close to 98%.</li>
	<li>Prepare two 50 mL conical tubes. Label one &#34;PEI&#34; and the other &#34;DNA&#34;.</li>
	<li>In the conical tube labeled PEI, dilute 1.2 mL of PEI (Polyethylenimine, see Table of Materials) to a total volume of 12.5 mL in OptiMEM media.</li>
	<li>In the conical tube labeled DNA, prepare an equimolar solution of plasmid DNA to a total of 500 &#181;g of DNA in a total volume of 12.5 mL in OptiMEM media.</li>
	<li>Add the DNA solution to the PEI solution, invert several times to thoroughly combine, and let incubate at room temperature for 10 min. 
	NOTE: It is critical that the DNA-PEI solution is allowed to incubate for the full 10 min in order for PEI to form charged complexes with the DNA.</li>
	<li>After the DNA-PEI solution has incubated for 10 min, use a serological pipet to slowly apply the DNA-PEImax solution to the Expi293 cells. Return the transfected cells to the incubator and let them incubate for 72 h (Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66460/66460fig01.jpg" /> 
Figure 1: Expi293 cells expressing GFP two days after transfection. After transfection with a plasmid containing a gene for GFP, the Expi293 cells transiently express eGFP. The cell morphology is round. The image was captured with a 15 ms exposure time. Microscope images are acquired using an inverted microscope equipped with epi-fluorescence illumination and a 10x/0.30 objective. Scale bar = 100 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66460/66460fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. rAAV vector purification
<ol>
	<li>After 72 h, transfer the cells in suspension to two 250 mL conical tubes and spin them down at 415 x g for 10 min at 4 &#176;C.</li>
	<li>Pour the supernatant media into a fresh 500 mL conical tube and store at 0 &#176;C on ice for later processing.</li>
	<li>Resuspend each cell pellet in 10 mL of AAV lysis buffer (Table 1). Pool the two lysates together in one of the tubes. Rinse the other tube with an additional 5 mL of AAV lysis buffer, then add it to the pooled lysates. Freeze at -70 &#176;C. 
	NOTE: The experiment can be paused at this point. Store the supernatant media at -70 &#176;C rather than on ice.</li>
	<li>Add 1:4 volume of AAV precipitation solution to the supernatant media from step 2.2. Invert several times to thoroughly mix and incubate at 0 &#176;C on ice for at least 2 h or overnight.</li>
	<li>Centrifugate the incubated solution at 3000 x g for 1 h at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet containing viral precipitate in 5 mL of AAV lysis buffer.</li>
	<li>Thaw the cell lysate at 37 &#176;C in a water bath.</li>
	<li>Pool the viral precipitate with the cell lysate. This is the crude lysate. Rinse the centrifugation tube that contained the viral precipitate with an additional 5 mL of AAV lysis buffer and pool with the crude lysate.</li>
	<li>Freeze the crude lysate to -70 &#176;C, then thaw at 37 &#176;C. Repeat this cycle once more. 
	NOTE: The crude lysate should not be left at 37 &#176;C for any longer than is necessary to thaw it.</li>
	<li>Once thawed for the third time, immediately add 4 &#181;L of benzonase to the crude lysate, invert to mix, and incubate for 30 min at 37 &#176;C.</li>
	<li>Centrifugate the crude lysate for 10 min at 650 x g at 18 &#176;C.</li>
	<li>Transfer the supernatant to a clean 50 mL conical tube. This is the crude virus. Discard the pellet.</li>
	<li>Centrifugate the crude virus for an additional 30 min at 3000 x g at 18 &#176;C to clear contaminating proteins.</li>
	<li>Transfer the supernatant to a clean 50 mL conical tube. This is the clarified virus. 
	NOTE: The experiment can be paused at this point. Freeze down the clarified virus to -70 &#176;C.</li>
	<li>Set up the multichannel peristaltic pump with four peristaltic tubes (see Table of Materials). Attach capillaries to both ends of each tube.</li>
	<li>Place the capillaries on the input side of the pump in a beaker filled with deionized water. Place the capillaries on the output side of the pump in an empty beaker. Run the peristaltic pump at 25.0 rpm to flush the tubing with deionized (DI) water.</li>
	<li>When thoroughly flushed, empty the peristaltic tubing by running the pump until the tubing is filled only with air. Lay all capillaries on a clean lint free wipe.</li>
	<li>Place four ultracentrifuge tubes in a rack on the output side of the pump.</li>
	<li>Use a 10 mL serological pipet to carefully dispense 10 mL of clarified virus into each ultracentrifuge tube. Take care not to introduce air bubbles. 
	NOTE: The volume of clarified virus in each tube can be stretched to up to 12 mL per tube, if necessary. Reduce the amount of 60% iodixanol appropriately in step 2.25 below.</li>
	<li>The clarified virus is underlaid with iodixanol fractions (Table 1) from lowest density to highest density using the peristaltic pump. The 15% fraction is added first, followed by the 25% fraction, the 40% fraction, and finally the 60% fraction. Transfer 22 mL (5.5 mL per ultracentrifuge tube) of the 15% iodixanol fraction into a clean 50 mL conical tube. Place the capillaries on the input side of the pump into the tube, taking care that all capillaries are touching the bottom of the tube.</li>
	<li>Start the pump and allow the tubing to fill with the iodixanol fraction. When the iodixanol fraction reaches the ends of the capillaries on the output side of the pump, stop the pump.</li>
	<li>Insert one output capillary into each ultracentrifuge tubes with clarified virus, taking care that the capillaries are touching the bottom of each ultracentrifuge tube. 
	NOTE: It is of critical importance that there is no air left in the capillaries. Iodixanol may flow through the peristaltic tubing at slightly different rates, so ensure that each individual output capillary is completely filled with iodixanol before inserting it into the clarified virus. It may be necessary to stop and start the pump several times.</li>
	<li>Start the pump and allow the ultracentrifuge tubes to fill. When the last of the 15% fraction is about to be taken into the input capillaries, stop the pump. It is critical that no air bubbles enter the peristaltic tubing. 
	NOTE: If air bubbles enter the input capillaries, the pump can be briefly run in the reverse direction to push them back out.</li>
	<li>Transfer 22 mL (5.5 mL per ultracentrifuge tube) of the 25% iodixanol fraction into the 50 mL conical tube. Take care that all capillaries are touching the bottom of the tube and start the pump. When the last of the 25% fraction is about to be taken into the input capillaries, stop the pump.</li>
	<li>Repeat step 2.24 with 20 mL (5 mL per ultracentrifuge tube) of the 40% iodixanol fraction and then with 24 mL (6 mL per ultracentrifuge tube) of the 60% fraction.</li>
	<li>If there is still unfilled volume left in the ultracentrifuge tube, continue to add more of the 60% fraction until the ultracentrifuge tubes are completely filled with liquid.</li>
	<li>Fill each tube until the lysate makes a dome over the top but does not overflow from the tube. Stop the pump and carefully remove each output capillary, taking care not to disturb the iodixanol gradient.</li>
	<li>Cap each ultracentrifuge tube with a spacer and load it into a Type 70 Ti rotor (see Table of Materials). 
	NOTE: Ensure that the rotor is appropriately balanced. Do not attempt to operate the ultracentrifuge without proper training.</li>
	<li>Load the Type 70 Ti rotor into the ultracentrifuge and centrifugate at 489,000 x g for 1 h at 18 &#176;C.</li>
	<li>After centrifugation, unload the rotor from the ultracentrifuge. Use needle nose pliers to carefully remove each ultracentrifuge tube from the rotor, taking care not to disturb the iodixanol gradient.</li>
	<li>Use a support ring stand with clamp to secure one ultracentrifuge tube.</li>
	<li>Attach a 20 GA needle to a 5 mL syringe and set aside. Use a lint-free wipe to remove the cap from the ultracentrifuge tube.</li>
	<li>Penetrate the wall of the ultracentrifuge tube with the needle about 3 mm below the 40%-60% iodixanol interface (Figure 2).</li>
	<li>Slowly aspirate the interface and part of the 40% fraction. Avoid aspirating the top of the 40% fraction, for a total draw of about 4 mL.</li>
	<li>Holding one finger over the open top of the ultracentrifuge tube, pull out the syringe and transfer the aspirated AAV fraction to a 50 mL conical tube. Discard the syringe in a sharps container. Discard the ultracentrifuge tube.</li>
	<li>Repeat steps 2.31-2.35 for each ultracentrifuge tube.</li>
	<li>Dilute the aspirated AAV fraction approximately two-fold in AAV lysis buffer to a volume of 40 mL.</li>
	<li>Flush the peristaltic tubing as described in steps 2.16-2.17. 
	NOTE: The experiment can be paused at this point. Store the diluted AAV fraction at 0 &#176;C overnight.</li>
	<li>Load 20 mL each of the diluted AAV fraction into two new ultracentrifuge tubes.</li>
	<li>For the second round of iodixanol density gradient centrifugation, the diluted AAV fraction is underlaid as described above using only 10 mL (5 mL per ultracentrifuge tube) of the 40% fraction and 14 mL (7 mL per ultracentrifuge tube) of the 60% fraction. Repeat steps 2.29-2.36 for ultracentrifugation and aspiration. 
	NOTE: Before storing the peristaltic pump and tubing, flush out the tubing with DI water as described in steps 2.16-2.17.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66460/66460fig02.jpg" /> 
Figure 2: Iodixanol gradient with 40%-60% iodixanol interface labeled. (A) First iodixanol gradient. Phenol red is used in the 40% iodixanol and 60% iodixanol fractions. It appears as a different color because of the difference in pH between the two fractions. The arrow indicates where the syringe should be inserted to harvest the rAAV fraction, just below the 40%-60% iodixanol interface. (B) Second iodixanol gradient. Only the 40% and 60% iodixanol fractions are used in this step. The arrow indicates where the syringe should be inserted to harvest the rAAV fraction. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66460/66460fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Buffer exchange and virus concentration
<ol>
	<li>After obtaining the AAV fraction from the second round of iodixanol density gradient centrifugation, dilute it two-fold with AAV buffer solution.</li>
	<li>Equilibrate the centrifugal filter by adding 20 mL of AAV buffer solution to the top of the filter. Centrifugate the apparatus at 3000 x g at 18 &#176;C for 5 min and discard flow through.</li>
	<li>Add the diluted AAV fraction to the centrifugal filter. Centrifugate the apparatus at 3000 x g at 18 &#176;C for 5 min and discard flow through.</li>
	<li>Add 20 mL of AAV buffer solution to the top of the filter. Centrifugate the apparatus at 3000 x g at 18 &#176;C for 5 min and discard flow through. 
	NOTE: If the centrifugal filter membrane becomes blocked, causing the solution to flow through inefficiently, carefully use a P200 micropipette to draw up and down the solution in the top of the filter. Take extreme care not to touch the filter with the micropipette tip.</li>
	<li>Repeat step 3.4 twice more. 
	NOTE: Ensure that the AAV is not overconcentrated by maintaining at least 1 mL of volume in the top of the filter. It may be necessary to adjust centrifugation times to prevent overconcentration. If AAV is overconcentrated, precipitation can occur.</li>
	<li>For a final titer in the range of 1012 vg/mL, spin down the virus to a final volume of about 1 mL. 
	NOTE: Depending on the serotype backbone used and the packaging efficiency of the virus, you may have to concentrate the virus in a smaller volume.</li>
	<li>Use a p1000 micropipette to transfer the concentrated AAV from the top of the centrifugal filter into a microcentrifuge tube. Use a p200 micropipette to wash the centrifugal filter with 50 &#181;L of AAV buffer to collect any remaining virus and pool with the rest of the concentrated AAV.</li>
	<li>Set aside a 2 &#181;L aliquot of the concentrated AAV for titering. See Supplementary Table 1 to view ITR primers that can be used for qPCR.</li>
	<li>Pass the concentrated AAV through a 0.2 &#181;m syringe filter to sterilize it. 
	NOTE: When sterilizing a small volume, take care to select a syringe filter with a small diameter in order to minimize virus loss. Using a low-protein binding filter of a small diameter will result in minimal viral loss (data not shown).</li>
	<li>The sterilized AAV can be used immediately to transduce cells or stored at 4 &#176;C for use within four weeks. It should be stored at -70 &#176;C for longer-term storage.</li>
</ol>

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+in+adenovirus+type+3+conjunctivitis.">Adeno-associated virus in adenovirus type 3 conjunctivitis.</a> Infect Immun. 11 (6), 1362-1370 (1975).</li><li>Grimm, D., Kern, A., Rittner, K., Kleinschmidt, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+recombinant+adeno-associated+virus+vectors+are+biologically+active+and+free+of+detectable+helper+and+wild-type+viruses.">Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.</a> Hum Gene Ther. 10 (6), 1031-1039 (1999).</li><li>Debelak, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cation-exchange+high-performance+liquid+chromatography+of+recombinant+adeno-associated+virus+type+2.">Cation-exchange high-performance liquid chromatography of recombinant adeno-associated virus type 2.</a> J Chromatogr B Biomed Sci App. 740 (2), 195-202 (2000).</li><li>Burova, E., Ioffe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+purification+of+recombinant+adenoviral+and+adeno-associated+viral+vectors:+methods+and+implications.">Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications.</a> Gene Ther. 12 (1), S5-S17 (2005).</li><li>Adams, B., Bak, H., Tustian, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+from+the+bench+towards+a+large+scale,+industrial+platform+process+for+adeno-associated+viral+vector+purification.">Moving from the bench towards a large scale, industrial platform process for adeno-associated viral vector purification.</a> Biotechnol Bioeng. 117 (10), 3199-3211 (2020).</li><li>Grieger, J. C., Choi, V. W., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+adeno-associated+viral+vectors.">Production and characterization of adeno-associated viral vectors.</a> Nat Protoc. 1 (3), 1412-1428 (2006).</li><li>Florea, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+purification+of+divergent+AAV+serotypes+using+AAVX+affinity+chromatography.">High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography.</a> Mol Ther Methods Clin Dev. 28, 146-159 (2022).</li><li>Chamberlain, K., Riyad, J. M., Weber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expressing+transgenes+that+exceed+the+packaging+capacity+of+adeno-associated+virus+capsids.">Expressing transgenes that exceed the packaging capacity of adeno-associated virus capsids.</a> Hum Gene Ther Methods. 27 (1), 1-12 (2016).</li><li>Green, E. A., Hamaker, N. K., Lee, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+vector+elements+and+process+conditions+in+transient+and+stable+suspension+HEK293+platforms+using+SARS-CoV-2+receptor+binding+domain+as+a+model+protein.">Comparison of vector elements and process conditions in transient and stable suspension HEK293 platforms using SARS-CoV-2 receptor binding domain as a model protein.</a> BMC Biotechnol. 23 (1), 7 (2023).</li><li>Erbacher, P., Zou, S., Bettinger, T., Steffan, A. M., Remy, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan-based+vector/DNA+complexes+for+gene+delivery:+Biophysical+characteristics+and+transfection+ability.">Chitosan-based vector/DNA complexes for gene delivery: Biophysical characteristics and transfection ability.</a> Pharm Res. 15 (9), 1332-1339 (1998).</li><li>Vandenberghe, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+serotype-dependent+release+of+functional+vector+into+the+culture+medium+during+adeno-associated+virus+manufacturing.">Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing.</a> Hum Gene Ther. 21 (10), 1251-1257 (2010).</li><li>Summerford, C., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-associated+heparan+sulfate+proteoglycan+is+a+receptor+for+adeno-associated+virus+type+2+virions.">Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.</a> J Virol. 72 (2), 1438-1445 (1998).</li><li>Wright, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+factors+that+contribute+to+recombinant+AAV2+particle+aggregation+and+methods+to+prevent+its+occurrence+during+vector+purification+and+formulation.">Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation.</a> Mol Ther. 12 (1), 171-178 (2005).</li><li>Gruntman, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+and+compatibility+of+recombinant+adeno-associated+virus+under+conditions+commonly+encountered+in+human+gene+therapy+trials.">Stability and compatibility of recombinant adeno-associated virus under conditions commonly encountered in human gene therapy trials.</a> Hum Gene Ther Methods. 26 (2), 71-76 (2015).</li><li>Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationale+and+strategies+for+the+development+of+safe+and+effective+optimized+AAV+vectors+for+human+gene+therapy.">Rationale and strategies for the development of safe and effective optimized AAV vectors for human gene therapy.</a> Mol Ther Nucleic Acids. 32, 949-959 (2023).</li><li>Mullard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+approves+first+gene+therapy+for+Duchenne+muscular+dystrophy,+despite+internal+objections.">FDA approves first gene therapy for Duchenne muscular dystrophy, despite internal objections.</a> Nat Rev Drug Discov. 22 (8), 610-610 (2023).</li><li>Center for Biologics Evaluation and Research. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approved+Cellular+and+Gene+Therapy+Products.">Approved Cellular and Gene Therapy Products.</a> US Food Drug Adm. , (2023).</li><li>Kang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+vectors+applied+to+the+treatment+of+CNS+disorders:+Clinical+status+and+challenges.">AAV vectors applied to the treatment of CNS disorders: Clinical status and challenges.</a> J Control Release Off J Control Release Soc. 355, 458-473 (2023).</li><li>De Wolf, D., Singh, K., Chuah, M. K., VandenDriessche, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemophilia+gene+therapy:+The+end+of+the+beginning.">Hemophilia gene therapy: The end of the beginning.</a> Hum Gene Ther. 34 (17-18), 782-792 (2023).</li><li>Simons, E. J., Trapani, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+opportunities+and+challenges+of+gene+therapy+for+treatment+of+inherited+forms+of+vision+and+hearing+loss.">The opportunities and challenges of gene therapy for treatment of inherited forms of vision and hearing loss.</a> Hum Gene Ther. 34 (17-18), 808-820 (2023).</li></ol>

The authors have no disclosures to report.

The double iodixanol density gradient purification protocol is the universal method because it is applicable to any AAV mutant variants, regardless of their receptor specificity. Early methods of AAV purification relied on particle density and included isopycnic centrifugation in CsCl and continuous sucrose density gradient centrifugation19. Later, serotype-specific approaches were developed, which made use of monoclonal antibodies bound to Sepharose columns20. A novel dens...

Recombinant adeno-associated viral (rAAV) vectors are widely used tools for the treatment of genetic diseases, including spinal muscular atrophy, retinal dystrophy, and hemophilia A1,2,3. rAAV vectors are engineered to lack viral genes present in wild-type AAV4, a small, non-envelope icosahedral virus with a linear single-stranded 4.7 kb DNA genome. AAV was first discovered in the 1960s as a contaminant of adenovirus preparations5. Despite its small capsid size, which limits the size of the transgene that can be packaged to a maximum of 4.9 kb excluding ITRs6, AAV is useful for transgene delivery because it is non-pathogenic in humans, allows expression of transgene in many dividing and nondividing cell types, and has limited immunogenic effects7.
As members of the genus dependoparvovirus, the production of rAAVs relies on the expression of helper genes present in adenovirus or herpes simplex virus8. Several strategies to produce rAAV have been developed, but production in HEK293 cells transformed with the adenoviral E1A/E1B helper genes is the most established method used today9. The general approach of rAAV production begins with the transfection of HEK293 cells with three plasmids containing the transgene within inverted terminal repeats (ITRs), AAV rep and cap genes, and additional adenoviral helper genes, respectively. Seventy-two hours after transfection, cells are harvested and processed to purify rAAV containing the transgene.
In the development of new rAAV vectors for therapeutic purposes, a major goal is producing vectors with increased transduction efficiency. An increase in the transduction efficiency of target cells would mean a decrease in the necessary clinical dose of rAAV, thus decreasing the likelihood of adverse immunogenic effects ranging from antibody-mediated neutralization to acute toxicities10,11. To improve the transduction efficacy of rAAV vectors, alterations can be made to the packaged genome or to the capsid. Viable methods to tune transduction efficacy via packaged genome design include the incorporation of strong and tissue-specific promoters, thoughtful selection of mRNA processing elements, and coding sequence optimization to improve translation efficiency12. Alterations to the capsid are made with the goal of increasing tropism for target human cell types. Efforts towards developing new rAAV transgene delivery vector capsids are generally characterized by a focus on either rational design of AAV capsids with specific mutations targeting specific cell receptors or directed evolution to identify capsids with tropism for specific cell types from high-complexity combinatorial capsid libraries without targeting one specific receptor (although some groups combine these approaches)13,14,15. In the directed evolution approach, combinatorial capsid libraries are constructed using a particular serotype backbone with mutated variable regions on the capsid exterior16. Combinatorial capsid libraries are often constructed from AAV serotypes not originating in humans, decreasing the risk of preexisting immunity during clinical use10. Therefore, purification methods that can be applied to any serotype are ideal to eliminate the need for serotype-specific optimization for the less commonly used serotypes serving as backbones for these libraries.
Iodixanol density gradient centrifugation is utilized to purify high titers of rAAV with high infectivity17. In this protocol, rAAV is produced in suspension cell culture to decrease the amount of labor needed to produce large titers of AAV. A centrifugation step is also included to clear cell lysate to reduce the presence of contaminating proteins and decrease the risk of virus precipitation. This protocol is a cost-effective method to produce preparations of high-purity rAAV suitable for pre-clinical use.

<table><tbody><tr><td>5810 R benchtop centrifuge</td><td>Eppendorf</td><td>22625501</td><td></td></tr><tr><td>8-channel peristaltic pump&amp;nbsp;</td><td>Watson-Marlow</td><td>020.3708.00A</td><td></td></tr><tr><td>Automated cell counter&amp;nbsp;</td><td>NanoEntek</td><td>EVE-MC</td><td></td></tr><tr><td>Avanti J-E high-speed centrifuge</td><td>Beckman Coulter</td><td>369001</td><td></td></tr><tr><td>Benzonase</td><td>Thermo Scientific</td><td>88701</td><td></td></tr><tr><td>Biological safety cabinet</td><td>Labconco</td><td>322491101</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator with shaker&amp;nbsp;</td><td></td><td></td><td>Set at 8% CO&lt;sub&gt;2&lt;/sub&gt; and 37 &amp;deg;C</td></tr><tr><td>Conical centrifuge tubes</td><td>Thermo Scientific</td><td>339652</td><td>50 mL</td></tr><tr><td>Conical centrifuge tubes</td><td>Thermo Scientific</td><td>339650</td><td>15 mL</td></tr><tr><td>Disposable micro-pipets</td><td>Fisherbrand</td><td>21-164-2G</td><td>Capillaries</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline without CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp; (DPBS) (10x)</td><td>Sigma-Aldrich</td><td>D1408</td><td></td></tr><tr><td>ECLIPSE Ts2R-FL inverted microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Expi293 Expression Medium</td><td>Gibco</td><td>A1435101</td><td></td></tr><tr><td>Expi293F cells</td><td>Gibco</td><td>A14527</td><td></td></tr><tr><td>Filter tips</td><td>USA Scientific</td><td>1126-7810</td><td>1000 &amp;micro;L</td></tr><tr><td>Filter tips</td><td>USA Scientific</td><td>1120-8810</td><td>200 &amp;micro;L</td></tr><tr><td>Filter tips</td><td>USA Scientific</td><td>1120-1810</td><td>20 &amp;micro;L</td></tr><tr><td>Filter tips</td><td>USA Scientific</td><td>1121-3810</td><td>10 &amp;micro;L</td></tr><tr><td>Hypodermic needles</td><td>Tyco Healthcare</td><td>820112</td><td>20 GA x 1-1/2 A</td></tr><tr><td>Ice bucket with lid</td><td>VWR</td><td>10146-184</td><td></td></tr><tr><td>JS-5.3 rotor</td><td>Beckman Coulter</td><td>368690</td><td></td></tr><tr><td>Magnesium chloride solution (1 M)</td><td>Millipore Sigma</td><td>M1028-100ML</td><td></td></tr><tr><td>Metal stand and clamp&amp;nbsp;</td><td>Fisherbrand</td><td>05-769-6Q</td><td></td></tr><tr><td>Microcentrifuge tubes</td><td>Eppendorf</td><td>22600028</td><td>1.5 mL</td></tr><tr><td>Needle nose pliers</td><td></td><td></td><td></td></tr><tr><td>Optima XE-90 ultracentrifuge</td><td>Beckman Coulter</td><td>A94471</td><td></td></tr><tr><td>Opti-MEM I Reduced-Serum Medium</td><td>Gibco</td><td>31985062</td><td></td></tr><tr><td>OptiPrep density gradient media (iodixanol)</td><td>Serumwerk</td><td>AXS-1114542</td><td>60% iodixanol solution</td></tr><tr><td>P1000 Pipet</td><td>Gilson</td><td>F144059M</td><td></td></tr><tr><td>P2 Pipet</td><td>Gilson</td><td>F144054M</td><td></td></tr><tr><td>P20 Pipet</td><td>Gilson</td><td>F144056M</td><td></td></tr><tr><td>P200 Pipet</td><td>Gilson</td><td>F144058M</td><td></td></tr><tr><td>Phenol red&amp;nbsp;solution</td><td>Sigma-Aldrich</td><td>P0290</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4474</td><td></td></tr><tr><td>Pipet-Aid XP pipette controller</td><td>Drummond Scientific</td><td>4-000-101</td><td></td></tr><tr><td>Plasmid pCapsid</td><td>De novo or Addgene, etc.&amp;nbsp;</td><td>N/A</td><td>We used pACGrh74.&amp;nbsp;</td></tr><tr><td>Plasmid pHelper</td><td>Addgene</td><td>112867</td><td></td></tr><tr><td>Plasmid pTransgene</td><td>De novo or Addgene, etc.&amp;nbsp;</td><td>N/A</td><td>We used pdsAAV-GFP.</td></tr><tr><td>Pluronic F-68 polyol solution (10%)</td><td>Mp Biomedicals</td><td>92750049</td><td></td></tr><tr><td>Polyethylene glycol 8000</td><td>Research Products International</td><td>P48080-500.0</td><td></td></tr><tr><td>Polyethylenimine HCl Max (PEI-Max)</td><td>Polysciences</td><td>NC1038561</td><td>Dilute in water to 40 &amp;mu;M</td></tr><tr><td>Polypropylene centrifuge tubes, sterile</td><td>Corning</td><td>431123</td><td>500 mL</td></tr><tr><td>Polypropylene centrifuge tubes, sterile</td><td>Corning</td><td>430776</td><td>250 mL</td></tr><tr><td>Polypropylene Optiseal tubes</td><td>Beckman Coulter</td><td>361625</td><td></td></tr><tr><td>Serological pipettes</td><td>Alkali Scientific</td><td>SP250-B</td><td>50 mL</td></tr><tr><td>Serological pipettes</td><td>Alkali Scientific</td><td>SP225-B</td><td>25 mL</td></tr><tr><td>Serological pipettes</td><td>Alkali Scientific</td><td>SP210-B</td><td>10 mL</td></tr><tr><td>Serological pipettes</td><td>Alkali Scientific</td><td>SP205-B</td><td>5 mL</td></tr><tr><td>Shaker flasks</td><td>Fisherbrand</td><td>PBV1000</td><td>1 L</td></tr><tr><td>Shaker flasks</td><td>Fisherbrand</td><td>PBV50-0</td><td>500 mL</td></tr><tr><td>Shaker flasks</td><td>Fisherbrand</td><td>PBV250</td><td>250 mL</td></tr><tr><td>Shaker flasks</td><td>Fisherbrand</td><td>PBV12-5</td><td>125 mL</td></tr><tr><td>Sodium chloride solution (5 M)</td><td>Fisher Scientific</td><td>NC1752640</td><td></td></tr><tr><td>Sterile syringes</td><td>Fisherbrand</td><td>14-955-458</td><td>5 mL</td></tr><tr><td>Syringe filter</td><td>Millipore</td><td>SLGV013SL</td><td>0.22 micron</td></tr><tr><td>Tris-HCl pH 8.5 (1 M)</td><td>Kd Medical</td><td>RGE3363</td><td></td></tr><tr><td>Trypan blue solution</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Tube rack assembly</td><td>Beckman Coulter</td><td>361646</td><td></td></tr><tr><td>Tube spacers (x4)</td><td>Beckman Coulter</td><td>361669</td><td></td></tr><tr><td>Tubing for peristaltic pump</td><td>Fisher Scientific</td><td>14190516</td><td></td></tr><tr><td>Type 70 Ti fixed-angle titanium rotor</td><td>Beckman Coulter</td><td>337922</td><td></td></tr><tr><td>Ultra low temperature freezer</td><td></td><td></td><td>Set at -70 &amp;deg;C</td></tr><tr><td>Vivaspin 20 centrifugal concentrator</td><td>Sartorius</td><td>VS2041</td><td></td></tr><tr><td>Water bath&amp;nbsp;</td><td></td><td></td><td>Set at 37 &amp;deg;C</td></tr></tbody></table>

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.

This method can be used to obtain titers of at least 1012 viral particles per mL. A titer can be obtained (Figure 3) by qPCR using the ITR primers provided in Supplementary Table 1, by ddPCR, or by any other titering method. Suboptimal titers could result from using a cap gene encoding a capsid with poor packaging efficiency.
Another possible source of suboptimal results is the poor transduction efficiency of Expi293 cells. It ...

Watch this Scientific Journal Video about Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications at JoVE.com

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.

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 ...

suspension-culture-production-purification-adeno-associated-virus

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

1.9K Views.  University of Florida. 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.

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

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

Cancer Research

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

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

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

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

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

Neuroscience

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

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

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

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

Immunology and Infection

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, ...

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.