Amira D. Rghei, Brenna A. Y. Stevens, Sylvia P. Thomas, and Jacob G. E. Yates were recipients of Ontario Veterinary College Student Stipends as well as Ontario Graduate Scholarships. Amira D. Rghei was the recipient of a Mitacs Accelerate Studentship. This work was funded by the Canadian Institutes for Health Research (CIHR) Project Grant (#66009) and a Collaborative Health Research Projects (NSERC partnered) grant (#433339) to SKW.

1. Double plasmid transfection of HEK293 cells in cell stacks
<ol>
	<li>Thaw a cryo-vial of HEK293 cells in a bead bath set at 37 &#176;C. 
	NOTE: Pre-warm complete DMEM to 37 &#176;C while cells are thawing to ensure the cold temperature does not shock cells when plating. Ensure the cells have a low passage number, ideally less than 20, to ensure optimal growth and transfection efficiency. Ensure that the cells are certified to be mycoplasma-free.</li>
	<li>Transfer the contents of the cryo-vial dropwise into a 1...

<ol>
	<li>Hastie, E., 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=Adeno-associated+virus+at+50:+A+golden+anniversary+of+discovery,+research,+and+gene+therapy+success-a+personal+perspective.">Adeno-associated virus at 50: A golden anniversary of discovery, research, and gene therapy success-a personal perspective.</a> Human Gene Therapy. 26 (5), 257-265 (2015).</li><li>Wang, D., Tai, P. W. L., Gao, G. <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+vector+as+a+platform+for+gene+therapy+delivery.">Adeno-associated virus vector as a platform for gene therapy delivery.</a> Nature Reviews Drug Discovery. 18 (5), 358-378 (2019).</li><li>Nathwani, A. C., 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=Long-term+safety+and+efficacy+of+factor+IX+gene+therapy+in+hemophilia+B.">Long-term safety and efficacy of factor IX gene therapy in hemophilia B.</a> The New England Journal of Medicine. 371 (21), 1994-2004 (2014).</li><li>Kuzmin, D. A., 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=The+clinical+landscape+for+AAV+gene+therapies.">The clinical landscape for AAV gene therapies.</a> Nature Reviews Drug Discovery. 20 (3), 173-174 (2021).</li><li>Yl&#228;-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endgame:+glybera+finally+recommended+for+approval+as+the+first+gene+therapy+drug+in+the+European+union.">Endgame: glybera finally recommended for approval as the first gene therapy drug in the European union.</a> Molecular Therapy: The Journal of the American Society of Gene Therapy. 20 (10), 1831-1832 (2012).</li><li>FDA approves novel gene therapy to treat patients with a rare form of inherited vision loss. FDA News Release. FDA Available from: <a target="_blank" href="https://www.fda.gov/news-events/press-announcements/fda-approves-novel-gene-therapy-treat-patients-rare-form-inherited-vision-loss">https://www.fda.gov/news-events/press-announcements/fda-approves-novel-gene-therapy-treat-patients-rare-form-inherited-vision-loss</a> (2020)</li><li>FDA approves innovative gene therapy to treat pediatric patients with spinal muscular atrophy, a rare disease and leading genetic cause of infant mortality. FDA News Release. 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H., Gilbert, L. B., Weitzman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+identifies+a+cellular+regulator+of+adeno-associated+virus.">A genetic screen identifies a cellular regulator of adeno-associated virus.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (26), 14991-14996 (2001).</li><li>McCarty, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-complementary+AAV+vectors;+advances+and+applications.">Self-complementary AAV vectors; advances and applications.</a> Molecular Therapy. 16 (10), 1648-1656 (2008).</li><li>Aschauer, D. F., Kreuz, S., Rumpel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+transduction+efficiency,+tropism+and+axonal+transport+of+aav+serotypes+1,+2,+5,+6,+8+and+9+in+the+mouse+brain.">Analysis of transduction efficiency, tropism and axonal transport of aav serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain.</a> PLOS One. 8 (9), 76310 (2013).</li><li>Zincarelli, C., Soltys, S., Rengo, G., Rabinowitz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+AAV+serotypes+1-9+mediated+gene+expression+and+tropism+in+mice+after+systemic+injection.">Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.</a> Molecular Therapy. 16 (6), 1073-1080 (2008).</li><li>Pillay, S., 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=Adeno-associated+virus+(AAV)+serotypes+have+distinctive+interactions+with+domains+of+the+cellular+AAV+receptor.">Adeno-associated virus (AAV) serotypes have distinctive interactions with domains of the cellular AAV receptor.</a> Journal of Virology. 91 (18), (2017).</li><li>Merkel, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+adeno-associated+virus+vectors+across+a+model+of+the+blood-brain+barrier;+a+comparative+study+of+transcytosis+and+transduction+using+primary+human+brain+endothelial+cells.">Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.</a> Journal of Neurochemistry. 140 (2), 216-230 (2017).</li><li>van Lieshout, L. P., 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=A+novel+triple-mutant+AAV6+capsid+induces+rapid+and+potent+transgene+expression+in+the+muscle+and+respiratory+tract+of+mice.">A novel triple-mutant AAV6 capsid induces rapid and potent transgene expression in the muscle and respiratory tract of mice.</a> Molecular Therapy. Methods &amp; Clinical Development. 9, 323-329 (2018).</li><li>Wu, Z., Asokan, A., Grieger, J. C., Govindasamy, L., Agbandje-McKenna, M., Samulski, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+tools+for+production+and+purification+of+recombinant+adeno-associated+virus+vectors.">Novel tools for production and purification of recombinant adeno-associated virus vectors.</a> Human Gene Therapy. 9 (18), 2745-2760 (1998).</li><li>Kimura, T., 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=Production+of+adeno-associated+virus+vectors+for+in+vitro+and+in+vivo+applications.">Production of adeno-associated virus vectors for in vitro and in vivo applications.</a> Scientific Reports. 9 (1), 13601 (2019).</li><li>Aurnhammer, C., 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=Universal+real-time+PCR+for+the+detection+and+quantification+of+adeno-associated+virus+serotype+2-derived+inverted+terminal+repeat+sequences.">Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences.</a> Human Gene Therapy Methods. 23 (1), 18-28 (2012).</li><li>. Paramyxoviridae: The viruses and their replication. Fields Virology Available from: <a target="_blank" href="https://www.scholars.northwestern.edu/en/publications/paramyxoviridae-the-viruses-and-their-replication">https://www.scholars.northwestern.edu/en/publications/paramyxoviridae-the-viruses-and-their-replication</a> (1996)</li><li>Boussif, O., 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=A+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (16), 7297-7301 (1995).</li><li>Kaludov, N., Brown, K. E., Walters, R. W., Zabner, J., Chiorini, J. 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The production of recombinant AAV (rAAV) vectors described in this paper uses common materials, reagents, and equipment found in majority of the molecular biology research labs and facilities. This paper allows for high-quality in vitro and in vivo grade rAAV to be produced by the reader. Above all, this protocol for rAAV production, compared to more tedious protocols involving cesium chloride purification, is efficient and avoids the use of ultracentrifugation. Once HEK293 cells have been transfected, ...

Gene therapy utilizing adeno-associated virus (AAV) vectors has made huge strides over the past three decades1,2. Demonstrated improvements in a diverse range of genetic diseases, including congenital blindness, hemophilia, and diseases of the musculoskeletal and central nervous system, have brought AAV gene therapy to the forefront of clinical research3,4. In 2012, the European Medicines Agency (EMA) approved Glybera, an AAV1 vector expressing lipoprotein lipase (LPL) for the treatment of LPL deficiency, making it the first marketing authorization for a gene therapy treatment in either Europe or the United States5. Since then, two additional AAV gene therapies, Luxturna6 and Zolgensma7, have received FDA approval, and the market is expected to expand quickly over the next 5 years with as many as 10-20 gene therapies expected by 20258. Available clinical data indicate that AAV gene therapy is a safe, well-tolerated, and efficacious modality making it one of the most promising viral vectors, with above 244 clinical trials involving AAV registered with ClinicalTrials.gov. The increasing interest in clinical applications involving AAV vectors requires robust and scalable production methods to facilitate the evaluation of AAV therapies in large animal models, as this is a critical step in the translational pipeline9.
For AAV vector production, the two main requirements are the AAV genome and the capsid. The genome of wild-type (wt)-AAV is single-stranded DNA that is approximately 4.7 kb in length10. The wt-AAV genome comprises inverted terminal repeats (ITRs) found at both ends of the genome, which are important for packaging, and the rep and cap genes11. The rep and cap genes, necessary for genome replication, assembly of the viral capsid, and encapsulation of the genome into the viral capsid, are removed from the viral genome and provided in trans for AAV vector production12. The removal of these genes from the viral genome provides room for therapeutic transgenes and all the necessary regulatory elements, including the promoter and polyA signal. The ITRs remain in the vector genome to ensure proper genome replication and viral encapsulation13,14. To improve the kinetics of transgene expression, AAV vector genomes can be engineered to be self-complementary, which mitigates the need for conversion from single-stranded to double-stranded DNA conversion during AAV genome replication, but reduces the coding capacity to ~2.4 kb15.
Beyond AAV genome design, capsid serotype selection determines the tissue and cell tropism of the AAV vector in vivo2. In addition to tissue tropism, different AAV serotypes have been shown to display different gene expression kinetics16. For example, Zincarelli et al.17&#160;classified different AAV serotypes into low expression serotypes (AAV2, 3, 4, 5), moderate expression serotypes (AAV1, 6, 8), and high expression serotypes (AAV7 and 9). They also categorized AAV serotypes into slow-onset expression (AAV2, 3, 4, 5) or rapid-onset expression (AAV1, 6, 7, 8, and 9). These divergent tropisms and gene expression kinetics are due to amino acid variations in the capsid proteins, capsid protein formations, and interactions with host cell receptors/co-receptors18. Some AAV capsids have additional beneficial characteristics such as the ability to cross the blood-brain barrier following intravascular administration (AAV9) or reside in long-living muscle cells for durable transgene expression (AAV6, 6.2FF, 8, and 9)19,20.
This paper aims to detail a cost-effective method for producing high-purity, high-titer, research-grade AAV vectors for use in preclinical large animal models. Production of AAV using this protocol is achieved using dual-plasmid transfection into adherent human embryonic kidney (HEK)293 cells grown in cell stacks. Furthermore, the study describes a protocol for heparin sulfate affinity chromatography purification, which can be used for AAV serotypes that contain heparin-binding domains, including AAV2, 3, 6, 6.2FF, 13, and DJ21,22.
A number of packaging systems are available for the production of AAV vectors. Among these, the use of a two-plasmid co-transfection systems, in which the Rep and Cap genes and Ad helper genes (E1A, E1B55K, E2A, E4orf6, and VA RNA) are contained within one plasmid (pHelper), has some practical advantages over the common three-plasmid (triple) transfection method, including reduced cost for plasmid production23,24. The AAV genome plasmid containing the transgene expression cassette (pTransgene), must be flanked by ITRs, and must not exceed ~4.7 kb in length. Vector titer and purity can be affected by the transgene due to potential cytotoxic effects during transfection. Assessment of vector purity is described herein. Vectors produced using this method, which yield a 1 x 1013 vg/mL for each, were evaluated in mice, hamsters, and ovine animal models.
Table 1: Composition of required solutions. Necessary information, including percentages and volumes, of components needed for various solutions throughout the protocol. <a href="https://www.jove.com/files/ftp_upload/62727/Table 1- 62727_R2.xlsx" target="_blank">Please click here to download this Table.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Millipore Sigma</td>
			<td>S2GPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Fisher Scientific</td>
			<td>SM2001C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Butanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A399-4</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>10 chamber cellstack</td>
			<td>Corning</td>
			<td>3271</td>
			<td></td>
		</tr>
		<tr>
			<td>1L PETG bottle</td>
			<td>Thermo Fisher Scientific</td>
			<td>2019-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/Bis Solution</td>
			<td>Bio-Rad</td>
			<td>1610158</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well skirted plate</td>
			<td>FroggaBio</td>
			<td>FS-96</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive plate seals</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-408-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Blood and Tissue Clean up Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td>Use for DNA clean up in section 4.6 of protocol</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Fisher Scientific</td>
			<td>B392-5</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Cell Culture Dishes</td>
			<td>Greiner bio-one</td>
			<td>7000232</td>
			<td>15 cm plates</td>
		</tr>
		<tr>
			<td>Culture Conical Tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>339650</td>
			<td>15 mL conical tube</td>
		</tr>
		<tr>
			<td>Culture Conical Tube</td>
			<td>Fisher Scientific</td>
			<td>14955240</td>
			<td>50 mL conical tube</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) with 1000 mg/L D-glucose, L-glutamine</td>
			<td>Cytiva Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Western Blotting Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>32209</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Greenfield</td>
			<td>P016EA95</td>
			<td>Dilute ethyl alcohol(95% vol) to 20% for section 3.7.4 and 70% for section 6.1.1.1</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>A38-500</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with Mg2+ and Ca2+</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH302268.02</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS without Mg2+ and Ca2+</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30588.02</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>American Tissue Culture Collection</td>
			<td>CRL-1573</td>
			<td>Upon receipt, thaw the cells and culture as described in manufacturer&#8217;s protocol. Once cells have been minimally passaged and are growing well, freeze a subfraction for future in aliquots and store in liquid nitrogen. Always use cells below passage number 30. Once cultured cells have been passaged more than 30 times, it is recommended to restart a culture from the stored aliquots</td>
		</tr>
		<tr>
			<td>HEK293 host cell protein ELISA kit</td>
			<td>Cygnus Technologies</td>
			<td>F650S</td>
			<td>Follow manufacturer&#8217;s instructions</td>
		</tr>
		<tr>
			<td>Heparin sulfate column</td>
			<td>Cytiva Life Sciences</td>
			<td>17040703</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Thermo Fisher Scientific</td>
			<td>KC34120</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;L-glutamine (200 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30034.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Volume Centrifuge Tube Support Cushion</td>
			<td>Corning</td>
			<td>CLS431124</td>
			<td>Support cushion must be used with large volume centrifuge tubes uless the centrifuge rotor has the approriate V-bottom cushions</td>
		</tr>
		<tr>
			<td>Large Volume Centrifuge Tubes</td>
			<td>Corning</td>
			<td>CLS431123-6EA</td>
			<td>500 mL centrifuge tubes</td>
		</tr>
		<tr>
			<td>MgCl2&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td>1.5 mL microcentrifuge tube, sterilize prior to use</td>
		</tr>
		<tr>
			<td>Molecular Grade Water</td>
			<td>Cytiva Life Sciences</td>
			<td>SH30538.03</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>L5125</td>
			<td>CAUTION. Wear gloves</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP35810</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimem, reduced serum medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td>Sterilize prior to use</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td>Dilute to 1x for use on cells</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Cytiva Life Sciences</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>pHelper plasmid</td>
			<td>De novo design or obtained from plasmid repository</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet basin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-681-502</td>
			<td>Purchase sterile pipet basins</td>
		</tr>
		<tr>
			<td>Polyethylene glycol tert-octylphenyl ether (Triton X-100)</td>
			<td>Thermo Fisher Scientific</td>
			<td>9002-93-1</td>
			<td>CAUTION. Wear gloves</td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Polyscience</td>
			<td>24765-1</td>
			<td>Follow manufacturer&#8217;s instructions to produce a 1L solution. 0.22&#956;m filter and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Polypropylene semi-skirted PCR Plate</td>
			<td>FroggaBio</td>
			<td>WS-96</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween 20)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP337-100</td>
			<td>CAUTION. Wear gloves</td>
		</tr>
		<tr>
			<td>polyvinylidene difluoride (PVDF) membrane</td>
			<td>Cytiva Life Sciences</td>
			<td>10600023</td>
			<td>Use forceps to manipulate. Wear gloves.</td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td>Progen</td>
			<td>65158</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Ladder</td>
			<td>FroggaBio</td>
			<td>PM008-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2546</td>
			<td></td>
		</tr>
		<tr>
			<td>pTrangene plasmid</td>
			<td>De novo design or obtained from plasmid repository</td>
			<td>NA</td>
			<td>Must contain SV40 polyA in genome to be compatible with AAV titration in section 5.0</td>
		</tr>
		<tr>
			<td>Pump tubing</td>
			<td>Cole-Parmer</td>
			<td>RK-96440-14</td>
			<td>Optimize length of tubing and containment of virus in fractions E1-E5</td>
		</tr>
		<tr>
			<td>RQ1 Dnase 10 Reaction Buffer</td>
			<td>Promega</td>
			<td>M6101</td>
			<td>Use at 10x concentration in protocol from section 4.0</td>
		</tr>
		<tr>
			<td>RQ1 Rnase-free Dnase</td>
			<td>Promega</td>
			<td>M6101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample dilutent</td>
			<td>Cygnus Technologies</td>
			<td>I700</td>
			<td>Must be purchased separately for use with HEK293 host cell protein ELISA kit</td>
		</tr>
		<tr>
			<td>Secondary antibody, HRP</td>
			<td>Thermo Fisher Scientific</td>
			<td>G-21040</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Oxoid</td>
			<td>LP0033B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>28312</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SS266-4</td>
			<td></td>
		</tr>
		<tr>
			<td>SV40 polyA primer probe</td>
			<td>IDT</td>
			<td></td>
			<td>Use sequence in Table X for quote from IDT for synthesis</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15524010</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Bio-Rad</td>
			<td>1450021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Filter</td>
			<td>Millipore Sigma</td>
			<td>UFC810024</td>
			<td>Ultra-4 Centrifugal 10K device must be used, as it has a 10000 molecular weight cutoff</td>
		</tr>
		<tr>
			<td>Universal Nuclease for cell lysis</td>
			<td>Thermo Fisher Scientific</td>
			<td>88702</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal qPCR master mix</td>
			<td>NEB</td>
			<td>M3003L</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Paper</td>
			<td>Millipore Sigma</td>
			<td>WHA1001325</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Fisher Scientific</td>
			<td>21985023</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>CAUTION:&#160;Refer to the Materials Table for guidelines on the use of dangerous chemicals.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Translation from small rodent models to larger animal models and eventual clinical application presents a significant challenge due to the large amount of AAV required to transduce larger animals and achieve therapeutic effects. To compare transduction efficiency of the rationally designed AAV6.2FF capsid, previously demonstrated a 101-fold increase in transduction efficiency in murine muscle cells compared to AAV63, mice, hamsters, and lambs were all administered AAV6.2FF expressing a human monoc...

Watch this Scientific Journal Video about Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models at JoVE.com

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

This protocol for production of AAV viral vectors is cost-effective and yields high purity, high titer, research-grade AAV for use in preclinical, large animal models. This technique uses adherent HEK293 cells in cell culture chambers, which is time efficient and less hands-on, allowing for fewer disruptions to the cells. This protocol can greatly aid in viral vector production for the field of gene therapy, and can also be used for the production of other AAV serotypes that also bind heparan sulfate.To begin, collect HEK293 cells from the culture when it reaches 80%confluency. Aspirate the media before gently washing the cell culture plate with three milliliters of PBS. Then aspirate PBS and add three milliliters of trypsin.Incubate the plates for two minutes at 37 degrees Celsius. After incubation, neutralize trypsin, by adding seven milliliters of complete DMEM to the plate, and collect the supernatant in 50 milliliter conical tubes. Gently invert the tubes to ensure the cells are homogenous.To determine the cell density, mix 10 microliters of the cell samples with 10 microliters of trypan blue. And add the mixture to a cell counting slide to analyze in the cell counter. Mix the cell suspension with one liter of pre warmed complete DMEM to see the culture chamber.Gently rotate the chamber to distribute the cells evenly. Incubate the chamber at 37 degrees Celsius with 5%carbon dioxide. In addition to the cell culture chamber, culture cells at 10 to the fourth cells per centimeter squared in a 15 centimeter plate as a reference for confluency.After 65 hours of incubation, when the cells are 80 to 90%confluent, slowly add the polyethyleneimine-DNA and DMEM mixture into the cell culture chamber port. Distribute the liquid evenly to all the rows before incubating at 37 degrees Celsius, and 5%carbon dioxide for 72 hours. After incubation, shake the cell culture chamber vigorously to dislodge the cells until the media appears cloudy, and pour it into four or 500 milliliter centrifuge tubes.Pellet the cells by centrifugation at 18, 000 times G for 30 minutes at four degrees Celsius. Pour the clarified supernatant into a one-liter PETG bottle, and resuspend the cell pellets in 50 milliliters of licensed buffer in 500 milliliter centrifuge tubes. Incubate the tubes for 60 minutes at 37 degrees Celsius.After incubation, centrifuge the tubes at 18, 000 times G for 30 minutes, and transfer the supernatant into the same one-liter PETG bottle. Store at minus 80 degrees Celsius. Thaw the crude lysate at four degrees Celsius overnight.Once thawed, filter it using a 0.22 micron filter. Immediately before the purification steps, passivate the centrifugal concentrator by adding four milliliters of filter pretreatment buffer for each heparin-Sepharose column being used. Place the tubing in a peristaltic pump.Sequentially run the solutions and media. Prepare for chromatography by attaching a five milliliter heparin-Sepharose column to the tubing, and running 25 milliliters of Bazell DMEM through the column. Then load 0.2 micromolar of the filtered crude lysate onto the column at a flow rate of one to two drops per second.Wash the column sequentially to elute five milliliters five times with 300 millimolar of sodium chloride HBSS, with magnesium and calcium, and label the five elutions as E1 to E5.When ready, spin the centrifugal concentrator containing the pretreatment buffer at 900 times G for two minutes. Discard the flow-through. Wash the centrifugal concentrator filter with four milliliters of HBSS with magnesium and calcium by spinning at 1000 times G for two minutes.Then add the elution E2 to the centrifugal concentrator, and spin at 1000 times G for five minutes. Discard the flow-through. Similarly spin the elution E3 into the centrifugal concentrator until the concentrated virus is approximately one milliliter.Remove the concentrated virus from the centrifugal concentrator using a P200 filtered tip, and collect it into a sterile 1.5 milliliter centrifuge tube. After collection of the virus, rinse the centrifugal concentrator with 200 microliters of HBSS with magnesium and calcium. Pipette up and down vigorously for 30 seconds to dislodge any virus adhered to the membrane, and collect the concentrated virus in the same 1.5 milliliter centrifuge tube.Mix the tube well. Wash the column and saturate with 20%ethanol before storing at four degrees Celsius. Store the pump tubing in one molar sodium hydroxide.The transduction efficiency of the AAV6.2FF capsid was compared in mice, hamsters, and lambs for 28 days upon intramuscular administration. Mice administered five times 10 to the 12th vector genomes per kilogram of AAV6.2FF human IgG, expressed between 171 to 237 micrograms per milliliter of human IgG in the serum. Hamster is administered at two times 10 to the 13th vector genomes per kilogram of AAV6.2FF human IgG, expressed much higher levels of human IgG than the mice.While the large animal model was able to express an average 35 micrograms per milliliter of human IgG levels in the plasma. Large animal models can determine the expression of the AAV transgene in vivo, and determine if the transgenes have therapeutic effect against the disorder or disease of interest. The study has showcased the great transduction ability of AAV6.2FF, a novel AAV 6 serotype vector in the skeletal muscle, as well as the low immunogenicity of AAV viral vectors in vivo.

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Research

JoVE Journal

Immunology and Infection

5.6K visualizações.  University of Guelph. Este protocolo de produção de vetores virais AAV é econômico e produz alta pureza, alto título, AAV de grau de pesquisa para uso em modelos animais pré-clínicos e grandes. Esta técnica utiliza células HEK293 aderentes em câmaras de cultura celular, que é eficiente no tempo e menos prática, permitindo menos interrupções nas células. Este protocolo pode ajudar muito na produção de vetores virais para o campo da terapia genética, e também pode ser usado para a produção de ou...