This work was supported financially by grants from INSERM, the CNRS, the Fondation pour la Recherche M&#233;dicale and the Agence Nationale pour la Recherche (ANR- 10-Blanc-1322) to D.G-D; The European Commission&#39;s Seventh Framework Program to B.K.; M.S. was supported by a postdoctoral fellowship from FRM (programme &#34;Equipe FRM 2009&#34;); C.B. was supported by a postdoctoral fellowship from UE, under grant agreement number 245266 (Orbivac).

1. Construction of a Nonreplicative CAV2 Genome Plasmid
Note: all plasmids described in this protocol have been described previously7 and are available upon request.
<ol>
	<li>Clone the GOI into the pShuttle plasmid, using appropriate restriction enzymes, to generate pShuttle-GOI (Figure 1A).</li>
	<li>Digest 2 &#956;g of pShuttle-GOI by AscI/PacI. The digestion releases the Shuttle-GOI fragment (~2.9 kbp + GOI ORF sequence) from the plasmid backbone (2.8 kbp).</li>
	<li>Purify the Shuttle-GOI fragment after agarose gel electrophoresis, using NucleoSpin Gel and PCR Clean-up kit.</li>
	<li>In parallel, digest 1 &#956;g of pCAV2 genomic plasmid by SwaI. The pCAV2 plasmid contains a unique SwaI site located within the E1 region (Figure 1B). Heat-inactivate this restriction enzyme at 65 &#176;C for 20 min.</li>
	<li>Transform competent E. coli BJ5183 bacteria with 500 ng of purified Shuttle-GOI fragment and 100 ng of linearized pCAV2 plasmid for homologous recombination (Figure 1B). Spread on LB-Amp plates. Incubate plates at 37 &#176;C for at least 20 hr.</li>
	<li>Perform PCR analysis directly on bacterial colonies using EmeraldAmp master mix with forward (5&#39;-CACGAGGCCCTTTCGTCTTCAA-3&#39;) and reverse (5&#39;-GCGGTAGTTTATCACAGTTAAATTGC-3&#39;) primers, under the following cycling conditions: 10 min at 95 &#176;C (for one cycle), 95 &#176;C for 1 min; 58 &#176;C for 1 min; 72 &#176;C for 1 min/kbp (for 39 cycles), and finally 5 min at 72 &#176;C. A product of 1 kbp + GOI ORF sequence is amplified from pCAV-GOI positive clones, whereas no PCR product can be detected with negative clones.</li>
	<li>Inoculate at least two positive colonies overnight each in 5 ml LB-amp medium at 37 &#176;C in a shaker (220 rpm).</li>
	<li>Extract plasmid DNAs using Nucleospin plasmid kit. Transform competent E. coli DH5&#945; bacteria with an aliquot of each DNA prep. Spread bacterial suspension on LB-Amp plates and incubate overnight at 37 &#176;C.</li>
	<li>Inoculate two colonies of each positive clone overnight in 3 ml LB-amp medium at 37 &#176;C in a shaker (220 rpm). Extract plasmid DNA using Nucleospin plasmid kit.</li>
	<li>Digest the resulting DNA and the parental pCAV2 plasmid (as a control) with NdeI. The restriction pattern of parental pCAV2 is 14.8 kbp, 9 kbp, 7.9 kbp, and 1.6 kbp. The GOI expression cassette is inserted within the 7.9 kbp fragment and contains at least one NdeI site, i.e. one in the cassette and the potential sites present in the GOI.</li>
	<li>Select positive constructions and proceed to step 2 with at least 2 independent clones.</li>
</ol>
2. Transfection of Recombinant CAV2 Genome Plasmid into DK-E1 Cells
Note: CAV2-derived non replicative vectors are genetically modified organisms classified as Biosafety Level 2 (BSL-2), whereas their use on animal models is classified as BSL-1. Please use proper containment and waste handling measures, including personal protective equipment, and work under BSL-2 laminar flow hoods.
<ol>
	<li>A day before transfection, seed DK-E1 cells on a 6-well plate. Grow cells at 37 &#176;C and 5% CO2, in Dulbecco&#39;s Modified Eagle Medium (DMEM) with high glucose, containing 7% heat-inactivated fetal calf serum (FCS), 1 mM sodium pyruvate, and 100 U/ml penicillin/100 &#956;g/ml streptomycin. Cells should be 70-80% confluent for transfection.</li>
	<li>Digest 2 &#956;g of pCAV-GOI plasmid with AscI, to release the nonviral sequence of the plasmid. Check the resulting restriction pattern by 0.8% agarose gel electrophoresis. Digestion should yield a ~31 kbp fragment containing the recombinant genome and a 2 kbp fragment corresponding to the plasmid backbone.</li>
	<li>Mix digested DNA with 200 &#956;l of Jet Prime Buffer by vortexing for 10 sec. Add 4 &#956;l of Jet Prime and mix by vortexing for 10 sec. Spin briefly to remove droplets and incubate for 10 min at room temperature.</li>
	<li>Add the transfection mix dropwise onto cells. Gently shake plates to evenly distribute the mix and incubate at 37 &#176;C.</li>
</ol>
3. Propagation of Recombinant CAV2
<ol>
	<li>One week after transfection, collect cells and culture medium in a 15 ml polypropylene tube. Disrupt cells by three freeze-thaw cycles (-80 &#176;C/37 &#176;C) and clear the lysate by centrifugation for 10 min at 1,800 x g.</li>
	<li>Collect supernatant and store at -20 &#176;C for subsequent virus propagation.</li>
	<li>Infect an 80-90% confluent monolayer of DK-E1 cells grown in one well of 6-well plate with 0.5 ml of the virus-containing supernatant. Incubate at 37 &#176;C for 1 hr, under mild agitation. Remove inoculum and replace it with 1.5 ml of complete DMEM containing 5% heat-inactivated FCS.</li>
	<li>Incubate cells at 37 &#176;C for 3-4 days. Cells should be monitored daily for appearance of a cytopathic effect (CPE, Figures 2A-B). If no clear CPE is visible, harvest cells after 4 days of culture and repeat steps 3.1-3.4.</li>
	<li>When a clear CPE affects the majority of cells, usually after 2-4 viral amplification rounds, collect cells with culture medium and freeze-thaw 3x as described above. Infect 80-90% confluent DK-E1 cells grown in three 10 cm diameter tissue culture dishes, using each time 0.5 ml of virus-containing supernatant.</li>
	<li>After 3-4 days (when CPE is complete), harvest cells from these 10 cm dishes, again disrupt them by three freeze-thaw cycles, clear the lysate by centrifugation for 10 min at 1,800 x g, collect supernatant and store at -20 &#176;C for large-scale CAV2 amplification.</li>
</ol>
4. Large-scale CAV2 Purification
<ol>
	<li>Infect 80-90% confluent monolayers of DK-E1 cells grown in forty 10 cm diameter tissue culture dishes. For each dish, use 0.1 ml of virus-containing supernatant diluted in 1 ml of complete DMEM without FCS. Incubate cells at 37 &#176;C for 3 to 4 hr under mild agitation. Add 5 ml of complete DMEM supplemented with 5% FCS.</li>
	<li>Incubate for 3 days. Harvest infected cells from the forty 10 cm dishes in 50 ml polypropylene tubes. Pellet cells at 1,200 x g for 10 min at 4 &#176;C and resuspend them in 15 ml of DMEM medium.</li>
	<li>Disrupt cells by three freeze-thaw cycles (-80 &#176;C/37 &#176;C). Remove cell debris by centrifuging at 1,800 x g for 10 min and store supernatant at -20 &#176;C prior to purification of viral particles.</li>
	<li>Prepare a discontinuous CsCl gradient in a 14 ml Ultra-Clear tube. First, pour 2 ml of CsCl solution (1.40 g/ml&#160;in phosphate buffered saline) at the bottom of the tube. Then, add slowly 2 ml of CsCl solution (1.25 g/ml&#160;in phosphate buffered saline) on top of the first solution.</li>
	<li>Load carefully the viral supernatant on top of the CsCl gradient. Fill the tube with mineral oil up to 2-3 mm from the top. Centrifuge for 1.5 hr at 130,000 x g and 18 &#176;C in a swinging SW40 rotor.</li>
	<li>After centrifugation, two white bands are clearly visible at the interface between the 2 CsCl solution layers (Figure 2C). Using a 21 G needle and a syringe, collect by side puncture the lower band containing mature CAV2 particles. Try as much as possible to avoid collecting the upper band, which corresponds to empty viral particles.</li>
	<li>Prepare a continuous CsCl gradient in a 14 ml transparent tube by mixing the collected viral particles with a CsCl solution (1.34 g/ml&#160;in phosphate buffered saline). Fill the tube with mineral oil up to 2-3 mm from the top. Centrifuge for 18 hr at 130,000 x g and 18 &#176;C in a swinging SW40 rotor.</li>
	<li>After centrifugation, collect the white CAV2-containing band by side puncture with a syringe as described above. Again, try to avoid collecting the remaining upper band. This should be easier in this continuous gradient, which separates the two bands better. The total volume of collected suspension should not exceed 2 ml.</li>
	<li>Equilibrate a Sephadex G-25 PD-10 column with 30 ml of PBS.</li>
	<li>Load the viral suspension and discard the flow-through.</li>
	<li>Elute with fractions of 500 &#956;l of PBS. Collect fractions 5-7, which usually contain the desalted CAV2 particles. The virus-containing fractions can easily be identified because they are opalescent. Add 150 &#956;l of glycerol to the 1.5 ml of CAV2 suspension and store in aliquots at -80 &#176;C.</li>
</ol>
5. Titration of CAV2 by End-point Dilution
<ol>
	<li>Thaw an aliquot of purified virus on ice and perform 10-fold serial dilutions (ranging from 10-2-10-12) in serum-free DMEM.</li>
	<li>Add 50 &#956;l of each viral dilution in 5 wells of a 96-well plate. Add 1.5 x 104 DK-E1 cells in each well. Incubate the plate at 37 &#176;C and 5% CO2 for 5 days.</li>
	<li>At day 5 post-infection, monitor CPE appearance by microscopic observation (Figures 2A-B). Infectious titers are determined as median tissue culture infectious doses (TCID50), using the Reed and Muench statistical method13.</li>
</ol>

<ol>
	<li>Kaufmann, J. K., Nettelbeck, 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=Virus+chimeras+for+gene+therapy,+vaccination,+and+oncolysis:+adenoviruses+and+beyond.">Virus chimeras for gene therapy, vaccination, and oncolysis: adenoviruses and beyond.</a> Trends Mol. Med. 18, 365-376 (2012).</li><li>Curr, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Topics Microbiol. Immunol. 343, 195-224 (2010).</li><li>Zak, D. E., 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=Merck+Ad5/HIV+induces+broad+innate+immune+activation+that+predicts+CD8(+)+T-cell+responses+but+is+attenuated+by+preexisting+Ad5+immunity.">Merck Ad5/HIV induces broad innate immune activation that predicts CD8(+) T-cell responses but is attenuated by preexisting Ad5 immunity.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 3503-3512 (2012).</li><li>Paillard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+non-human+adenoviruses+versus+human+adenoviruses.">Advantages of non-human adenoviruses versus human adenoviruses.</a> Hum. Gene Ther. 8, 2007-2009 (1997).</li><li>Ackermann, A., Guelzow, T., Staeheli, P., Schneider, U., Heimrich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+viral+dissemination+in+the+mouse+nervous+system,+using+a+green+fluorescent+protein-expressing+Borna+disease+virus+vector.">Visualizing viral dissemination in the mouse nervous system, using a green fluorescent protein-expressing Borna disease virus vector.</a> J. Virol. 84, 5438-5442 (2010).</li><li>Bru, T., Salinas, S., Kremer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+canine+adenovirus+type+2+and+its+vectors.">An update on canine adenovirus type 2 and its vectors.</a> Viruses. 2, 2134-2153 (2010).</li><li>Bouet-Cararo, 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=Canine+adenoviruses+elicit+both+humoral+and+cell-mediated+immune+responses+against+rabies+following+immunisation+of+sheep.">Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep.</a> Vaccine. 29, 1304-1310 (2011).</li><li>Soudais, C., Skander, N., Kremer, E. J. <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+in+vivo+transduction+of+neurons+throughout+the+rat+CNS+using+novel+helper-dependent+CAV-2+vectors.">Long-term in vivo transduction of neurons throughout the rat CNS using novel helper-dependent CAV-2 vectors.</a> FASEB J. 18, 391-393 (2004).</li><li>Deinhardt, K., 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=Rab5+and+Rab7+control+endocytic+sorting+along+the+axonal+retrograde+transport+pathway.">Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway.</a> Neuron. 52, 293-305 (2006).</li><li>Soudais, C., Laplace-Builhe, C., Kissa, K., Kremer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+transduction+of+neurons+by+canine+adenovirus+vectors+and+their+efficient+retrograde+transport+in+vivo.">Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo.</a> FASEB J. 15, 2283-2285 (2001).</li><li>Peltekian, E., Garcia, L., Danos, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotropism+and+retrograde+axonal+transport+of+a+canine+adenoviral+vector:+a+tool+for+targeting+key+structures+undergoing+neurodegenerative+processes.">Neurotropism and retrograde axonal transport of a canine adenoviral vector: a tool for targeting key structures undergoing neurodegenerative processes.</a> Mol. Ther. 5, 25-32 (2002).</li><li>Chartier, 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=Efficient+generation+of+recombinant+adenovirus+vectors+by+homologous+recombination+in+Escherichia+coli.">Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli.</a> J. Virol. 70, 4805-4810 (1996).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> Am. J. Hyg. 27, 493-497 (1938).</li><li>Segura, M. M., Puig, M., Monfar, M., Chillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatography+purification+of+canine+adenoviral+vectors.">Chromatography purification of canine adenoviral vectors.</a> Human Gene Ther. Methods. 23, 182-197 (2012).</li><li>Segura, M. 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=A+real-time+PCR+assay+for+quantification+of+canine+adenoviral+vectors.">A real-time PCR assay for quantification of canine adenoviral vectors.</a> Journal of virological. 163, 129-136 (2010).</li><li>Fernandes, 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=Bioprocess+development+for+canine+adenovirus+type+2+vectors.">Bioprocess development for canine adenovirus type 2 vectors.</a> Gene Ther. , (2012).</li><li>Rinne, A., Littwitz, C., Bender, K., Kienitz, M. C., Pott, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+delivery+of+short+hairpin+RNA+(shRNA)+mediates+efficient+gene+silencing+in+terminally+differentiated+cardiac+myocytes.">Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.</a> Methods Mol. Biol. 515, 107-123 (2009).</li><li>Huang, B., Kochanek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+silencing+of+huntingtin+expression+by+shRNA.">Adenovirus-mediated silencing of huntingtin expression by shRNA.</a> Hum. Gene Ther. 16, 618-626 (2005).</li><li>Tordo, N., 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=Canine+adenovirus+based+rabies+vaccines.">Canine adenovirus based rabies vaccines.</a> Dev. Biol. 131, 467-476 (2008).</li></ol>

Authors have nothing to disclose.

The simple method described herein, adapted from the well-documented Ad5 technology, allows the efficient production of nonreplicative CAV2 vectors with a typical cloning capacity of ~4.2 kbp. Regarding time considerations, a recombinant CAV2 genome is usually generated in two weeks, whereas it will take an additional 5 weeks to amplify, purify and titer viral suspensions.
Although the production of the first batch of CAV2-derived vectors may appear time-consuming, it should be reminded that C...

Over the past decades, adenoviruses (Ads) derived vectors have proven their efficacy for gene therapy and vaccination, as well as in oncolytic virotherapy 1. Ads are nonenveloped icosahedral viruses of the Adenoviridae family, which have been isolated from mammals, birds, reptiles, amphibians and fish. Since their discovery in 1953, Ads have been intensively studied as models to study virus/cell interactions and, more recently, as vector-based gene delivery systems 2. Indeed, Ads-based vectors offer many advantages, such as their broad host range and well-characterized biology, together with the ease with which they can be genetically manipulated and amplified for large-scale production.
In order to overcome clinical difficulties related to pre-existing immunity in human populations towards vectors derived from human Ads 3, we and others began to derive vectors from nonhuman Ads 4. In addition, nonhuman adenoviral vectors appear to be more adapted for mass vaccination in veterinary medicine than the classically used adenovirus type 5 (Ad5), since they should be more compliant with safety and security requirements during the risk assessment by the national regulatory authorities. In the late 1990s, we described the first recombinant CAV2 as a nonhuman alternative to vectors derived from Ad5 5. Since then, numerous studies have confirmed the potential of CAV2 vectors for therapeutic or antigenic gene transfer (for reviews 6,7). Like other Ads, CAV2-derived vectors are stable and can be produced at high titers, which facilitates their in vivo use. They are also safe, since they are not integrative, although they allow long lasting transgene expression in vivo (&#62;1 year) 8. Remarkably, and better than human Ads serotypes, CAV2 vectors have been shown to be highly neurotropic, with a very efficient retrograde transport in axons 9,10. This intrinsic property brought the idea of using recombinant CAV2 vectors to transduce neurons of specific brain areas that are hardly accessible to lentiviral vectors, so far commonly used for gene transfer into the central nervous system 11.
Here, we describe a simple and classical protocol to construct, produce and purify nonreplicative CAV2 vectors derived from the Manhattan strain. Recombinant CAV2 genomes are constructed by cloning the desired gene of interest (GOI) in a cassette downstream of the cytomegalovirus (CMV) early promoter, which provides a ubiquitous expression. CAV2 vectors have a cloning capacity of ~4.2 kbp, which allows expressing large cDNA. In a second step, this expression cassette is inserted by homologous recombination in place of the E1 region of the CAV2 genome, leading to a nonreplicative virus (dlE1), as described initially by Chartier et al.12&#160;Finally, viral particles are produced upon transfection of the genomic plasmid into a CAV2-E1-expressing canine kidney cell line (DK-E1) and are serially amplified prior to concentration and purification of viral stocks. The method described here uses a two-step ultracentrifugation in cesium chloride (CsCl) gradients, which enables enrichment of infectious particles with a high final titer (usually over 1010 infectious particles per ml). The viral suspension is then desalted by chromatography through disposable Sephadex G-25 columns so that it is ultimately pure enough to be directly used in vivo.

<table><tbody><tr><td>Restriction enzymes</td><td>New England Biolabs</td><td>Various</td><td></td></tr><tr><td>Jet Prime transfection kit</td><td>Polyplus</td><td>114-01</td><td></td></tr><tr><td>DMEM</td><td>Gibco (Life Technologies)</td><td>10567014</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BJ5183 bacteria</td><td>Agilent</td><td>200154</td><td></td></tr><tr><td>PD-10 desalting column</td><td>GE Healthcare</td><td>17-0851-01</td><td></td></tr><tr><td>EmeraldAmp polymerase</td><td>Takara</td><td>RR320A</td><td></td></tr><tr><td>Nucleospin plasmid kit</td><td>Macherey-Nagel</td><td>740558.250</td><td></td></tr><tr><td>NucleoSpin Gel and PCR Clean-up</td><td>Macherey-Nagel</td><td>740609.50</td><td></td></tr><tr><td>14 ml tubes, Ultra Clear</td><td>Beckman</td><td>344060</td><td></td></tr></tbody></table>

production and purification of non replicative canine adenovirus type 2 derived vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

Production of recombinant CAV2 vectors relies on basic but important molecular biology techniques. Prior to the production of viral vectors, it is indeed necessary to construct a recombinant CAV2 genome bearing an expression cassette for the transgene. This construction involves two steps: first, the gene of interest (GOI) is cloned in a shuttle plasmid as an expression cassette (i.e.&#160;with promoter and polyadenylation signal). This functional element is flanked by two CAV2-derived genomic sequences, represe...

Watch this Scientific Journal Video about Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors at JoVE.com

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

production-purification-non-replicative-canine-adenovirus-type-2

Research

JoVE Journal

Immunology and Infection

7.1K Views.  Université Toulouse 3. These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Video: Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

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

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

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

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

Bioengineering

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

An Efficient Method for Adenovirus Production

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

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

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

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

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

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

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

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. ...

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an ...

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. ...

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. ...

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

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

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.