This work was supported by DFG grant EH 192/5-1 (A.E.), the EU (E-rare-2) project Transposmart (A.E.), the UWH Forschungsf&#246;rderung (E.S. and W.Z), and the PhD programme of the University Witten/Herdecke (P.B.). J.L. was supported by a stipend of the Chinese Scholarship council and T.B. and M.G by the Else Kr&#246;ner-Fresenius foundation (EKFS).

<ol>
	<li>Benihoud, K., Yeh, P., Perricaudet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+vectors+for+gene+delivery.">Adenovirus vectors for gene delivery.</a> Curr Opin Biotechnol. 10 (5), 440-447 (1999).</li><li>Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus:+the+first+effective+in+vivo+gene+delivery+vector.">Adenovirus: the first effective in vivo gene delivery vector.</a> Hum Gene Ther. 25 (1), 3-11 (2014).</li><li>Parks, R. J., 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+helper-dependent+adenovirus+vector+system:+removal+of+helper+virus+by+Cre-mediated+excision+of+the+viral+packaging+signal.">A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.</a> Proc Natl Acad Sci U S A. 93 (24), 13565-13570 (1996).</li><li>Palmer, D., Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+system+for+helper-dependent+adenoviral+vector+production.">Improved system for helper-dependent adenoviral vector production.</a> Mol Ther. 8 (5), 846-852 (2003).</li><li>Morral, 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=High+doses+of+a+helper-dependent+adenoviral+vector+yield+supraphysiological+levels+of+alpha1-antitrypsin+with+negligible+toxicity.">High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity.</a> Hum Gene Ther. 9 (18), 2709-2716 (1998).</li><li>Muruve, 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=Helper-dependent+adenovirus+vectors+elicit+intact+innate+but+attenuated+adaptive+host+immune+responses+in+vivo.">Helper-dependent adenovirus vectors elicit intact innate but attenuated adaptive host immune responses in vivo.</a> J Virol. 78 (11), 5966-5972 (2004).</li><li>Ehrhardt, 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=A+gene-deleted+adenoviral+vector+results+in+phenotypic+correction+of+canine+hemophilia+B+without+liver+toxicity+or+thrombocytopenia.">A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia.</a> Blood. 102 (7), 2403-2411 (2003).</li><li>Cots, D., Bosch, A., 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=Helper+dependent+adenovirus+vectors:+progress+and+future+prospects.">Helper dependent adenovirus vectors: progress and future prospects.</a> Curr Gene Ther. 13 (5), 370-381 (2013).</li><li>Mizuguchi, H., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+construction+of+a+recombinant+adenovirus+vector+by+an+improved+in+vitro+ligation+method.">Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method.</a> Hum Gene Ther. 9 (17), 2577-2583 (1998).</li><li>Mizuguchi, H., Kay, M. A. <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+for+constructing+E1-+and+E1/E4-deleted+recombinant+adenoviral+vectors.">A simple method for constructing E1- and E1/E4-deleted recombinant adenoviral vectors.</a> Hum Gene Ther. 10 (12), 2013-2017 (1999).</li><li>Ehrhardt, A., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+adenoviral+helper-dependent+vector+results+in+long-term+therapeutic+levels+of+human+coagulation+factor+IX+at+low+doses+in+vivo.">A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo.</a> Blood. 99 (11), 3923-3930 (2002).</li><li>Jager, 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=A+rapid+protocol+for+construction+and+production+of+high-capacity+adenoviral+vectors.">A rapid protocol for construction and production of high-capacity adenoviral vectors.</a> Nat Protoc. 4 (4), 547-564 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. 1-3, (2001).</li></ol>

The authors declare that they have no competing financial interests.

The protocol presented here allows purification of HCAdV vectors based on human adenovirus type 5 based on previously described procedures 4,12. The HCAdV genome within the pAdFTC plasmid is devoid of all adenovirus genes and only carries the 5&#39;- and 3&#39;- ITRs and the packaging signal. In this strategy the HV AdNG163R-2 4 provides all necessary genes for efficient virus production in trans. This offers a packaging capacity of up to 35 kb, which clearly outcompetes first and second ge...

For gene therapeutic applications it is of great importance to avoid cytotoxic and immunogenic side effects caused by expression of viral proteins, the transgene itself, or by incoming viral proteins. Adenovirus vectors (AdV) are widely used to introduce foreign DNA into a wide variety of cells to investigate the impact of transgene expression 1,2. The most advanced version of AdV is represented by high-capacity adenovirus vectors (HCAdV) lacking all viral coding sequences 3,4 and thereby offering a packaging capacity up to 35 kb combined with low immunogenicity and low toxicity 5-8. Due to their high packaging capacity they allow delivery of large or multiple transgenes using a single vector dose. Therefore, they represent a valuable tool for the research community.
In contrast to first- or second-generation AdV lacking the early genes E1 and/or E3 that can be easily produced using commercial kits, vector genome construction and virus production of HCAdV is more complex. The system for the construction of HCAdV genomes is based on the plasmid pAdFTC carrying a HCAdV genome devoid of all viral coding sequences and the shuttle plasmid pHM5 9-12. Any gene of interest of up to 14 kilobases (kb) can be cloned into the shuttle vector pHM5 in which the multiple cloning site is flanked by recognition/cleaving sites of the homing endonucleases PI-SceI and I-CeuI. Therefore, a cloned gene of interest can be released by consecutive PI-SceI and I-CeuI digests for subsequent directed insertion into the same restriction sites present in the HCAdV genome contained in the plasmid pAdFTC. In pAdFTC the transgene insertion site located between the PI-SceI and I-CeuI cleavage sites is flanked by stuffer DNA and the noncoding adenoviral sequences required for genome packaging such as the 5&#39; and 3&#39; inverted terminal repeats (ITRs) at both ends and the packaging signal downstream of the 5&#39;ITR. The additional stuffer DNA provides optimal size of the final HCAdV genome ranging from 27 to 36 kb to ensure efficient packaging during virus production. Since pAdFTC is a large plasmid with up to 45 kb (dependent on the size of the inserted transgene) and the usage of homing endonucleases with comparably long DNA recognition sites exhibits strong DNA binding, several cleanup steps are necessary during transfer of the transgene from pHM5 to pAdFTC. Careful handling avoiding shearing forces is recommended.
The ITRs of the HCAdV genome are flanked by NotI restriction enzyme recognition sites located directly upstream of the 5&#39;ITR and downstream of the 3&#39;ITR 12. Therefore, HCAdV can be released by NotI digest for subsequent transfection of the viral genome into the HCAdV producer cell line. Note that the usage of the restriction enzyme NotI for release of the viral genome from the plasmid pAdFTC implies that the inserted transgene is devoid of NotI DNA recognition sites. The HEK293 cell based producer cells (116 cells) stably express Cre recombinase. For virus amplification 116 cells are co-infected with a helper virus (HV) providing all AdV genes needed for replication and packaging in trans 3,4. The HV is a first-generation AdV with a floxed packing signal which is removed during virus amplification by Cre recombinase expressed in 116 cells 4. This ensures that predominantly HCAdV genomes containing an intact packaging signal are encapsidated.
Pre-amplification of the HCAdV is performed by conducting serial passaging steps in 116 cells grown on surfaces in tissue culture dishes. After each passage viral particles are released from infected cells by conducting three consecutive freeze-thaw steps. With every passage increasing numbers of cells are infected with 1/3 of cell lysate from the preceding passage. Finally lysate from the last pre-amplification step is used to infect producer cells grown in suspension in a spinner flask for large scale amplification. Virions are purified from the suspension cells by performing ultracentrifugation in a cesium chloride density gradient 4,12. With this procedure empty particles and fully assembled particles are separated into two distinct bands. To further concentrate the HCAdV particles a second non-gradual ultracentrifugation step is performed. Subsequently the resulting band containing the HCAdV is collected and dialyzed against a physiological buffer. Final vector preparations are characterized with respect to numbers of absolute viral particles, infectious particles and HV contamination levels. Absolute viral particles can be determined by lysing viral particles and measuring the absorbance at 260 nm or by performing quantitative real-time PCR (qPCR) 12. Infectivity of the purified virus particles can be determined by qPCR measuring HCAdV genomes present within infected cells 3 hr post-infection.

<table><tbody><tr><td>I-CeuI</td><td>New England Biolabs</td><td>R0699S</td><td>restriction digest</td></tr><tr><td>PI-SceI</td><td>New England Biolabs</td><td>R0696S</td><td>restriction digest</td></tr><tr><td>T4 Ligase</td><td>New Engand Biolabs</td><td>M0202S</td><td>ligation</td></tr><tr><td>SwaI</td><td>New England Biolabs</td><td>R0604S</td><td>restriction digest</td></tr><tr><td>NotI</td><td>New England Biolabs</td><td>R0189S</td><td>restriction digest</td></tr><tr><td>Calf Intestinal Alkaline Phosphatase (CIP)</td><td>New England Biolabs</td><td>M0290S</td><td>dephosphorylation of digested plasmids</td></tr><tr><td>Hygromycin B</td><td>PAN Biotech</td><td>P02-015</td><td>selection of CRE expressing 116 cells</td></tr><tr><td>DMEM</td><td>PAN Biotech</td><td>P04-03590&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Hek293T cell culture medium</td></tr><tr><td>Minimal Essential Medium (MEM) Eagle</td><td>PAN Biotech</td><td>P04-08500</td><td>116 cell culture medium&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffer saline (DPBS)</td><td>PAN Biotech</td><td>P04-36500</td><td>washing of cells, resuspension of cells</td></tr><tr><td>250-ml storage bottle</td><td>Sigma</td><td>CLS430281-24EA</td><td>infection of 116 cells grown in suspension</td></tr><tr><td>500-ml PP CentrifugeTubes</td><td>Sigma</td><td>CLS431123-36EA</td><td>sedimentation of cells from suspension culture</td></tr><tr><td>Spinner flask</td><td>Bellco</td><td>1965-61030</td><td>growth of 116 cells in suspension</td></tr><tr><td>Ultra Clear Ultracentrifuge tubes</td><td>Beckmann Coulter</td><td>344059</td><td>density gradient centrifugation</td></tr><tr><td>Ultracentrifuge</td><td>Beckmann Coulter</td><td></td><td>density gradient centrifugation</td></tr><tr><td>SW-41 rotor</td><td>Beckmann Coulter</td><td></td><td>density gradient centrifugation</td></tr><tr><td>Spectrum Laboratories Spectrapor Membrane</td><td>VWR</td><td>132129</td><td>dialysis tubing</td></tr><tr><td>ready-to-use dialysis cassettes&amp;nbsp;</td><td>Thermo</td><td>66383</td><td>dialysis</td></tr><tr><td>one shot DH10B electrocompetent &lt;i&gt;E. coli&lt;/i&gt;</td><td>invitrogen</td><td>C4040-52</td><td>transformation of ligation reactions</td></tr><tr><td>PureYield Plasmid Midiprep System</td><td>Promega</td><td>A2495</td><td>midiprep</td></tr><tr><td>peqGOLD Tissue DNA Mini Kit&amp;nbsp;</td><td>Peqlab</td><td>12-3396-02</td><td>isolation of genomic DNA</td></tr><tr><td>SuperFect Transfection Reagent</td><td>Qiagen</td><td>301305</td><td>tranfection of 116 cells</td></tr><tr><td>opti MEM (10% FBS)</td><td>Gibco</td><td>31985-062</td><td>transfection of 116 cells</td></tr><tr><td>iQ SYBR Green Supermix</td><td>BioRad</td><td>&amp;nbsp;170-8882</td><td>q-PCR</td></tr><tr><td>CFX 96 C1000 touch&amp;nbsp;</td><td>Biorad</td><td></td><td>qPCR machine</td></tr><tr><td>Phenol/Chloroform/Isoamyl alcohol&amp;nbsp;</td><td>Carl Roth</td><td>A156.1</td><td>purification of DNA</td></tr><tr><td>Cesium chloride</td><td>Carl Roth</td><td>8627.1</td><td>density gradient centrifugation</td></tr><tr><td>sodium acetate 99%</td><td>Carl Roth</td><td>6773.2</td><td>DNA precipitation</td></tr><tr><td>LB medium&amp;nbsp;</td><td>Carl Roth</td><td>X968.3</td><td>bacterial growth medium</td></tr><tr><td>ethanol&amp;nbsp; 99.8% pure</td><td>Carl Roth</td><td>9065.5&amp;nbsp;</td><td>DNA precipitation and washing</td></tr><tr><td>SDS 99.5%</td><td>Carl Roth</td><td>2326.2</td><td>lysis&amp;nbsp; buffer</td></tr><tr><td>EDTA</td><td>Carl Roth</td><td>8043.2</td><td>lysis&amp;nbsp; buffer</td></tr><tr><td>Tris-HCl 99%</td><td>Carl Roth</td><td>9090.3</td><td>dialysis buffer/ lysis&amp;nbsp; buffer</td></tr><tr><td>glycerol 99.5%</td><td>Carl Roth</td><td>3783.1</td><td>dialysis buffer</td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;98.5%</td><td>Carl Roth</td><td>KK36.2</td><td>dialysis buffer</td></tr><tr><td>NaCl</td><td>Carl Roth</td><td>3957.1</td><td>optional dialysis buffer</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth</td><td>3904.2</td><td>optional dialysis buffer</td></tr><tr><td>sucrose</td><td>Carl Roth</td><td>9286.1</td><td>optional dialysis buffer</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;x2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>4984.2</td><td>optional dialysis buffer</td></tr><tr><td>1.5-ml tubes</td><td>sarstedt</td><td>72,730,005</td><td>storage of virus preparations at -80 &amp;deg;C</td></tr></tbody></table>

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.

Here representative examples for cloning, amplification and purification of HCAdV preparations are shown. An overview of the cloning strategy (Figure 1) and representative examples for cloning and release of the HCAdV genome by restriction enzyme digest are provided (Figure 2). A typical restriction pattern after release of the GOI-expression cassette from pHM5 by PI-SceI and I-CeuI digest and subsequent phenol-chloroform extraction and ...

Watch this Scientific Journal Video about Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5 at JoVE.com

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

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 ...

cloning-large-scale-production-high-capacity-adenoviral-vectors-based

Research

JoVE Journal

Bioengineering

14.3K Views.  University Witten/Herdecke. 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. 

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

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

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 ...

Immunology and Infection

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.

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 ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

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 ...

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, ...