Name: efficient recombinant parvovirus production with the help of adenovirus-derived systems
Uploaded: 2012-04-23T12:00:00
Description: Watch this Scientific Journal Video about Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems at JoVE.com

We thank the team of the DKFZ virus production and development unit, in particular Marcus M&uuml;ller, Silvia M&uuml;nstermann, Barbara Liebetrau, and Mandy Roscher. This study has been partly supported by grants from the Federal Ministry of Education and Research (BMBF) and the Helmholtz Association in the framework of the Deutsches Krebsforschungszentrum / Canc&eacute;rop&ocirc;le du Grand-Est joint Programme in Applied Tumour Virology.

Note that a laboratory with a biosafety level 2 is required for the production of recombinant parvoviruses (recPV).
The protocol is subdivided in two main parts. The first part (production of recPV via transfection) is required to produce the minimal amount of recPVs that serves as inoculum in the second part of the protocol (production of recPVs via infection). Once a small amount of recPVs is produced, the first part of the protocol can be omitted, and the recombinant parvovirus can be ampli...

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We have shown that recPV production can be enhanced by the presence of adenoviral genomic elements. We have increased the recPV yields by more than 10 fold (from 0.3 to 5 TU/cell) by providing adenovirus genomic element via transfection and by more than 100 fold co-infecting the cells with Ad-VP-helper in combination with the recPV in comparison to conventional protocols. The protocol described here can be further optimized by determining the most appropriate timing for the delivery of the adenovirus genomic elements and...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM </td>
 <td>Sigma </td>
 <td>D5796</td>
 </tr>
 <tr>
 <td>MEM</td>
 <td>Sigma</td>
 <td>M4655</td>
 </tr>
 <tr>
 <td>Foetal Bovine Serum</td>
 <td>PAA</td>
 <td>A15-101</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Gibco</td>
 <td>25030-024</td>
 </tr>
 <tr>
 <td>Fugene</td>
 <td>Roche</td>
 <td>047097050001</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA</td>
 <td>Invitrogen</td>
 <td>25300062</td>
 </tr>
 <tr>
 <td>Benzonase Nuclease</td>
 <td>Sigma</td>
 <td>E8263</td>
 </tr>
 <tr>
 <td>Adeno-X Rapid Titer Kit </td>
 <td>Clontech</td>
 <td>632250</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems at JoVE.com

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

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 ...

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