The capacity to efficiently transduce nondividing cells, shuttle small as well large genetic elements, and maintain stable long-term transgene expression are properties that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications.

Three major properties are the basis of these previous capacities:

1. First, retroviral vector integrate its genome into the target cell DNA and allow long term expression even if no specific integration sequence has been shown.
2. Second, retroviral vectors exhibit a relatively large  capacity of cDNA cloning close to 8 kb.
3. The final integrated sequence that encodes for the gene of interest do not encode for any viral protein derived from the packaging parental virus.                    

Here you can find the state of the art technology for lentiviral vector production by transient transfection or packaging cell lines and the practices used by Vectalys for concentration, purification, titering, and determining the safety of a vector stock.

We provide:

• Stock products expressing reporter genes like GFP, LacZ or luciferase
• If you have developed your own construct, we can manufacture it for you on a totally custom base, depending on the aim of your project
• Lentiviral vectors  based on custom vectors on the following basic backbones described in the "gene work" part.

Viral vector production:

Retroviral or lentiviral vectors are produced by transient transfection of 293T cells by plasmids carrying packaging functions and the recombinant construct with the cDNA or shRNA sequence of interest. To produce a lentiviral or retroviral particle, several packaging functions are necessary:

• the enzymatic products of the pol gene and the structural proteins encoded by the gag and env genes lead to the budding at the plasma membrane of the producer cell line. After packaging, all these functions will contribute to the efficiency of the early steps of viral cycle: cell entry, reverse transcription, nuclear translocation and integration.  Here, the parental or viral replicative cycle is finished and the following events are only transcription and translation of foreign inserts without any viral proteins synthesis.  
• Only for lentiviruses, a virally-encoded post-transcriptional activator called Rev is necessary for efficient gene expression.    
• The parental envelope protein can be substituted by the corresponding protein of another virus to modify the tropism of the virion. The G protein of vesicular stomatisis virus (VSV-G) is classically used to pseudotype lentiviral as well oncoretroviral particles. 

Viral supernatant is harvested directly at a titer of about 10E6 TU/ml and can by concentrated and purified by one or several rounds of tangential filtration to reach a titer of about 10E9 TU/ml.

Vectalys provides several batches of retroviral or lentiviral vectors including low titer stocks at 10E6 TU/ml (200 ml). This concentration is high enough for certain ex vivo applications like tumoral cell or keratinocytes transduction. However, concentration of vector stocks is required for most cell transduction like hematopoïetic stem cells, dendritic cells or neurons in order to increase cell transduction efficiencies. The following concentration and volume are available but keep in mind that your needs are determining the batch's specifications:

•  10E6 TU/ml: 50 ml to 200 ml (or more)
• 10E7 TU/ml : 2 or 50 ml
• 10E9 TU/ml : 1 to 2 ml
• 10E11 TU batch

Viral vector titering:

Titers of viral vectors like viruses critically depend on the method used for titration. The titers can be determined by two different approachs:

• The quantification of vector particles capable of achieving all steps between cell binding to transgene expression. In this case the titer is expressed by the number of transducing units per milliliter (TU/ml)
• The determination of the total particle concentration that reflects the efficiency of vector production and packaging but do not give the infectious titer. This titer is expressed as the number of viral matrix protein (P24) per ml.

The transducing titer implies the used of target cells and the method used to measure the completion of the pre integrative and integrative steps will depend of the nature of the insert. Vectors containing reporter genes like luciferase, LacZ or now GFP can be quantified by using the intrinsic property of each insert, but for others constructs we use an universal method that target specific viral derived sequence by QPCR. Whatever the transgene, measured titers vary with the conditions used for titrations and the transgene expression depends from the transcriptional and translational regulatory elements that drive the protein expression. For instance, volume of samples, cells used as well the number of cells during vector-cell incubation may have a strong influence on the measured vector titer. To obtain more information about the technology used by vectalys, ask us more details by sending a mail to : Cet e-mail est protégé contre les robots collecteurs de mails, votre navigateur doit accepter le Javascript pour le voir

Viral vector safety:

During vector stock production the emergence of replication- competent recombinants (RCRs) have to be avoided and must be checked to permit their manipulation in L2 safety rooms as recommended by the “Commission de Génie Génétique, (CGG)”.
Biosafety is best reached by distributing the various viral sequences encoding the functional and structural proteins necessary from particles packaging to DNA integration as many independent expression units. This system allows to maximize the number of recombination events that would be required to obtain a recombinant vector able to replicate.

Vectalys used a lentiviral vector system based on the generation of vector particles from three or four separate plasmids to ensure that only replication-defective vectors are produced. Moreover, the use of self-inactivating (SIN) vector is developed and used. It means that the viral U3 sequence of the LTR able to drive the residual viral DNA expression is deleted. For this reason, the presence of an  internal promoter to drive specifically the transgene expression is required.

Furthermore, each vector batch is checked for the absence of replication- competent recombinants by P24 assay on transduced cell supernatant. Permissive cells are transduced at MOI10 and after 10 days of transduction, cell supernatants are harvested and tested for P24 detection. These assay's specifications have been validated by the results summarized in the following table:

To obtain more information about the technology used by vectalys, ask us more details by sending a mail to : Cet e-mail est protégé contre les robots collecteurs de mails, votre navigateur doit accepter le Javascript pour le voir