Technologies

Lentiviral vector technology

GEG Tech know-how is based on more 20 years of experience in vectorology and has led to the design of new generations of vectors. GEG Tech expertise has enabled the design and development of the Lenti-ONE technology platform which covers almost all needs within the gene transfer domain.
Technologies -

Depending on your needs, the Lenti-ONE platform is available in three versions: DNA integrating, DNA non-integrating and RNA, all three of which are derived from HIV-1 and take the advantages of the lentiviral vectors: Efficacy, Stability, and high design flexibility with a high level of biosafety.

DNA integrating systems

HOW DOES IT WORK ?

Technologies -
  • Long term or transient expression
  • Genome editing – CRISPR or ZFN
  • High level of biosafety
  • Vaccination, Gene & Cell therapies

GEG Tech DNA integrating systems are derived from lentivirus and are used for a long-term and high expression in dividing and non-dividing cells in vitro as well as in vivo or for transgenesis with a high efficacy rate. Furthermore, lentiviral vectors have a high flexibility of design, allowing them to vectorize large and complex systems. They offer the possibility of achieving envelop engineering, thus allowing them to target specific cell populations.

GEG Tech know-how allows to design and produce these vectors with the highest efficacy and quality for R&D applications compare to competitors. The design and process of production are suitable with GLP and GMP production for clinical projects.

GEG Tech has designed several integrating systems which divide in two different classes according the type of vector integrase:

  • The system including the wild type integrase (IN-WT) of the lentivirus: the integration occurs randomly but preferentially in active genome regions. This system is the most efficient to transfer and express a sequence of interest but it can potentially induce genotoxic side effects which could be undesirable in some projects.
  • The systems including a mutated integrase inhibiting LEDGF interactions (IN-R166A or IN-Q168A). Usually, the vector genome integration in the host cell chromatin is subject to a bias toward transcriptionally active regions due to an interaction between the integrase and LEDGF, an ubiquitous transcription factor. These two mutations are expected to abolish Integrase–LEDGF interactions and make the vector genome integration profile more random in the target cell genome and decrease the potential genotoxic side effects.

DNA Non integrating systems

HOW DOES IT WORK ?

Technologies -
  • Long term and robust expression

  • Cell and Animal models

  • Cancer immunotherapy – CAR cells

  • Gene & Cell therapies

GEG Tech DNA non-integrating systems are derived from lentivirus for which the integrase has been mutated. The implemented mutations prevent the integration of the DNA vector into the cell genome which remains in the episomal form and limit the potential genotoxic effects observed with integrating lentiviral vectors. Consequently, these systems offer a better level of biosafety than DNA integrating vectors.

These non-integrating systems are used for a short-term expression in dividing cells or a long-term expression non-dividing cells without integration step in vitro as well as  in vivo or for transgenesis. The design and process of production are suitable with GLP and GMP production for clinical projects.

These non integrating systems take advantage of lentiviral vectors and provide a good alternative to AAV because they are less immunogenic, their cargo capacity is higher, and their flexibility of design are better, notably the envelop engineering enabling them to target specific cell populations.

GEG Tech has designed three non-integrating systems according the type of integrase mutation:

The integrase mutations  almost completely disable the integration capabilities; however, there remains a residual activity estimated around 0,1%. The three different mutations designed by GEG Tech are IN(D64V), IN(N) and IN(LQ). These three mutations can exhibit different profiles of expression or level of integrase residual activity depending  the experimental context. GEG Tech recommends to test them to determine which one is best suited to the specific project.

RNA systems

HOW DOES IT WORK ?

Technologies -
  • Transient expression

  • Genome editing – CRISPR or ZFN

  • Very high level of biosafety

  • Vaccination, Gene & Cell therapies

GEG Tech RNA systems are derived from lentivirus form which the reverse transcriptase has been mutated. The mutations implemented prevent the conversion of RNA vector genomes in DNA vector genomes. Consequently, with the appropriate design, these systems can be taken in charge by cells as mRNAs coding transgenes of interest. These RNA systems induce no genotoxic effects and have a high level of biosafety combined with a high efficacy, close to that of the integrating lentiviral vectors and even higher in several specific contexts.

These non integrating systems and are used for a short-term expression in dividing and non-dividing cells in vitro as well as in vivo or for transgenesis. The design and process of production are suitable with GLP and GMP production for clinical projects.

These RNA systems take advantage of lentiviral vectors and provide a good alternative to mRNA transfection because they are more efficient, more stable in vivo and their flexibility of design are better, notably the envelop engineering allowing to target specific cell populations.

GEG Tech has designed three RNA systems according the type of reverse transcriptase mutation:

RT(D110E), RT(E478Q) and RT(D1003/E478Q) are three mutation versions that disable the reverse-transcriptase of the vector, making the expression highly transient from RNA molecules. The three mutations exhibit a full inactivation of the reverse transcription capabilities, no residual activity has been observed. However, these three mutations can exhibit different profiles of expression depending  the experimental context. GEG Tech recommends to test them to determine which one is the best suited according the project.

Live tracking of Lenti-ONE in living cells

The ANCHOR breakthrough technology developed by NeoVirTech allows real time visualisation and quantification of virus and viral vector infection/transduction and replication in living cells. This innovative system allows to tag the virus genomes and to visualize their cycle with an extreme accuracy.

GFP spots in living cells

GEG Tech has successfully integrated the ANCHOR system in their lenti-ONE lines. It is now possible to easily and precisely live visualize the transduction step of a Lenti-ONE, containing standard transgenes or your sequence of interest, to evaluate precisely the efficiency of the transduction in your experimental context to find the best conditions.

Furthermore, ANCHOR system allows the precise determination and without difficulty of the number of integration of lenti-ONE genomes by cell.This way, you can screen and easily select clonal populations with the wanted number of integration.

As you can see on the video below, ANCHOR technology is based on two elements, the fusion of OR protein with a florescent reporter and the ANCH sequence which binds OR. GEG Tech provides ANCHOR technology either through two vectors system (OR and ANCH in two different Lenti-ONE) or in all-in-one system with OR and ANCH in the same Lenti-ONE.

The ANCH sequence contains nucleation seeds for the binding of OR proteins. Insertion of the sequence in the vector genome does not disturb the expression of Lenti-ONE nor induces genotoxicity.The OR-Fluorescent Protein expressing gene specifically recognizes and spreads on the ANCH sequence, triggering the formation of a fluorescent focus easily detectable and quantifiable by microscopy (either classical or High content). The strong accumulation of OR-FP protein onto its cognate ANCH sequence is far enough to detect single DNA molecules. Therefore, fluorescent spot corresponds to the position of a single vector DNA copy.

Click on the miniature to see the full-size image or here to download the PDF.

Don’t hesitate to ask more informations about ANCHOR-Lenti-ONE. We also can include it in your system.