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Directed evolution of genomic regions based on lentiviral vectors

Référence

03894-01

Statut des brevets

Priority patent application EP09290856.5 filed on November 10th, 2009 entitled “Lentiviral-based vector and its use in directed evolution of genomic regions, genes and polynucleotides”
PCT/EP2010/067246 filed on November 10th, 2010
WO 2011/058081 published on May 19th, 2011

Inventeurs

Matteo NEGRONI
Sarah GALLOIS-MONTBRUN
Paola ROSSOLILLO
Vicenzo DI BARTOLO
Gilles UZE
Etienne SIMON-LORIERE
Roland MARQUET
Valérie VIVET-BOUDOU

Statut commercial

Exclusive or non-exclusive licenses, Collaborative agreement

Laboratoire

Architecture et réactivité de l’ARN, a CNRS laboratory (UPR 9002) in Strasbourg, France, http://www-ibmc.u-strasbg.fr/arn/

Description

CONTEXT

Randomised mutagenesis of genes of interest is nowadays commonly used to solve molecular genetic engineering problems. In the existing prior art, the method consists in (1) the generation of a library of mutated genes and (2) the screening of this library for the presence of mutants possessing a given property, either in vitro or in bacterial cells, if possible.
However, when eukaryotic proteins are targeted, the properties observed in vitro for a given mutant, often do not result in the desired phenotype when introduced in eukaryotic cells. There is thus a need for a simple and effective method to create genetic diversity, adapted to mammalian or human cell applications.

TECHNICAL DESCRIPTION

The present invention concerns a new method of directing evolution of a target genomic region (or gene) of interest for obtaining variants of this target genomic region, that confer a desired phenotype to target mammalian, and in particular human, cells.
It concerns an in vitro method to generate genetic variability by preparing a cell library as well as a method to isolate variants of the genomic region or protein able to impact the phenotype of a cell, and to identify these targeted variants. The cell library is obtained through repeated transductions by a population of conditional replication-defective lentiviral particles. This procedure allows to mutate and screen the clones in the same system.

BENEFITS

  • Generation of the library of genomic variants directly in the biological vector (lentiviral particles) that is used to deliver efficiently and in a controlled manner the target genomic region to the target cell
  • Generation of genetic variability by mutations and recombination events
  • A single copy of the genetic variant in each clone – very stable integration
  • Method adapted to work on mammalian (particularly human) proteins and applied to a wide range of cell types
  • Simpler, easier and more efficient (no false positive of desired phenotypes) than commonly used methods – no cytopathic effect induced with respect to previously described lentiviral based systems

INDUSTRIAL APPLICATIONS

This innovation is a powerful tool to generate a genetic variability, that is useful in molecular engineering, to design a desired activity in a genomic construct.

For further information, please contact us (Ref 03894-01)

 


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