New Relevant Animal Model for AlzheimerÂ’s Disease and Cholinergic Impairment Study

CONTEXT Cholinergic neurons are involved in numerous physiological functions in mammals including motor activity, sleep-wakefulness (vigilance) and cognitive behavior (cognition). The dramatic consequences of the loss of cholinergic neurons is associated with AlzheimerÂ’s disease, but impairment of the cholinergic system has been also reported in other pathologies, for example in certain forms of epilepsy and in schizophrenia. One way to investigate the function of cholinergic structures is to analyze the consequences of their impairment. Until recently, the generation of such a loss-of-function phenotype relied on the destruction of cholinergic neurons by methods which are not specific for these neurons (excitotoxins, electrolytic lesions). Moreover, constitutive knockout in the mouse of the choline acetyltransferase (ChAT) gene, which is the enzyme of acetylcholine biosynthesis, is further lethal at birth and thus cannot be used to investigate cholinergic function in the adult, while conditional gene knockout requires the slow and costly generation of mice with loxP sites and is restricted to date to this animal species.    The invention relates to methods and compositions for modulating (e.g. reducing or inhibiting) the expression of the choline acetyltransferase. This is achieved by knocking down the expression of ChAT by lentivirus-mediated RNA interference. BENEFITS Inventors provide new and powerful tools that are able to achieve effective and specific inhibition of choline acetyltransferase expression, and thus inhibition of acetylcholine synthesis, which can be spatially and/or timely controlled. The method according to the present invention now obviates the generation of knock-out mice and further allows studies in species other than the mouse.

DIRECTED EVOLUTION OF GENOMIC REGIONS BASED ON LENTIVIRAL VECTORS

 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.     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. The method is especially suited for genomic regions coding proteins from the following groups : -       Structural proteins ; -       signal transduction proteins, receptors or receptor-binding proteins ; -       proteins with enzymatic activity (i.e. kinase, pro-drug activation enzymes) -       secreted proteins (i.e. interferon) Â… 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