Sandrine Baghdoyan: Research Engineer (Inserm)
Jacqueline Gide: Associate engineer (CECS)
Aims and background:
Identification of new pathological biomarkers for monogenic diseases using a functional genomic approach.
Monogenic diseases are often lacking of physiological human cellular models dedicated to the study of the molecular mechanism implicated in the development of the pathology. When they exist, they often consist in patient’s samples difficult to amplify and providing a high variability between donors. Human Embryonic Stem (hES) cells derived from Preimplantation Genetic Diagnosed (PGD)-embryos offer a new alternative source of cellular model as they can be largely expanded, differentiated in several cell types and harbors “naturally” the causative mutation of the pathology. Such cell lines are now opening the opportunity to carry out “large scale” functional genomics screens in order to identify the genes able to revert the mutant phenotype of the cells.
The proof of concept of such approach has been initiated in the team on a PGD-derived hES cells carrying the causative mutation for Myotonic Dystrophy type 1 (DM1). We perform the transient silencing of several genes in parallel by RNA interference in order to identify those able to modify two types of DM1 biomarkers expressed by one DM1-hES cell line progeny : the pathological intranuclear ribonucleoaggregates called foci and the insulin receptor splicing defect.
Our aim is to highlight the genetic networks that can impact on DM1 biomarkers to provide new therapeutic targets for treatments by chemical compounds.
In the future, such strategy of highly parallel gene silencing will then be adapted to progenies derived from other-PGD-derived hES cells for which biomarkers are identified.
Strategy, means and methods:
To transiently silence in parallel several genes, we take advantage of “home designed” or commercially defined sets of siRNA that we transfect in one mesodermal progeny derived from DM1-PGD-hES cells in 96- or 384-well plates by the use of the Bravo automate from Velocity 11.
Results and future prospects:
Using a small set of « home designed » siRNA we have identified one “modifier” gene whose downregulation or functional inactivation by chemical compounds is able to correct the splicing defect of the insulin receptor in DM1mutated cells.
The ability of this gene to modify the other DM1 biomarkers will be explored in various cell types by classical experimental approaches and the molecular mechanism implicated to correct the DM1 missplicings will be analyzed. Finally, the functional inactivation of our target gene by one identified pharmaceutical agent will be tested in DM1 mouse model in order to monitor its ability to impact in vivo on DM1 molecular and physiological features.
The perspectives are to upgrade the scale of the functional genomic screen to a genome wide study to get an highlight of all of the biological networks relevant to be targeted for DM1.