title: Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1.
abstract: Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1-MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, we have generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. We then developed an innovative 96-well plate screening platform to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of the hits, which corroborated the feasibility of the approach described herein to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery.
authors.affiliation: Valentia BioPharma, Scientific Park of the University of Valencia, Paterna, Valencia 46980, Spain. Department of Genetics, University of Valencia, Burjassot, Valencia 46010, Spain.
authors.forename: Irma
authors.initials: I
authors.lastname: García-Alcover
authors.affiliation: Valentia BioPharma, Scientific Park of the University of Valencia, Paterna, Valencia 46980, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Colonques-Bellmunt
authors.affiliation: Valentia BioPharma, Scientific Park of the University of Valencia, Paterna, Valencia 46980, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Garijo
authors.affiliation: Valentia BioPharma, Scientific Park of the University of Valencia, Paterna, Valencia 46980, Spain.
authors.forename: José R
authors.initials: JR
authors.lastname: Tormo
authors.affiliation: Department of Genetics, University of Valencia, Burjassot, Valencia 46010, Spain. INCLIVA Health Research Institute, Valencia 46010, Spain.
authors.forename: Rubén
authors.initials: R
authors.lastname: Artero
authors.affiliation: Valentia BioPharma, Scientific Park of the University of Valencia, Paterna, Valencia 46980, Spain.
authors.forename: Mari Carmen
authors.initials: MC
authors.lastname: Álvarez-Abril
authors.affiliation: Valentia BioPharma, Scientific Park of the University of Valencia, Paterna, Valencia 46980, Spain. a.lopez@valentiabiopharma.com.
authors.forename: Arturo
authors.initials: A
authors.lastname: López Castel
authors.affiliation: Valentia BioPharma, Scientific Park of the University of Valencia, Paterna, Valencia 46980, Spain. Department of Genetics, University of Valencia, Burjassot, Valencia 46010, Spain. INCLIVA Health Research Institute, Valencia 46010, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Pérez-Alonso
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D004331:Drosophila melanogaster; D057166:High-Throughput Screening Assays; D009223:Myotonic Dystrophy / Q000235:genetics*
keywords: In vivo screening; Luciferase; Minigene; Myotonic dystrophy; Splicing