title: Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa.
abstract: The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.
authors.affiliation: Centre de recherche Institut de la Vision, Sorbonne Universités, Université Pierre et Marie Curie UM80, INSERM U968, and CNRS UMR 7210, Paris, France.
authors.forename: Adeline
authors.initials: A
authors.lastname: Berger
authors.affiliation: Centre de recherche en Myologie, Sorbonne Universités, Université Pierre et Marie Curie, UM76, INSERM U974 and CNRS FRE 3617, Paris, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Lorain
authors.affiliation: Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Département des Sciences du Vivant (DSV), Institut d'Imagerie Biomédicale (I2BM), MIRCen, Fontenay-aux-Roses, France; Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud, Université Paris-Saclay, UMR9199, Neurodegenerative Diseases Laboratory, Fontenay-aux-Roses, France.
authors.forename: Charlène
authors.initials: C
authors.lastname: Joséphine
authors.affiliation: Centre de recherche Institut de la Vision, Sorbonne Universités, Université Pierre et Marie Curie UM80, INSERM U968, and CNRS UMR 7210, Paris, France.
authors.forename: Melissa
authors.initials: M
authors.lastname: Desrosiers
authors.affiliation: Centre de recherche en Myologie, Sorbonne Universités, Université Pierre et Marie Curie, UM76, INSERM U974 and CNRS FRE 3617, Paris, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Peccate
authors.affiliation: Centre de recherche en Myologie, Sorbonne Universités, Université Pierre et Marie Curie, UM76, INSERM U974 and CNRS FRE 3617, Paris, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Voit
authors.affiliation: UFR des sciences de la santé Simone Veil, Université Versailles Saint-Quentin, Montigny-le-Bretonneux, France.
authors.forename: Luis
authors.initials: L
authors.lastname: Garcia
authors.affiliation: Centre de recherche Institut de la Vision, Sorbonne Universités, Université Pierre et Marie Curie UM80, INSERM U968, and CNRS UMR 7210, Paris, France; Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France; Fondation Ophtalmologique Adolphe de Rothschild, Paris, France; Institute of Ophthalmology, University College of London, London, UK.
authors.forename: José-Alain
authors.initials: JA
authors.lastname: Sahel
authors.affiliation: Centre de recherche Institut de la Vision, Sorbonne Universités, Université Pierre et Marie Curie UM80, INSERM U968, and CNRS UMR 7210, Paris, France; Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Département des Sciences du Vivant (DSV), Institut d'Imagerie Biomédicale (I2BM), MIRCen, Fontenay-aux-Roses, France; Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud, Université Paris-Saclay, UMR9199, Neurodegenerative Diseases Laboratory, Fontenay-aux-Roses, France. Electronic address: alexis.bemelmans@cea.fr.
authors.forename: Alexis-Pierre
authors.initials: AP
authors.lastname: Bemelmans
mesh_terms: D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D015870:Gene Expression; D005799:Genes, Dominant; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007438:Introns; D051379:Mice; D008822:Mice, Transgenic; D010641:Phenotype; D010786:Photoreceptor Cells / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D021381:Protein Transport; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000628:therapy; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020040:Trans-Splicing; D014161:Transduction, Genetic
chemical_list: D012322:RNA Precursors; D012333:RNA, Messenger; D012243:Rhodopsin