title: In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2.
abstract: Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.
authors.affiliation: Hereditary Cancer Program, Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Spain.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Castellanos
authors.forename: Imma
authors.initials: I
authors.lastname: Rosas
authors.forename: Ares
authors.initials: A
authors.lastname: Solanes
authors.forename: Isabel
authors.initials: I
authors.lastname: Bielsa
authors.forename: Conxi
authors.initials: C
authors.lastname: Lázaro
authors.forename: Cristina
authors.initials: C
authors.lastname: Carrato
authors.forename: Cristina
authors.initials: C
authors.lastname: Hostalot
authors.forename: Pepe
authors.initials: P
authors.lastname: Prades
authors.forename: Francesc
authors.initials: F
authors.lastname: Roca-Ribas
authors.forename: Ignacio
authors.initials: I
authors.lastname: Blanco
authors.forename: Eduard
authors.initials: E
authors.lastname: Serra
mesh_terms: D000293:Adolescent; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D018095:Germ-Line Mutation / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D060172:Morpholinos / Q000008:administration & dosage* / Q000235:genetics / Q000627:therapeutic use; D016518:Neurofibromatosis 2 / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D025581:Neurofibromin 2 / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics
chemical_list: D060172:Morpholinos; D025581:Neurofibromin 2; D016372:RNA, Antisense