title: Ex vivo treatment with a novel synthetic aminoglycoside NB54 in primary fibroblasts from Rett syndrome patients suppresses MECP2 nonsense mutations.
abstract: BACKGROUND
  Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2) comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT). Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro.
  
  
  RESULTS
  We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X) common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%), R270X (27%) and R294X (18%). In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF).
  
  
  CONCLUSIONS
  Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.
authors.affiliation: Sagol Neuroscience Center, Sheba Medical Center, Tel Hashomer, Israel.
authors.forename: Manuela
authors.initials: M
authors.lastname: Vecsler
authors.forename: Bruria
authors.initials: B
authors.lastname: Ben Zeev
authors.forename: Igor
authors.initials: I
authors.lastname: Nudelman
authors.forename: Yair
authors.initials: Y
authors.lastname: Anikster
authors.forename: Amos J
authors.initials: AJ
authors.lastname: Simon
authors.forename: Ninette
authors.initials: N
authors.lastname: Amariglio
authors.forename: Gideon
authors.initials: G
authors.lastname: Rechavi
authors.forename: Timor
authors.initials: T
authors.lastname: Baasov
authors.forename: Eva
authors.initials: E
authors.lastname: Gak
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology*; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D018389:Codon, Nonsense / Q000235:genetics*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D005839:Gentamicins / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015518:Rett Syndrome / Q000235:genetics* / Q000473:pathology*
chemical_list: C540944:5-O-(5-amino-5-deoxyribofuranosyl)-1N-(4-amino-2-hydroxybutanoyl)paromamine; D000617:Aminoglycosides; D019208:Brain-Derived Neurotrophic Factor; D018389:Codon, Nonsense; D005839:Gentamicins; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D012333:RNA, Messenger