טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSabbah Annie
SubjectEvaluating Suppression of Nonsense Mutations of the PCDH15
Gene, Underlying Type 1 Usher Syndrome, by
Aminoglycoside Antibiotics
DepartmentDepartment of Medicine
Supervisor Professor Tamar Ben-Yosef
Full Thesis textFull thesis text - English Version


Abstract

Type I Usher syndrome (USH1) is a recessively-inherited condition, characterized by profound prelingual deafness, vestibular areflexia, and prepubertal onset of retinitis pigmentosa (RP) which to date has no effective treatment. USH1 is genetically heterogeneous and can be caused by mutations in each of at least seven genes. While truncating mutations of certain genes cause USH1, missense mutations of some of the same genes cause nonsyndromic deafness, suggesting that partial or low level activity of the encoded proteins may be sufficient for normal retinal function although not for normal hearing. Interventions to enable at least some translation of full-length protein, may delay the onset and/or progression of RP in individuals with USH1 due to nonsense mutations. One such possible therapeutic approach is suppression of nonsense mutations by small molecules such as aminoglycoside antibiotics. Suppression of nonsense mutations by commercial aminoglycosides was initially demonstrated in vitro, using a transcription/ translation assay of a reporter plasmid harboring various nonsense mutations of USH1C, CDH23 and PCDH15, underlying USH1C, USH1D and USH1F, respectively. I also demonstrated ex vivo suppression, by the same aminoglycosides, of the p.R245X mutation, which was recently identified as a major cause of USH1 in Ashkenazi Jews. Qualitative ex vivo suppression of p.W1764X, a nonsense mutation underlying USH1D, was proved using the same expression system. In parallel, in collaboration with Prof. Baasov at the chemistry Faculty, Technion, we are developing a series of new aminoglycoside-derived compounds, which will maintain their suppressive activity, while having reduced toxicity. We have recently characterized NB30, a derivative of the commercial aminoglycoside paromomycin. NB30 promotes dose-dependent suppression of different nonsense mutations underlying USH1 with lower potency than the commercial aminoglycosides gentamicin and paromomycin. To further improve its capacities, a second generation derivative named NB54 was synthesized. We show that NB54 has significantly reduced cell, cochlear and acute toxicities and has substantially higher stop codon read-through potency in both in vitro and ex vivo studies than those of gentamicin, a currently clinically used aminoglycoside. The research described here will have important implications for development of targeted interventions that are effective for patients with USH1 and nonsyndromic RP caused by various nonsense mutations.