Aminoglycosides were previously suggested as potential therapeutics for the read-through of nonsense mutations leading to various genetic diseases.
13,16 Here we demonstrate the ability of a series of aminoglycosides to induce read-through in the disease causing p.R31X nonsense mutation in USH1C both in vitro and in cell culture assays along within retinal cells. We observed substantial differences in read-through efficiency between in vitro translation assays and cellular systems that have previously been reported for various other systems.
13,16 Possible reasons for these differences are the cytotoxicity of the tested compound and the different length of the recovered polypeptide in the test systems. With regard to the latter, in the present in vitro experiments a short 35-kDa reporter polypeptide is recovered, whereas in cell culture experiments an 80-kDa full-length tagged harmonin a1 was recovered. Nevertheless, in both cases the highest rate of read-through was induced with G418, followed by gentamicin, NB30, and paromomycin. These data are consistent with the previously reported data gathered on other mutations.
20 –22,25,46 It is noteworthy that the read-through efficacy of the novel designed aminoglycoside NB30 corresponds to the data recently obtained for the investigational new read-through–inducing drug PTC124 in cell culture and retinal explants (Goldmann et al., manuscript submitted). Based on the promising preclinical results, the latter drug is currently applied in clinical trials for the treatment of patients with cystic fibrosis caused by nonsense mutations.
47 The impact of the tested aminoglycosides by the read-through of the
USH1C-p.R31X nonsense mutation is further demonstrated by showing restoration of the harmonin scaffold function. The recovered harmonin protein after the treatments with G418, gentamicin, and NB30 exhibited 75% binding activity to the tail of USH2a molecules compared with the wt harmonin protein. Considering the observed read-through efficiencies of 22% by G418 and 2.6% by NB30 (
Table 1), the binding activity data correlate to a restoration of approximately 16.5% and 2% activity of harmonin scaffolding function in the cell by G418 and NB30, respectively (
Table 1). The aminoglycoside-induced production of 1% to 3% full-size normal protein in a recessive disease may be sufficient to stop or slow down the progression of the disease,
19,48 particularly in USH1C, in which protein expression approaches zero and in which even such a low protein level is sufficient to restore a near-normal or at least a clinically less severe phenotype.
26,49,50 Interestingly, nonsense mutations in
USH1C leading to the expression of truncated harmonin cause combined deaf-blindness (USH1), whereas certain missense mutations introducing a punctual incorrect amino acid in the protein sequence lead to nonsyndromic deafness not associated with RP.
2,11,12 The transcriptional read-through of a nonsense mutation such as the
USH1C-p.R31X mutation can introduce a correct or an incorrect amino acid. In any case, the read-through–induced restoration of full-length harmonin of NB30 should be sufficient for retinal function.