Abstract
ELX-02 is a clinical stage, small-molecule eukaryotic ribosomal selective glycoside acting to induce read-through of premature stop codons (PSCs) that results in translation of full-length protein. However, improved read-through at PSCs has raised the question of whether native stop codon (NSC) fidelity would be impacted. Here, we compare read-through by ELX-02 in PSC and NSC contexts. DMS-114 cells containing a PSC in the TP53 gene were treated with ELX-02 and tested for increased nuclear p53 protein expression while also monitoring two other proteins for NSC read-through. Additionally, blood samples were taken from healthy subjects pre- and post-treatment with ELX-02 (0.3-7.5 mg/kg). These samples were processed to collect white blood cells and then analyzed by western blot to identify native and potentially elongated proteins from NSC read-through. In a separate experiment, lymphocytes cultivated with vehicle or ELX-02 (20 and 100 μg/ml) were subjected to proteomic analysis. We found that ELX-02 produced significant read-through of the PSC found in TP53 mRNA in DMS-114 cells, resulting in increased p53 protein expression and consistent with decreased nonsense-mediated mRNA degradation. NSC read-through protein products were not observed in either DMS-114 cells or in clinical samples from subjects dosed with ELX-02. The number of read-through proteins identified by using proteomic analysis was lower than estimated, and none of the NSC read-through products identified with >2 peptides showed dose-dependent responses to ELX-02. Our results demonstrate significant PSC read-through by ELX-02 with maintained NSC fidelity, thus supporting the therapeutic utility of ELX-02 in diseases resulting from nonsense alleles. SIGNIFICANCE STATEMENT: ELX-02 produces significant read-through of premature stop codons leading to full-length functional protein, demonstrated here by using the R213X mutation in the TP53 gene of DMS-114 cells. In addition, three complementary techniques suggest that ELX-02 does not promote read-through of native stop codons at concentrations that lead to premature stop codon read-through. Thus, ELX-02 may be a potential therapeutic option for nonsense mutation-mediated genetic diseases.
Highlights
10–12% of all mutations are single-point mutations that lead to an in-frame UAA, UAG, or UGA codon in messenger ribonucleic acid (Krawczak et al, 2000; Mort et al, 2008), thereby introducing a stop signal
The studies reported here demonstrate that ELX-02 produced significant premature stop codon (PSC) read-through of an exemplar nonsense mutation in the TP53 gene of a cancer cell line but did not increase the presence of extended protein variants indicative of native stop codon (NSC) read-through in either DMS-114 cells or peripheral blood mononuclear cells (PBMC) derived from normal healthy human subjects. These studies build on previous work testing the potential for translational read-through of nonsense mutations by ELX-02 using in vitro models (Kandasamy et al, 2012; Xue et al, 2014; Bidou et al, 2017; Friesen et al, 2018; Brasell et al, 2019) and in vivo mouse models of cystic fibrosis and cystinosis (Xue et al, 2014; Brasell et al, 2019)
These studies show that ELX-02 restores functional activity to proteins encoded by genes containing a PSC yet has limited impact on NSCs and supports its therapeutic potential in diseases resulting from nonsense alleles
Summary
10–12% of all mutations are single-point mutations that lead to an in-frame UAA, UAG, or UGA codon in messenger ribonucleic acid (mRNA) (Krawczak et al, 2000; Mort et al, 2008), thereby introducing a stop signal. These “nonsense” mutations that result in premature stop codons (PSCs) are the cause of a significant number of genetic diseases (Linde and Kerem, 2008), including cystic fibrosis, cystinosis, Usher syndrome, primary ciliary dyskinesia, polycystic kidney disease, mucopolysaccharidosis I, and a variety of cancers (Bordeira-Carrico et al, 2012). Curative therapies do not exist for most nonsense mutation diseases, and treatment options are needed beyond palliative care
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