Aminoglycoside-induced mutation suppression (stop codon readthrough) as a therapeutic strategy for Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is the most common, lethal, X-linked genetic disease, affecting 1 in 3500 newborn males. It is caused by mutations in the DMD gene. Owing to the large size of the gene, the mutation rate in both germline and somatic cells is very high. Nearly 13-15% of DMD cases are caused by nonsense mutations leading to premature termination codons in the reading frame that results in truncated dystrophin protein. Currently there is no cure for DMD. The only available treatment is the use of glucocorticoids that have modest beneficial effects accompanied by significant side effects. Different therapeutic strategies have been developed ranging from gene therapy to exon skipping and nonsense mutation suppression to produce the full-length protein. These strategies have shown promise in the mdx mouse model of muscular dystrophy where they have been reported to ameliorate the dystrophic phenotype and correct the physiological defects in the membrane. Each of these molecular approaches are being investigated in clinical trials. Here we review nonsense mutation suppression by aminoglycosides as a therapeutic strategy to treat DMD with special emphasis on gentamicin-induced readthrough of disease-causing premature termination codons.

Keywords: DMD; dystrophinopathy; gentamicin; mutation suppression; readthrough.

Figures

Figure 1.

Induction of protein translation by gentamicin. (A) Normal protein synthesis. The ribosome binds to the mRNA and the recognition occurs between mRNA codon and amino-acyl-tRNA at the A site and an amino acid is inserted by the corresponding t-RNA. As the ribosome moves along the mRNA, the polypeptide chain is moved to the P site. This process continues until the normal stop codon is reached where the release factors bind to the mRNA and polypeptide is released. (B) Readthrough of premature termination codons (PTCs) in the presence of gentamicin. In PTC-bearing transcripts, the translation halts at the PTC. However, in the presence of gentamicin, a conformation change occurs at the decoding site which leads to reduced discrimination of near-cognate tRNAs. A near-cognate amino acid is thus inserted at the stop site and the protein synthesis continues to the normal stop codon of the gene making the full-length protein.

Figure 2.

4,6-disubstituted 2-deoxystreptoamine (2-DOS) structure of gentamicin (adapted from Hermann, 2007). The differences between three variants of gentamicin are at R1 and R2 position and are highlighted with red boxes. These slight differences have been proposed to contribute to readthrough potential of gentamicin.

Figure 3.

Serum creatine kinase (CK) level in Duchenne muscular dystrophy (DMD) subjects bearing stop codons treated with gentamicin daily for 14 days. A statistically significant change compared with baseline was observed at day 14 (*p = 0.007). CK level gradually returned to baseline as seen at day 28 and day 42 reflecting the long half-life of dystrophin produced by readthrough.

Figure 4.

Dystrophin expression in muscle biopsies pretreatment and posttreament with gentamicin measured by immunofluorescence. (A), (B) Pretreatment and posttreatment muscle biopsy sections stained with NCL-Dys2 antibody in patient 6 (Table 1) treated with gentamicin for 6 months (once a week). Dystrophin levels increased from 4.5% to 15.44% posttreatment. (C), (D) Pretreatment and posttreatment muscle biopsy sections stained with NCL-Dys2 antibody in patient 10 (Table 1) treated with gentamicin for 6 months (twice a week). Dystrophin levels increased from 2.52% to 13.0% posttreatment. Scale bar = 100 µm.

Figure 5.

(A) Dystrophin-specific T-cell immunity. Peripheral blood mononuclear cells (PBMCs) collected from Subject 11 (Table 1) during gentamicin treatment (6 months, twice a week) at day 180 and 4 months after the end of the treatment (day 300) were stimulated with minidystrophin peptide pools MDP-1, MDP-2, and MDP-3. Immune response was observed in MDP3 stimulated PBMCs at day 180 but not at day 300. (B) Day 180 PBMCs were tested against 14 intersecting subpools of MDP-3 to identify single 20 amino acid peptide targeted by the T cells. Only two pools (F & I) that had peptide 126 in common were recognized localizing the epitope to dystrophin amino acid 3325–3344.